Question: Autologous transplantation of mesenchymal stem cells causes fewer opportunistic infections than induction therapy with anti-interleukin-2 receptor antibodies.

Title: Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial. Content: CONTEXT Antibody-based induction therapy plus calcineurin inhibitors (CNIs) reduce acute rejection rates in kidney recipients; however, opportunistic infections and toxic CNI effects remain challenging. Reportedly, mesenchymal stem cells (MSCs) have successfully treated graft-vs-host disease. OBJECTIVE To assess autologous MSCs as replacement of antibody induction for patients with end-stage renal disease who undergo ABO-compatible, cross-match-negative kidney transplants from a living-related donor. DESIGN, SETTING, AND PATIENTS One hundred fifty-nine patients were enrolled in this single-site, prospective, open-label, randomized study from February 2008-May 2009, when recruitment was completed. INTERVENTION Patients were inoculated with marrow-derived autologous MSC (1-2 x 10(6)/kg) at kidney reperfusion and two weeks later. Fifty-three patients received standard-dose and 52 patients received low-dose CNIs (80% of standard); 51 patients in the control group received anti-IL-2 receptor antibody plus standard-dose CNIs. MAIN OUTCOME MEASURES The primary measure was 1-year incidence of acute rejection and renal function (estimated glomerular filtration rate [eGFR]); the secondary measure was patient and graft survival and incidence of adverse events. RESULTS Patient and graft survival at 13 to 30 months was similar in all groups. After 6 months, 4 of 53 patients (7.5%) in the autologous MSC plus standard-dose CNI group (95% CI, 0.4%-14.7%; P = .04) and 4 of 52 patients (7.7%) in the low-dose group (95% CI, 0.5%-14.9%; P = .046) compared with 11 of 51 controls (21.6%; 95% CI, 10.5%-32.6%) had biopsy-confirmed acute rejection. None of the patients in either autologous MSC group had glucorticoid-resistant rejection, whereas 4 patients (7.8%) in the control group did (95% CI, 0.6%-15.1%; overall P = .02). Renal function recovered faster among both MSC groups showing increased eGFR levels during the first month after surgery than the control group. Patients receiving standard-dose CNI had a mean difference of 6.2 mL/min per 1.73 m(2) (95% CI, 0.4-11.9; P=.04) and those in the low-dose CNI of 10.0 mL/min per 1.73 m(2) (95% CI, 3.8-16.2; P=.002). Also, during the 1-year follow-up, combined analysis of MSC-treated groups revealed significantly decreased risk of opportunistic infections than the control group (hazard ratio, 0.42; 95% CI, 0.20-0.85, P=.02) CONCLUSION Among patients undergoing renal transplant, the use of autologous MSCs compared with anti-IL-2 receptor antibody induction therapy resulted in lower incidence of acute rejection, decreased risk of opportunistic infection, and better estimated renal function at 1 year. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00658073.

Title: The antidiabetic effect of mesenchymal stem cells is unrelated to their transdifferentiation potential but to their capability to restore Th1/Th2 balance and to modify the pancreatic microenvironment. Content: Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease that results from cell-mediated autoimmune destruction of insulin-producing cells. In T1DM animal models, it has been shown that the systemic administration of multipotent mesenchymal stromal cells, also referred as to mesenchymal stem cells (MSCs), results in the regeneration of pancreatic islets. Mechanisms underlying this effect are still poorly understood. Our aims were to assess whether donor MSCs (a) differentiate into pancreatic β-cells and (b) modify systemic and pancreatic pathophysiologic markers of T1DM. After the intravenous administration of 5 × 10(5) syngeneic MSCs, we observed that mice with T1DM reverted their hyperglycemia and presented no donor-derived insulin-producing cells. In contrast, 7 and 65 days post-transplantation, MSCs were engrafted into secondary lymphoid organs. This correlated with a systemic and local reduction in the abundance of autoaggressive T cells together with an increase in regulatory T cells. Additionally, in the pancreas of mice with T1DM treated with MSCs, we observed a cytokine profile shift from proinflammatory to antinflammatory. MSC transplantation did not reduce pancreatic cell apoptosis but recovered local expression and increased the circulating levels of epidermal growth factor, a pancreatic trophic factor. Therefore, the antidiabetic effect of MSCs intravenously administered is unrelated to their transdifferentiation potential but to their capability to restore the balance between Th1 and Th2 immunological responses along with the modification of the pancreatic microenvironment. Our data should be taken into account when designing clinical trials aimed to evaluate MSC transplantation in patients with T1DM since the presence of endogenous precursors seems to be critical in order to restore glycemic control.

Title: Comparison of allogeneic vs autologous bone marrow–derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial. Content: CONTEXT Mesenchymal stem cells (MSCs) are under evaluation as a therapy for ischemic cardiomyopathy (ICM). Both autologous and allogeneic MSC therapies are possible; however, their safety and efficacy have not been compared. OBJECTIVE To test whether allogeneic MSCs are as safe and effective as autologous MSCs in patients with left ventricular (LV) dysfunction due to ICM. DESIGN, SETTING, AND PATIENTS A phase 1/2 randomized comparison (POSEIDON study) in a US tertiary-care referral hospital of allogeneic and autologous MSCs in 30 patients with LV dysfunction due to ICM between April 2, 2010, and September 14, 2011, with 13-month follow-up. INTERVENTION Twenty million, 100 million, or 200 million cells (5 patients in each cell type per dose level) were delivered by transendocardial stem cell injection into 10 LV sites. MAIN OUTCOME MEASURES Thirty-day postcatheterization incidence of predefined treatment-emergent serious adverse events (SAEs). Efficacy assessments included 6-minute walk test, exercise peak VO2, Minnesota Living with Heart Failure Questionnaire (MLHFQ), New York Heart Association class, LV volumes, ejection fraction (EF), early enhancement defect (EED; infarct size), and sphericity index. RESULTS Within 30 days, 1 patient in each group (treatment-emergent SAE rate, 6.7%) was hospitalized for heart failure, less than the prespecified stopping event rate of 25%. The 1-year incidence of SAEs was 33.3% (n = 5) in the allogeneic group and 53.3% (n = 8) in the autologous group (P = .46). At 1 year, there were no ventricular arrhythmia SAEs observed among allogeneic recipients compared with 4 patients (26.7%) in the autologous group (P = .10). Relative to baseline, autologous but not allogeneic MSC therapy was associated with an improvement in the 6-minute walk test and the MLHFQ score, but neither improved exercise VO2 max. Allogeneic and autologous MSCs reduced mean EED by −33.21% (95% CI, −43.61% to −22.81%; P < .001) and sphericity index but did not increase EF. Allogeneic MSCs reduced LV end-diastolic volumes. Low-dose concentration MSCs (20 million cells) produced greatest reductions in LV volumes and increased EF. Allogeneic MSCs did not stimulate significant donor-specific alloimmune reactions. CONCLUSIONS In this early-stage study of patients with ICM, transendocardial injection of allogeneic and autologous MSCs without a placebo control were both associated with low rates of treatment-emergent SAEs, including immunologic reactions. In aggregate, MSC injection favorably affected patient functional capacity, quality of life, and ventricular remodeling. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01087996.

Title: Immunogenicity of induced pluripotent stem cells Content: Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.

Title: Expandable Cardiovascular Progenitor Cells Reprogrammed from Fibroblasts. Content: Stem cell-based approaches to cardiac regeneration are increasingly viable strategies for treating heart failure. Generating abundant and functional autologous cells for transplantation in such a setting, however, remains a significant challenge. Here, we isolated a cell population with extensive proliferation capacity and restricted cardiovascular differentiation potentials during cardiac transdifferentiation of mouse fibroblasts. These induced expandable cardiovascular progenitor cells (ieCPCs) proliferated extensively for more than 18 passages in chemically defined conditions, with 10(5) starting fibroblasts robustly producing 10(16) ieCPCs. ieCPCs expressed cardiac signature genes and readily differentiated into functional cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) in vitro, even after long-term expansion. When transplanted into mouse hearts following myocardial infarction, ieCPCs spontaneously differentiated into CMs, ECs, and SMCs and improved cardiac function for up to 12 weeks after transplantation. Thus, ieCPCs are a powerful system to study cardiovascular specification and provide strategies for regenerative medicine in the heart.

Title: A more efficient method to generate integration-free human iPS cells Content: We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match ∼20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.

Title: A crucial role for interleukin (IL)-1 in the induction of IL-17–producing T cells that mediate autoimmune encephalomyelitis Content: It was recently demonstrated that interleukin (IL)-23–driven IL-17–producing (ThIL-17) T cells mediate inflammatory pathology in certain autoimmune diseases. We show that the induction of antigen-specific ThIL-17 cells, but not T helper (Th)1 or Th2 cells, by immunization with antigens and adjuvants is abrogated in IL-1 receptor type I–deficient (IL-1RI−/−) mice. Furthermore, the incidence of experimental autoimmune encephalomyelitis (EAE) was significantly lower in IL-1RI−/− compared with wild-type mice, and this correlated with a failure to induce autoantigen-specific ThIL-17 cells, whereas induction of Th1 and Th2 responses was not substantially different. However, EAE was induced in IL-1RI−/− mice by adoptive transfer of autoantigen-specific cells from wild-type mice with EAE. IL-23 alone did not induce IL-17 production by T cells from IL-1RI−/− mice, and IL-23–induced IL-17 production was substantially enhanced by IL-1α or IL-1β, even in the absence of T cell receptor stimulation. We demonstrate essential roles for phosphatidylinositol 3-kinase, nuclear factor κB, and novel protein kinase C isoforms in IL-1– and IL-23–mediated IL-17 production. Tumor necrosis factor α also synergized with IL-23 to enhance IL-17 production, and this was IL-1 dependent. Our findings demonstrate that IL-1 functions upstream of IL-17 to promote pathogenic ThIL-17 cells in EAE.

Title: Stem cell transplantation in patients with autonomic neuropathy due to primary (AL) amyloidosis. Content: OBJECTIVES Patients with AL amyloidosis can benefit from high-dose chemotherapy and autologous stem cell transplantation (ASCT). Transplantation can be challenging due to fluid shifts, sepsis, and cardiac dysrhythmias. Amyloidosis may present with autonomic neuropathy (AN) that renders peritransplant care problematic. The purpose of this study was to determine the outcome of patients with AN during and after ASCT. METHODS We performed a case-control study of patients with AL amyloidosis with associated AN and compared them to a large matched cohort without AN who also underwent ASCT. RESULTS We identified 13 patients with AN who underwent ASCT and a matched control group of 95 patients without AN. Patients with AN had more organs involved (median 2.5 vs 1, p < 0.001) and the conditioning dose of melphalan was often reduced by 30% compared to controls without AN (p = 0.0015). Median duration of hospitalization was similar for both cohorts, as were engraftment kinetics. Atrial fibrillation occurred in all patients with AN but in only 1 control patient (p < 0.0001). Median overall survival (OS) for patients with AN was 29 months but >60 months for controls (p < 0.0001). On univariate analysis, cardiac involvement (p = 0.0132), AN (p = 0.0011), glomerular filtration rate (p = 0.038), number of organs involved (p = 0.0064), and NT-pro-BNP (p = 0.039) all had an impact on OS. On multivariate analysis, AN retained an independent adverse impact on OS. CONCLUSIONS Patients with autonomic neuropathy secondary to AL amyloidosis can undergo autologous stem cell transplantation with relative safety. Autonomic neuropathy is an independent, adverse determinant of survival in these patients.

Title: Senescent growth arrest in mesenchymal stem cells is bypassed by Wip1-mediated downregulation of intrinsic stress signaling pathways. Content: Human mesenchymal stem cells (hMSCs) have been widely studied as a source of primary adult stem cells for cell therapy because of their multidifferentiation potential; however, the growth arrest (also known as "premature senescence") often found in hMSCs cultured in vitro has been a major obstacle to the in-depth characterization of these cells. In addition, the inability to maintain constant cell growth hampers the development of additional genetic modifications aimed at achieving desired levels of differentiation to specific tissues; however, the molecular mechanisms that govern this phenomenon remain unclear, with the exception of a few studies demonstrating that induction of p16INK4a is responsible for this senescence-like event. Here, we observed that the premature growth arrest in hMSCs occurs in parallel with the induction of p16INK4a, following abrogation of inhibitory phosphorylation of retinoblastoma protein. These stress responses were concurrent with increased formation of reactive oxygen species (ROSs) from mitochondria and increased p38 mitogen-activated protein kinase (MAPK) activity. The introduction of Wip1 (wild-type p53 inducible phosphatase-1), a well-studied stress modulator, significantly lowered p16INK4a expression and led to p38 MAPK inactivation, although it failed to affect the levels of ROSs. Moreover, the suppression of stress responses by Wip1 apparently extended the life span of hMSCs, compared with control conditions, while maintaining their multilineage differentiation potential. Based on these results, we suggest that senescent growth arrest in hMSCs may result from activation of stress signaling pathways and consequent onset of stress responses, due in part to ROS production during prolonged in vitro culture.

Title: Differentiation of mesenchymal stem cells and embryonic stem cells into steroidogenic cells using steroidogenic factor-1 and liver receptor homolog-1. Content: Previously, we have demonstrated that mesenchymal stem cells could be differentiated into steroidogenic cells through steroidogenic factor-1 and 8bromo-cAMP treatment. Use of liver receptor homolog-1, another of the nuclear receptor 5A family nuclear receptors, with 8bromo-cAMP also resulted in the differentiation of human mesenchymal stem cells into steroid hormone-producing cells. The same approaches could not be applied to other undifferentiated cells such as embryonic stem cells or embryonal carcinoma cells, because the over-expression of the nuclear receptor 5A family is cytotoxic to these cells. We established embryonic stem cells carrying tetracycline-regulated steroidogenic factor-1 gene at the ROSA26 locus. The embryonic stem cells were first differentiated into a mesenchymal cell lineage by culturing on collagen IV-coated dishes and treating with pulse exposures of retinoic acid before expression of steroidogenic factor-1. Although the untreated embryonic stem cells could not be converted into steroidogenic cells by expression of steroidogenic factor-1 in the absence of leukemia inhibitory factor due to inability of the cells to survive, the differentiated cells could be successfully converted into steroidogenic cells when expression of steroidogenic factor-1 was induced. They exhibited characteristics of adrenocortical-like cells and produced a large amount of corticosterone. These results indicated that pluripotent stem cells could be differentiated into steroidogenic cells by the nuclear receptor 5A family of protein via the mesenchymal cell lineage. This approach may provide a source of cells for future gene therapy for diseases caused by steroidogenesis deficiencies.

Title: Tightrope act: autophagy in stem cell renewal, differentiation, proliferation, and aging Content: Autophagy is a constitutive lysosomal catabolic pathway that degrades damaged organelles and protein aggregates. Stem cells are characterized by self-renewal, pluripotency, and quiescence; their long life span, limited capacity to dilute cellular waste and spent organelles due to quiescence, along with their requirement for remodeling in order to differentiate, all suggest that they require autophagy more than other cell types. Here, we review the current literature on the role of autophagy in embryonic and adult stem cells, including hematopoietic, mesenchymal, and neuronal stem cells, highlighting the diverse and contrasting roles autophagy plays in their biology. Furthermore, we review the few studies on stem cells, lysosomal activity, and autophagy. Novel techniques to detect autophagy in primary cells are required to study autophagy in different stem cell types. These will help to elucidate the importance of autophagy in stem cells during transplantation, a promising therapeutic approach for many diseases.

Title: Epithelial to Mesenchymal Transition by TGFβ-1 Induction Increases Stemness Characteristics in Primary Non Small Cell Lung Cancer Cell Line Content: BACKGROUND Cancer Stem Cells (CSCs) hypothesis asserts that only a small subset of cells within a tumour is capable of both tumour initiation and sustainment. The Epithelial-Mesenchymal Transition (EMT) is an embryonic developmental program that is often activated during cancer invasion and metastasis. The aim of this study is to shed light on the relationship between EMT and CSCs by using LC31 lung cancer primary cell line. MATERIALS AND METHODS A549 and LC31 cell lines were treated with 2 ng/ml TGFβ-1 for 30 days, and 80 days, respectively. To evaluate EMT, morphological changes were assessed by light microscopy, immunofluorescence and cytometry for following markers: cytokeratins, e-cadherin, CD326 (epithelial markers) and CD90, and vimentin (mesenchymal markers). Moreover, RT-PCR for Slug, Twist and β-catenin genes were performed. On TGFβ-1 treated and untreated LC31 cell lines, we performed stemness tests such as pneumospheres growth and stem markers expression such as Oct4, Nanog, Sox2, c-kit and CD133. Western Blot for CD133 and tumorigenicity assays using NOD/SCID mice were performed. RESULTS TGFβ-1 treated LC31 cell line lost its epithelial morphology assuming a fibroblast-like appearance. The same results were obtained for the A549 cell line (as control). Immunofluorescence and cytometry showed up-regulation of vimentin and CD90 and down-regulation of cytocheratin, e-cadherin and CD326 in TGFβ-1 treated LC31 and A549 cell lines. Slug, Twist and β-catenin m-RNA transcripts were up-regulated in TGFβ-1 treated LC31 cell line confirming EMT. This cell line showed also over-expression of Oct4, Nanog, Sox2 and CD133, all genes of stemness. In addition, in TGFβ-1 treated LC31 cell line, an increased pneumosphere-forming capacity and tumours-forming ability in NOD/SCID mice were detectable. CONCLUSIONS The induction of EMT by TGFβ-1 exposure, in primary lung cancer cell line results in the acquisition of mesenchymal profile and in the expression of stem cell markers.

Title: Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells Content: The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

Title: IL6 derived from cancer-associated fibroblasts promotes chemoresistance via CXCR7 in esophageal squamous cell carcinoma Content: Various factors and cellular components in the tumor microenvironment are key drivers associated with drug resistance in many cancers. Here, we analyzed the factors and molecular mechanisms involved in chemoresistance in patients with esophageal squamous cell carcinoma (ESCC). We found that interleukin 6 (IL6) derived mainly from cancer-associated fibroblasts played the most important role in chemoresistance by upregulating C-X-C motif chemokine receptor 7 (CXCR7) expression through signal transducer and activator of transcription 3/nuclear factor-κB pathway. CXCR7 knockdown resulted in the inhibition of IL6-induced proliferation and chemoresistance. In addition, CXCR7 silencing significantly decreased gene expression associated with stemness, chemoresistance and epithelial–mesenchymal transition and suppressed the proliferation ability of ESCC cells in three-dimensional culture systems and angiogenesis assay. In clinical samples, ESCC patients with high expression of CXCR7 and IL6 presented a significantly worse overall survival and progression-free survival upon receiving cisplatin after operation. These results suggest that the IL6–CXCR7 axis may provide a promising target for the treatment of ESCC.

Title: Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration. Content: Mesenchymal stem cells (MSCs) commonly defined by in vitro functions have entered clinical application despite little definition of their function in residence. Here, we report genetic pulse-chase experiments that define osteoblastic cells as short-lived and nonreplicative, requiring replenishment from bone-marrow-derived, Mx1(+) stromal cells with "MSC" features. These cells respond to tissue stress and migrate to sites of injury, supplying new osteoblasts during fracture healing. Single cell transplantation yielded progeny that both preserve progenitor function and differentiate into osteoblasts, producing new bone. They are capable of local and systemic translocation and serial transplantation. While these cells meet current definitions of MSCs in vitro, they are osteolineage restricted in vivo in growing and adult animals. Therefore, bone-marrow-derived MSCs may be a heterogeneous population with the Mx1(+) population, representing a highly dynamic and stress responsive stem/progenitor cell population of fate-restricted potential that feeds the high cell replacement demands of the adult skeleton.

Title: Very small embryonic-like stem cells with maximum regenerative potential get discarded during cord blood banking and bone marrow processing for autologous stem cell therapy. Content: Very small embryonic-like stem cells (VSELs) are possibly lost during cord blood banking and bone marrow (BM) processing for autologus stem cell therapy mainly because of their small size. The present study was conducted on human umbilical cord blood (UCB, n=6) and discarded red blood cells (RBC) fraction obtained after separation of mononuclear cells from human BM (n=6), to test this hypothesis. The results show that VSELs, which are pluripotent stem cells with maximum regenerative potential, settle along with the RBCs during Ficoll-Hypaque density separation. These cells are very small in size (3-5 μm), have high nucleo-cytoplasmic ratio, and express nuclear Oct-4, cell surface protein SSEA-4, and other pluripotent markers such as Nanog, Sox-2, Rex-1, and Tert as indicated by immunolocalization and quantitative polymerase chain reaction (Q-PCR) studies. Interestingly, a distinct population of slightly larger, round hematopoietic stem cells (HSCs) with cytoplasmic Oct-4 were detected in the "buffy" coat, which usually gets banked or used during autologus stem cell therapy. Immunohistochemical studies on the umbilical cord tissue (UCT) sections (n=3) showed the presence of nuclear Oct-4-positive VSELs and many fibroblast-like mesenchymal stem cells (MSCs) with cytoplasmic Oct-4. These VSELs with nuclear Oct-4, detected in UCB, UCT, and discarded RBC fraction obtained after BM processing, may persist throughout life, maintain tissue homeostasis, and undergo asymmetric cell division to self-renew as well as produce larger progenitor stem cells, viz. HSCs or MSCs, which follow differentiation trajectories depending on the somatic niche. Hence, it can be concluded that the true stem cells in adult body tissues are the VSELs, whereas the HSCs and MSCs are actually progenitor stem cells that arise by asymmetric cell division of VSELs. The results of the present study may help explain low efficacy reported during adult autologous stem cell trials, wherein unknowingly progenitor stem cells are injected rather than the pluripotent stem cells with maximum regenerative potential.

Title: The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells Content: The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

Title: Islet-Like Clusters Derived from Mesenchymal Stem Cells in Wharton's Jelly of the Human Umbilical Cord for Transplantation to Control Type 1 Diabetes Content: BACKGROUND There is a widespread interest in developing renewable sources of islet-replacement tissue for type I diabetes mellitus. Human mesenchymal cells isolated from the Wharton's jelly of the umbilical cord (HUMSCs), which can be easily obtained and processed compared with embryonic and bone marrow stem cells, possess stem cell properties. HUMSCs may be a valuable source for the generation of islets. METHODOLOGY AND PRINCIPAL FINDINGS HUMSCs were induced to transform into islet-like cell clusters in vitro through stepwise culturing in neuron-conditioned medium. To assess the functional stability of the islet-like cell clusters in vivo, these cell clusters were transplanted into the liver of streptozotocin-induced diabetic rats via laparotomy. Glucose tolerance was measured on week 12 after transplantation accompanied with immunohistochemistry and electron microscopy analysis. These islet-like cell clusters were shown to contain human C-peptide and release human insulin in response to physiological glucose levels. Real-time RT-PCR detected the expressions of insulin and other pancreatic beta-cell-related genes (Pdx1, Hlxb9, Nkx2.2, Nkx6.1, and Glut-2) in these islet-like cell clusters. The hyperglycemia and glucose intolerance in streptozotocin-induced diabetic rats was significantly alleviated after xenotransplantation of islet-like cell clusters, without the use of immunosuppressants. In addition to the existence of islet-like cell clusters in the liver, some special fused liver cells were also found, which characterized by human insulin and nuclei-positive staining and possessing secretory granules. CONCLUSIONS AND SIGNIFICANCE In this study, we successfully differentiate HUMSCs into mature islet-like cell clusters, and these islet-like cell clusters possess insulin-producing ability in vitro and in vivo. HUMSCs in Wharton's Jelly of the umbilical cord seem to be the preferential source of stem cells to convert into insulin-producing cells, because of the large potential donor pool, its rapid availability, no risk of discomfort for the donor, and low risk of rejection.

Title: Liver receptor homolog-1 regulates the transcription of steroidogenic enzymes and induces the differentiation of mesenchymal stem cells into steroidogenic cells. Content: Steroidogenic factor-1 (SF-1, also known as Ad4BP) has been demonstrated to be a primary transcriptional regulator of steroidogenic-related genes. However, mRNA for liver receptor homolog-1 (LRH-1), which together with SF-1, belongs to the NR5A nuclear receptor family, is expressed at much higher levels than SF-1 mRNA in the human gonad. In our previous studies, we demonstrated that SF-1 induced the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into steroidogenic cells such as Leydig or adrenocortical cells. The introduction of LRH-1 into human MSCs (hMSCs) with the aid of cAMP also induced the expression of steroidogenic enzymes, including CYP17, and their differentiation into steroid hormone-producing cells. Promoter analysis, EMSA, and chromatin immunoprecipitation assay using LRH-1-transduced hMSCs indicated that three LRH-1 binding sites were responsible for CYP17 transactivation. Immunohistochemical studies showed that LRH-1 protein was expressed in human Leydig cells. The CYP17 promoter region was highly methylated in hMSCs, whereas it was demethylated by the introduction of LRH-1 and cAMP treatment. These results indicate that LRH-1 could represent another key regulator of the steroidogenic lineage in MSCs and play a vital role in steroid hormone production in human Leydig cells.

Title: Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML Content: Cancer stem cells have been proposed to be important for initiation, maintenance and recurrence of various malignancies, including acute myeloid leukemia (AML). We have previously reported that CD34+CD38− human primary AML stem cells residing in the endosteal region of the bone marrow are relatively chemotherapy resistant. Using a NOD/SCID/IL2rγnull mouse model of human AML, we now show that the AML stem cells in the endosteal region are cell cycle quiescent and that these stem cells can be induced to enter the cell cycle by treatment with granulocyte colony-stimulating factor (G-CSF). In combination with cell cycle-dependent chemotherapy, G-CSF treatment significantly enhances induction of apoptosis and elimination of human primary AML stem cells in vivo. The combination therapy leads to significantly increased survival of secondary recipients after transplantation of leukemia cells compared with chemotherapy alone.

Title: Transgenic Interleukin 10 Prevents Induction of Experimental Autoimmune Encephalomyelitis Content: The effectiveness of interleukin 10 (IL-10) in the treatment of autoimmune-mediated central nervous system inflammation is controversial. Studies of the model system, experimental autoimmune encephalomyelitis (EAE), using various routes, regimens, and delivery methods of IL-10 suggest that these variables may affect its immunoregulatory function. To study the influence of these factors on IL-10 regulation of EAE pathogenesis, we have analyzed transgenic mice expressing human IL-10 (hIL-10) transgene under the control of a class II major histocompatibility complex (MHC) promoter. The hIL-10 transgenic mice are highly resistant to EAE induced by active immunization, and this resistance appears to be mediated by suppression of autoreactive T cell function. Myelin-reactive T helper 1 cells are induced but nonpathogenic in the IL-10 transgenic mice. Antibody depletion confirmed that EAE resistance is dependent on the presence of the transgenic IL-10. Mice expressing the hIL-10 transgene but not the endogenous murine IL-10 gene demonstrated that transgenic IL-10 from MHC class II–expressing cells is sufficient to block induction of EAE. This study demonstrates that IL-10 can prevent EAE completely if present at appropriate levels and times during disease induction.

Title: the Development of Autoimmune Myocarditis in Mice by an Content: BACKGROUND Interleukin (IL)-12 exerts a potent proinflammatory effect by stimulating T-helper (Th) 1 responses. This effect is believed to be mediated primarily through the activation of STAT4 and subsequent production of interferon (IFN)-gamma. Methods and Results- We examined the role of IL-12 receptor (IL-12R) signaling in the development of murine experimental autoimmune myocarditis (EAM) induced by cardiac myosin immunization. Both IL-12Rbeta1-deficient mice and STAT4-deficient mice were resistant to the induction of myocarditis. Treatment with exogenous IL-12 exacerbated disease. We questioned whether IFN-gamma is required for the disease-promoting activity of IL-12. On the contrary, we found that IFN-gamma suppresses EAM. Lack of IFN-gamma due to either depletion with an antibody or a genetic deficiency exacerbated myocarditis. Spleens from IFN-gamma-deficient mice immunized with cardiac myosin showed increased cellularity; greater numbers of CD3+, CD4+, CD8+, and IL-2-producing cells; and heightened ability to produce cytokines on stimulation in vitro. Treatment of mice with recombinant IFN-gamma suppressed the development of myocarditis. CONCLUSIONS IL-12/IL-12R/STAT4 signaling promotes the development of EAM. In contrast, IFN-gamma plays a protective role. The disease-limiting effects of IFN-gamma might be explained by its ability to control the expansion of activated T lymphocytes.

Title: Induction of myelodysplasia by myeloid-derived suppressor cells. Content: Myelodysplastic syndromes (MDS) are age-dependent stem cell malignancies that share biological features of activated adaptive immune response and ineffective hematopoiesis. Here we report that myeloid-derived suppressor cells (MDSC), which are classically linked to immunosuppression, inflammation, and cancer, were markedly expanded in the bone marrow of MDS patients and played a pathogenetic role in the development of ineffective hematopoiesis. These clonally distinct MDSC overproduce hematopoietic suppressive cytokines and function as potent apoptotic effectors targeting autologous hematopoietic progenitors. Using multiple transfected cell models, we found that MDSC expansion is driven by the interaction of the proinflammatory molecule S100A9 with CD33. These 2 proteins formed a functional ligand/receptor pair that recruited components to CD33’s immunoreceptor tyrosine-based inhibition motif (ITIM), inducing secretion of the suppressive cytokines IL-10 and TGF-β by immature myeloid cells. S100A9 transgenic mice displayed bone marrow accumulation of MDSC accompanied by development of progressive multilineage cytopenias and cytological dysplasia. Importantly, early forced maturation of MDSC by either all-trans-retinoic acid treatment or active immunoreceptor tyrosine-based activation motif–bearing (ITAM-bearing) adapter protein (DAP12) interruption of CD33 signaling rescued the hematologic phenotype. These findings indicate that primary bone marrow expansion of MDSC driven by the S100A9/CD33 pathway perturbs hematopoiesis and contributes to the development of MDS.

Title: Conversion of vascular endothelial cells into multipotent stem-like cells Content: Mesenchymal stem cells can give rise to several cell types, but varying results depending on isolation methods and tissue source have led to controversies about their usefulness in clinical medicine. Here we show that vascular endothelial cells can transform into multipotent stem-like cells by an activin-like kinase-2 (ALK2) receptor–dependent mechanism. In lesions from individuals with fibrodysplasia ossificans progressiva (FOP), a disease in which heterotopic ossification occurs as a result of activating ALK2 mutations, or from transgenic mice expressing constitutively active ALK2, chondrocytes and osteoblasts expressed endothelial markers. Lineage tracing of heterotopic ossification in mice using a Tie2-Cre construct also suggested an endothelial origin of these cell types. Expression of constitutively active ALK2 in endothelial cells caused endothelial-to-mesenchymal transition and acquisition of a stem cell–like phenotype. Similar results were obtained by treatment of untransfected endothelial cells with the ligands transforming growth factor-β2 (TGF-β2) or bone morphogenetic protein-4 (BMP4) in an ALK2-dependent manner. These stem-like cells could be triggered to differentiate into osteoblasts, chondrocytes or adipocytes. We suggest that conversion of endothelial cells to stem-like cells may provide a new approach to tissue engineering.

Title: Human Cytomegalovirus-Encoded Interleukin-10 Homolog Inhibits Maturation of Dendritic Cells and Alters Their Functionality Content: Interleukin-10 (IL-10) suppresses the maturation and cytokine production of dendritic cells (DCs), key regulators of adaptive immunity, and prevents the activation and polarization of naïve T cells towards protective gamma interferon-producing effectors. We hypothesized that human cytomegalovirus (HCMV) utilizes its viral IL-10 homolog (cmvIL-10) to attenuate DC functionality, thereby subverting the efficient induction of antiviral immune responses. RNA and protein analyses demonstrated that the cmvIL-10 gene was expressed with late gene kinetics. Treatment of immature DCs (iDCs) with supernatant from HCMV-infected cultures inhibited both the lipopolysaccharide-induced DC maturation and proinflammatory cytokine production. These inhibitory effects were specifically mediated through the IL-10 receptor and were not observed when DCs were treated with supernatant of cells infected with a cmvIL-10-knockout mutant. Incubation of iDCs with recombinant cmvIL-10 recapitulated the inhibition of maturation. Furthermore, cmvIL-10 had pronounced long-term effects on those DCs that could overcome this inhibition of maturation. It enhanced the migration of mature DCs (mDCs) towards the lymph node homing chemokine but greatly reduced their cytokine production. The inability of mDCs to secrete IL-12 was maintained, even when they were restimulated by the activated T-cell signal CD40 ligand in the absence of cmvIL-10. Importantly, cmvIL-10 potentiates these anti-inflammatory effects, at least partially, by inducing endogenous cellular IL-10 expression in DCs. Collectively, we show that cmvIL-10 causes long-term functional alterations at all stages of DC activation.

Title: Engraftment of engineered ES cell–derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium Content: Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)–derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6–10-fold because of induction of proliferation on purification. Long-term engraftment (4–5 months) was observed when co-transplanting selected ES cell–derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell–derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells.

Title: Mesenchymal and haematopoietic stem cells form a unique bone marrow niche Content: The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin+ MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent ‘mesenspheres’ that can self-renew and expand in serial transplantations. Nestin+ MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or β3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin+ cells and favours their osteoblastic differentiation, in vivo nestin+ cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin+ MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin+ cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs.

Title: Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway. Content: CD4(+) helper T (Th) cells play a crucial role in the delicate balance between host defense and autoimmune disease. Two important populations of helper T cells are the proinflammatory, interleukin-17 (IL-17)-producing (Th17) cells and the anti-inflammatory forkhead box P3-positive (FoxP3(+)) T regulatory (Treg) cells. Here we show that all-trans retinoic acid (ATRA) and other agonists of the retinoic acid receptor alpha (RARalpha) inhibit the formation of Th17 cells and promote FoxP3 expression. Conversely, inhibition of retinoic acid signaling constrains transforming growth factor beta (TGF-beta1) induction of FoxP3. The effect of ATRA is mediated independently of IL-2, signal transducer and activator of transcription 5 (Stat5) and Stat3, representing a novel mechanism for the induction of FoxP3 in CD4 T cells. As previous studies have shown that vitamin A derivatives are protective in animal models of autoimmune disease, the current data suggest a previously unrecognized role for RARalpha in the regulation of CD4(+) T-cell differentiation and provide a mechanism for the anti-inflammatory effects of retinoic acid.

Title: Mesenchymal Inflammation Drives Genotoxic Stress in Hematopoietic Stem Cells and Predicts Disease Evolution in Human Pre-leukemia. Content: Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system, but the underlying molecular mechanisms and relevance to human disease remain poorly defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the pre-leukemic disorder Shwachman-Diamond syndrome (SDS) induces mitochondrial dysfunction, oxidative stress, and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the SDS mouse model and a range of human pre-leukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome (MDS), the principal leukemia predisposition syndrome. Collectively, our findings identify mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as a targetable determinant of disease outcome in human pre-leukemia.

Title: Reprogramming committed murine blood cells to induced hematopoietic stem cells with defined factors. Content: Hematopoietic stem cells (HSCs) sustain blood formation throughout life and are the functional units of bone marrow transplantation. We show that transient expression of six transcription factors Run1t1, Hlf, Lmo2, Prdm5, Pbx1, and Zfp37 imparts multilineage transplantation potential onto otherwise committed lymphoid and myeloid progenitors and myeloid effector cells. Inclusion of Mycn and Meis1 and use of polycistronic viruses increase reprogramming efficacy. The reprogrammed cells, designated induced-HSCs (iHSCs), possess clonal multilineage differentiation potential, reconstitute stem/progenitor compartments, and are serially transplantable. Single-cell analysis revealed that iHSCs derived under optimal conditions exhibit a gene expression profile that is highly similar to endogenous HSCs. These findings demonstrate that expression of a set of defined factors is sufficient to activate the gene networks governing HSC functional identity in committed blood cells. Our results raise the prospect that blood cell reprogramming may be a strategy for derivation of transplantable stem cells for clinical application.

Title: Extracellular vesicles derived from renal cancer stem cells induce a pro-tumorigenic phenotype in mesenchymal stromal cells Content: Renal carcinomas have been shown to contain a population of cancer stem cells (CSCs) that present self-renewing capacity and support tumor growth and metastasis. CSCs were shown to secrete large amount of extracellular vesicles (EVs) that can transfer several molecules (proteins, lipids and nucleic acids) and induce epigenetic changes in target cells. Mesenchymal Stromal Cells (MSCs) are susceptible to tumor signalling and can be recruited to tumor regions. The precise role of MSCs in tumor development is still under debate since both pro- and anti-tumorigenic effects have been reported. In this study we analysed the participation of renal CSC-derived EVs in the interaction between tumor and MSCs. We found that CSC-derived EVs promoted persistent phenotypical changes in MSCs characterized by an increased expression of genes associated with cell migration (CXCR4, CXCR7), matrix remodeling (COL4A3), angiogenesis and tumor growth (IL-8, Osteopontin and Myeloperoxidase). EV-stimulated MSCs exhibited in vitro an enhancement of migration toward the tumor conditioned medium. Moreover, EV-stimulated MSCs enhanced migration of renal tumor cells and induced vessel-like formation. In vivo, EV-stimulated MSCs supported tumor development and vascularization, when co-injected with renal tumor cells. In conclusion, CSC-derived EVs induced phenotypical changes in MSCs that are associated with tumor growth.

Title: Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis. Content: Myelin-directed autoimmunity is considered to play a key role in the pathogenesis of multiple sclerosis (MS). Increased production of both pro- and anti-inflammatory cytokines is a common finding in MS. Interleukin-17 (IL-17) is a recently described cytokine produced in humans almost exclusively by activated memory T cells, which can induce the production of proinflammatory cytokines and chemokines from parenchymal cells and macrophages. In situ hybridisation with synthetic oligonucleotide probes was adopted to detect and enumerate IL-17 mRNA expressing mononuclear cells (MNC) in blood and cerebrospinal fluid (CSF) from patients with MS and control individuals. Numbers of IL-17 mRNA expressing blood MNC were higher in patients with MS and acute aseptic meningoencephalitis (AM) compared to healthy individuals. Higher numbers of IL-17 mRNA expressing blood MNC were detected in MS patients examined during clinical exacerbation compared to remission. Patients with MS had higher numbers of IL-17 mRNA expressing MNC in CSF compared to blood. This increase in numbers of IL-17 mRNA expressing MNC in CSF was not observed in patients with AM. Our results thus demonstrate increased numbers of IL-17 mRNA expressing MNC in MS with higher numbers in CSF than blood, and with the highest numbers in blood during clinical exacerbations.

Title: Microvesicles derived from human umbilical cord mesenchymal stem cells stimulated by hypoxia promote angiogenesis both in vitro and in vivo. Content: Although mesenchymal stem cells (MSCs) have been increasingly trialed to treat a variety of diseases, the underlying mechanisms remain still elusive. In this study, human umbilical cord (UC)-derived MSCs were stimulated by hypoxia, and the membrane microvesicles (MVs) in the supernatants were collected by ultracentrifugation, observed under an electron microscope, and the origin was identified with the flow cytometric technique. The results showed that upon hypoxic stimulus, MSCs released a large quantity of MVs of ~100 nm in diameter. The MVs were phenotypically similar to the parent MSCs, except that the majority of them were negative for the receptor of platelet-derived growth factor. DiI-labeling assay revealed that MSC-MVs could be internalized into human UC endothelial cells (UC-ECs) within 8 h after they were added into the culture medium. Carboxyfluorescein succinimidyl ester-labeling technique and MTT test showed that MSC-MVs promoted the proliferation of UC-ECs in a dose-dependent manner. Further, MVs could enhance in vitro capillary network formation of UC-ECs in a Matrigel matrix. In a rat hindlimb ischemia model, both MSCs and MSC-MVs were shown to improve significantly the blood flow recovery compared with the control medium (P<0.0001), as assessed by laser Doppler imaging analysis. These data indicate that MV releasing is one of the major mechanisms underlying the effectiveness of MSC therapy by promoting angiogenesis.

Title: Quantitative assessment of T-cell repertoire recovery after hematopoietic stem cell transplantation Content: Delayed T cell recovery and restricted T cell receptor (TCR) diversity after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are associated with increased risks of infection and cancer relapse. Technical challenges have limited faithful measurement of TCR diversity after allo-HSCT. Here we combined 5' rapid amplification of complementary DNA ends PCR with deep sequencing to quantify TCR diversity in 28 recipients of allo-HSCT using a single oligonucleotide pair. Analysis of duplicate blood samples confirmed that we accurately determined the frequency of individual TCRs. After 6 months, cord blood-graft recipients approximated the TCR diversity of healthy individuals, whereas recipients of T cell-depleted peripheral-blood stem cell grafts had 28-fold and 14-fold lower CD4(+) and CD8(+) T cell diversities, respectively. After 12 months, these deficiencies had improved for the CD4(+) but not the CD8(+) T cell compartment. Overall, this method provides unprecedented views of T cell repertoire recovery after allo-HSCT and may identify patients at high risk of infection or relapse.

Title: A role for interleukin-1β in determining the lineage fate of embryonic rat hippocampal neural precursor cells. Content: Neurogenesis occurs in the hippocampus of the developing and adult brain due to the presence of multipotent stem cells and restricted precursor cells at different stages of differentiation. It has been proposed that they may be of potential benefit for use in cell transplantation approaches for neurodegenerative disorders and trauma. Prolonged release of interleukin-1β (IL-1β) from activated microglia has a deleterious effect on hippocampal neurons and is implicated in the impaired neurogenesis and cognitive dysfunction associated with aging, Alzheimer's disease and depression. This study assessed the effect of IL-1β on the proliferation and differentiation of embryonic rat hippocampal NPCs in vitro. We show that IL-1R1 is expressed on proliferating NPCs and that IL-1β treatment decreases cell proliferation and neurosphere growth. When NPCs were differentiated in the presence of IL-1β, a significant reduction in the percentages of newly-born neurons and post-mitotic neurons and a significant increase in the percentage of astrocytes was observed in these cultures. These effects were attenuated by IL-1 receptor antagonist. These data reveal that IL-1β exerts an anti-proliferative, anti-neurogenic and pro-gliogenic effect on embryonic hippocampal NPCs, which is mediated by IL-1R1. The present results emphasise the consequences of an inflammatory environment during NPC development, and indicate that strategies to inhibit IL-1β signalling may be necessary to facilitate effective cell transplantation approaches or in conditions where endogenous hippocampal neurogenesis is impaired.

Title: Bone progenitor dysfunction induces myelodysplasia and secondary leukemia Content: Mesenchymal cells contribute to the 'stroma' of most normal and malignant tissues, with specific mesenchymal cells participating in the regulatory niches of stem cells. By examining how mesenchymal osteolineage cells modulate haematopoiesis, here we show that deletion of Dicer1 specifically in mouse osteoprogenitors, but not in mature osteoblasts, disrupts the integrity of haematopoiesis. Myelodysplasia resulted and acute myelogenous leukaemia emerged that had acquired several genetic abnormalities while having intact Dicer1. Examining gene expression altered in osteoprogenitors as a result of Dicer1 deletion showed reduced expression of Sbds, the gene mutated in Schwachman-Bodian-Diamond syndrome-a human bone marrow failure and leukaemia pre-disposition condition. Deletion of Sbds in mouse osteoprogenitors induced bone marrow dysfunction with myelodysplasia. Therefore, perturbation of specific mesenchymal subsets of stromal cells can disorder differentiation, proliferation and apoptosis of heterologous cells, and disrupt tissue homeostasis. Furthermore, primary stromal dysfunction can result in secondary neoplastic disease, supporting the concept of niche-induced oncogenesis.

Title: An activator of the cAMP/PKA/CREB pathway promotes osteogenesis from human mesenchymal stem cells. Content: Mesenchymal stem cells (MSCs) are multipotent adult stem cells capable of differentiating along the osteoblast, adipocyte, and chondrocyte lineages. Regulation of MSCs differentiation may be a useful tool for regenerative medicine and cell-based therapy. The discovery of small molecule that activates the osteogenic differentiation of MSCs could aid in the development of a new anabolic drug for osteoporosis treatment. We identified CW008, a derivative of pyrazole-pyridine, that stimulates osteoblast differentiation of human MSCs and increases bone formation in ovariectomized mice. CW008 promotes osteogenesis by activating cAMP/PKA/CREB signaling pathway and inhibiting leptin secretion. These results suggest that CW008 is an agonist of cAMP/PKA/CREB pathway in osteogenic differentiation and that application of CW008 may be useful for the treatment of bone-related diseases and for the study of bone biology.

Title: Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming Content: We describe robust induction of autophagy during the reprogramming of mouse fibroblasts to induced pluripotent stem cells by four reprogramming factors (Sox2, Oct4, Klf4 and c-Myc), henceforth 4F. This process occurs independently of p53 activation, and is mediated by the synergistic downregulation of mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy-related genes. The 4F coordinately repress mTORC1, but bifurcate in their regulation of autophagy-related genes, with Klf4 and c-Myc inducing them but Sox2 and Oct4 inhibiting them. On one hand, inhibition of mTORC1 facilitates reprogramming by promoting cell reshaping (mitochondrial remodelling and cell size reduction). On the other hand, mTORC1 paradoxically impairs reprogramming by triggering autophagy. Autophagy does not participate in cell reshaping in reprogramming but instead degrades p62, whose accumulation in autophagy-deficient cells facilitates reprogramming. Our results thus reveal a complex signalling network involving mTORC1 inhibition and autophagy induction in the early phase of reprogramming, whose delicate balance ultimately determines reprogramming efficiency.

Title: On the origin and impact of mesenchymal stem cell heterogeneity: new insights and emerging tools for single cell analysis. Content: Mesenchymal stem cells (MSCs) display substantial cell-to-cell variation. This heterogeneity manifests among donors, among tissue sources, and within cell populations. Such pervasive variability complicates the use of MSCs in regenerative applications and may limit their therapeutic efficacy. Most conventional assays measure MSC properties in bulk and, as a consequence, mask this cell-to-cell variation. Recent studies have identified extensive variability amongst and within clonal MSC populations, in dimensions including functional differentiation capacity, molecular state (e.g. epigenetic, transcriptomic, and proteomic status), and biophysical properties. While the origins of these variations remain to be elucidated, potential mechanisms include in vivo micro-anatomical heterogeneity, epigenetic bistability, and transcriptional fluctuations. Emerging tools for single cell analysis of MSC gene and protein expression may yield further insight into the mechanisms and implications of single cell variation amongst these cells, and ultimately improve the clinical utility of MSCs in tissue engineering and regenerative medicine applications. This review outlines the dimensions across which MSC heterogeneity is present, defines some of the known mechanisms that govern this heterogeneity, and highlights emerging technologies that may further refine our understanding and improve our clinical application of this unique cell type.

Title: Parathyroid Hormone Directs Bone Marrow Mesenchymal Cell Fate. Content: Intermittent PTH administration builds bone mass and prevents fractures, but its mechanism of action is unclear. We genetically deleted the PTH/PTHrP receptor (PTH1R) in mesenchymal stem cells using Prx1Cre and found low bone formation, increased bone resorption, and high bone marrow adipose tissue (BMAT). Bone marrow adipocytes traced to Prx1 and expressed classic adipogenic markers and high receptor activator of nuclear factor kappa B ligand (Rankl) expression. RANKL levels were also elevated in bone marrow supernatant and serum, but undetectable in other adipose depots. By cell sorting, Pref1+RANKL+ marrow progenitors were twice as great in mutant versus control marrow. Intermittent PTH administration to control mice reduced BMAT significantly. A similar finding was noted in male osteoporotic patients. Thus, marrow adipocytes exhibit osteogenic and adipogenic characteristics, are uniquely responsive to PTH, and secrete RANKL. These studies reveal an important mechanism for PTH's therapeutic action through its ability to direct mesenchymal cell fate.

Title: Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells. Content: Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (Prf1) transcription, and repressed re-expression of memory CTL markers Bcl6 and IL-7Ralpha. Binding of Eomes and STAT5 to Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Ralpha and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Ralpha repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Ralpha-deficient effector CD8(+) T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1(+) and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.

Title: Reconstitution of the immune system after hematopoietic stem cell transplantation in humans Content: Hematopoietic stem cell transplantation is associated with a severe immune deficiency. As a result, the patient is at high risk of infections. Innate immunity, including epithelial barriers, monocytes, granulocytes, and NK cells recovers within weeks after transplantation. By contrast, adaptive immunity recovers much slower. B- and T-cell counts normalize during the first months after transplantation, but in particular, T-cell immunity may remain impaired for years. During the last decade, much of the underlying mechanisms have been identified. These insights may provide new therapies to accelerate recovery.

Title: Generating an iPSC bank for HLA-matched tissue transplantation based on known donor and recipient HLA types. Content: The likelihood for immunological rejection of Human Leukocyte Antigens (HLA)-mismatched induced pluripotent stem cells (iPSCs) limits their therapeutic potential. Here we show how a tissue bank from 150 selected homozygous HLA-typed volunteers could match 93% of the UK population with a minimal requirement for immunosuppression. Our model provides a practical approach for using existing HLA-typed samples to generate an iPSC stem cell bank that circumvents prospective typing of a large number of individuals.

Title: Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Content: The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model.

Title: Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming. Content: Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion.

Title: Recipient-type specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia. Content: CD4+CD25+ regulatory T cells (Treg's) play a pivotal role in preventing organ-specific autoimmune diseases and in inducing tolerance to allogeneic organ transplants. We and others recently demonstrated that high numbers of Treg's can also modulate graft-versus-host disease (GVHD) if administered in conjunction with allogeneic hematopoietic stem cell transplantation in mice. In a clinical setting, it would be impossible to obtain enough freshly purified Treg's from a single donor to have a therapeutic effect. Thus, we performed regulatory T cell expansion ex vivo by stimulation with allogeneic APCs, which has the additional effect of producing alloantigen-specific regulatory T cells. Here we show that regulatory T cells specific for recipient-type alloantigens control GVHD while favoring immune reconstitution. Irrelevant regulatory T cells only mediate a partial protection from GVHD. Preferential survival of specific regulatory T cells, but not of irrelevant regulatory T cells, was observed in grafted animals. Additionally, the use of specific regulatory T cells was compatible with some form of graft-versus-tumor activity. These data suggest that recipient-type specific Treg's could be preferentially used in the control of GVHD in future clinical trials.

Title: Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds Content: Reprogramming of mouse and human somatic cells can be achieved by ectopic expression of transcription factors, but with low efficiencies. We report that DNA methyltransferase and histone deacetylase (HDAC) inhibitors improve reprogramming efficiency. In particular, valproic acid (VPA), an HDAC inhibitor, improves reprogramming efficiency by more than 100-fold, using Oct4-GFP as a reporter. VPA also enables efficient induction of pluripotent stem cells without introduction of the oncogene c-Myc.

Title: Cancer cell–autonomous contribution of type I interferon signaling to the efficacy of chemotherapy Content: Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell–mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN-α and IFN-β receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN–related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy.

Title: Inducible caspase-9 suicide gene controls adverse effects from alloreplete T cells after haploidentical stem cell transplantation. Content: To test the feasibility of a single T-cell manipulation to eliminate alloreactivity while sparing antiviral and antitumor T cells, we infused 12 haploidentical hematopoietic stem cell transplant patients with increasing numbers of alloreplete haploidentical T cells expressing the inducible caspase 9 suicide gene (iC9-T cells). We determined whether the iC9-T cells produced immune reconstitution and if any resultant graft-versus-host disease (GVHD) could be controlled by administration of a chemical inducer of dimerization (CID; AP1903/Rimiducid). All patients receiving >10(4) alloreplete iC9-T lymphocytes per kilogram achieved rapid reconstitution of immune responses toward 5 major pathogenic viruses and concomitant control of active infections. Four patients received a single AP1903 dose. CID infusion eliminated 85% to 95% of circulating CD3(+)CD19(+) T cells within 30 minutes, with no recurrence of GVHD within 90 days. In one patient, symptoms and signs of GVHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of proinflammatory cytokines, and rash) resolved within 2 hours of AP1903 infusion. One patient with varicella zoster virus meningitis and acute GVHD had iC9-T cells present in the cerebrospinal fluid, which were reduced by >90% after CID. Notably, virus-specific T cells recovered even after AP1903 administration and continued to protect against infection. Hence, alloreplete iC9-T cells can reconstitute immunity posttransplant and administration of CID can eliminate them from both peripheral blood and the central nervous system (CNS), leading to rapid resolution of GVHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes. This trial was registered at www.clinicaltrials.gov as #NCT01494103.

Title: miR-205 hinders the malignant interplay between prostate cancer cells and associated fibroblasts. Content: AIMS Tumor microenvironment is a strong determinant for the acquisition of metastatic potential of cancer cells. We have recently demonstrated that cancer-associated fibroblasts (CAFs) elicit a redox-dependent epithelial-mesenchymal transition (EMT) in prostate cancer (PCa) cells, driven by cycloxygenase-2/hypoxia-inducible factor-1 (HIF-1)/nuclear factor-κB pathway and enhancing tumor aggressiveness. Here, we investigated the involvement of microRNAs (miRNAs) in tumor-stroma interplay to identify possible tools to counteract oxidative stress and metastasis dissemination. RESULTS We found that miR-205 is the most downmodulated miRNA in PCa cells upon CAF stimulation, due to direct transcriptional repression by HIF-1, a known redox-sensitive transcription factor. Rescue experiments demonstrated that ectopic miR-205 overexpression in PCa cells counteracts CAF-induced EMT, thus impairing enhancement of cell invasion, acquisition of stem cell traits, tumorigenicity, and metastatic dissemination. In addition, miR-205 blocks tumor-driven activation of surrounding fibroblasts by reducing pro-inflammatory cytokine secretion. INNOVATION Overall, such findings suggest miR-205 as a brake against PCa metastasis by blocking both the afferent and efferent arms of the circuit between tumor cells and associated fibroblasts, thus interrupting the pro-oxidant and pro-inflammatory circuitries engaged by reactive stroma. CONCLUSION The evidence that miR-205 replacement in PCa cells is able not only to prevent but also to revert the oxidative/pro-inflammatory axis leading to EMT induced by CAFs sets the rationale for developing miRNA-based approaches to prevent and treat metastatic disease.

Title: Cutting edge: interleukin 17 signals through a heteromeric receptor complex. Content: IL-17 is an inflammatory cytokine produced primarily by a unique lineage of CD4 T cells that plays critical roles in the pathogenesis of multiple autoimmune diseases. IL-17RA is a ubiquitously expressed receptor that is essential for IL-17 biologic activity. Despite widespread receptor expression, the activity of IL-17 is most classically defined by its ability to induce the expression of inflammatory cytokines, chemokines, and other mediators by stromal cells. The lack of IL-17 responsiveness in mouse stromal cells genetically deficient in IL-17RA is poorly complemented by human IL-17RA, suggesting the presence of an obligate ancillary component whose activity is species specific. This component is IL-17RC, a distinct member of the IL-17R family. Thus, the biologic activity of IL-17 is dependent on a complex composed of IL-17RA and IL-17RC, suggesting a new paradigm for understanding the interactions between the expanded family of IL-17 ligands and their receptors.

Title: Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Content: Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However, several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently, it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However, NPCs express Sox2 endogenously, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small-molecule combination, BIX-01294 and BayK8644, that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts, which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors, such as Sox2, and improve reprogramming efficiency.

Title: Type 2 cytokines: mechanisms and therapeutic strategies Content: Type 2 immune responses are defined by the cytokines interleukin-4 (IL-4), IL-5, IL-9 and IL-13, which can either be host protective or have pathogenic activity. Type 2 immunity promotes antihelminth immunity, suppresses type 1-driven autoimmune disease, neutralizes toxins, maintains metabolic homeostasis, and regulates wound repair and tissue regeneration pathways following infection or injury. Nevertheless, when type 2 responses are dysregulated, they can become important drivers of disease. Type 2 immunity induces a complex inflammatory response characterized by eosinophils, mast cells, basophils, type 2 innate lymphoid cells, IL-4-and/or IL-13-conditioned macrophages and T helper 2 (TH2) cells, which are crucial to the pathogenesis of many allergic and fibrotic disorders. As chronic type 2 immune responses promote disease, the mechanisms that regulate their maintenance are thought to function as crucial disease modifiers. This Review discusses the many endogenous negative regulatory mechanisms that antagonize type 2 immunity and highlights how therapies that target some of these pathways are being developed to treat type 2-mediated disease.

Title: Prostaglandin E2 as an inhibitory modulator of fibrogenesis in human lung allografts. Content: RATIONALE Donor mesenchymal stromal/stem cell (MSC) expansion and fibrotic differentiation is associated with development of bronchiolitis obliterans syndrome (BOS) in human lung allografts. However, the regulators of fibrotic differentiation of these resident mesenchymal cells are not well understood. OBJECTIVES This study examines the role of endogenous and exogenous prostaglandin (PG)E2 as a modulator of fibrotic differentiation of human lung allograft-derived MSCs. METHODS Effect of PGE2 on proliferation, collagen secretion, and α-smooth muscle actin (α-SMA) expression was assessed in lung-resident MSCs (LR-MSCs) derived from patients with and without BOS. The response pathway involved was elucidated by use of specific agonists and antagonists. MEASUREMENT AND MAIN RESULTS PGE2 treatment of LR-MSCs derived from normal lung allografts significantly inhibited their proliferation, collagen secretion, and α-SMA expression. On the basis of pharmacologic and small-interfering RNA approaches, a PGE2/E prostanoid (EP)2/adenylate cyclase pathway was implicated in these suppressive effects. Stimulation of endogenous PGE2 secretion by IL-1β was associated with amelioration of their myofibroblast differentiation in vitro, whereas its inhibition by indomethacin augmented α-SMA expression. LR-MSCs from patients with BOS secreted significantly less PGE2 than non-BOS LR-MSCs. Furthermore, BOS LR-MSCs were found to be defective in their ability to induce cyclooxygenase-2, and therefore unable to up-regulate PGE2 synthesis in response to IL-1β. BOS LR-MSCs also demonstrated resistance to the inhibitory actions of PGE2 in association with a reduction in the EP2/EP1 ratio. CONCLUSIONS These data identify the PGE2 axis as an important autocrine-paracrine brake on fibrotic differentiation of LR-MSCs, a failure of which is associated with BOS.

Title: Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity Content: Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately twofold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we show that engineered haplodeficiency of Il2 gene expression not only reduces T cell IL-2 production by twofold but also mimics the autoimmune dysregulatory effects of the naturally occurring susceptibility alleles of Il2. Reduced IL-2 production achieved by either genetic mechanism correlates with reduced function of CD4+ CD25+ regulatory T cells, which are critical for maintaining immune homeostasis.

Title: The NLRP12 Sensor Negatively Regulates Autoinflammatory Disease by Modulating Interleukin-4 Production in T Cells. Content: Missense mutations in the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing family of gene 12 (Nlrp12) are associated with periodic fever syndromes and atopic dermatitis in humans. Here, we have demonstrated a crucial role for NLRP12 in negatively regulating pathogenic T cell responses. Nlrp12(-/-) mice responded to antigen immunization with hyperinflammatory T cell responses. Furthermore, transfer of CD4(+)CD45RB(hi)Nlrp12(-/-) T cells into immunodeficient mice led to more severe colitis and atopic dermatitis. NLRP12 deficiency did not, however, cause exacerbated ascending paralysis during experimental autoimmune encephalomyelitis (EAE); instead, Nlrp12(-/-) mice developed atypical neuroinflammatory symptoms that were characterized by ataxia and loss of balance. Enhanced T-cell-mediated interleukin-4 (IL-4) production promotes the development of atypical EAE disease in Nlrp12(-/-) mice. These results define an unexpected role for NLRP12 as an intrinsic negative regulator of T-cell-mediated immunity and identify altered NF-κB regulation and IL-4 production as key mediators of NLRP12-associated disease.

Title: Reserves, functional, immunoregulatory, and cytogenetic properties of bone marrow mesenchymal stem cells in patients with myelodysplastic syndromes. Content: Defective hematopoiesis supporting capacity of bone marrow (BM) stroma has been implicated in the pathophysiology of myelodysplastic syndromes (MDS). The aim of this study is to explore whether the BM stroma progenitors, namely the mesenchymal stem cells (MSCs), are primarily affected in MDS by evaluating the reserves, the functional properties, as well as the cytogenetic characteristics, in comparison to BM hematopoietic cells, in patients with de novo MDS (n = 13). The number, differentiation potential toward adipocytes/chondrocytes/osteoblasts and immunosuppressive function in terms of inhibition of mitogen-induced T-cell proliferation did not differ significantly between patient and normal (n = 20) MSCs. Patient MSCs did not show any aberrations in the production of proinflammatory or growth-promoting cytokines and did not harbor the cytogenetic abnormalities present in hematopoietic cells. Occasional patient and normal MSC cultures, however, developed irrelevant chromosomal alterations (trisomies 5 and 7) with uncertain pathophysiologic significance. Compared to controls, patient MSCs displayed impaired proliferative and clonogenic potential through passages that might represent a nonspecific abnormality associated with the chronic inflammatory process present in patients' BM. These data suggest that BM MSCs from MDS patients do not belong to the abnormal clone and do not represent the main cellular source contributing to the inflammatory marrow microenvironment.

Title: An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Content: The generation of induced pluripotent stem cells (iPSCs) provides the opportunity to use patient-specific somatic cells, which are a valuable source for disease modeling and drug discovery. To promote research involving these cells, it is important to make iPSCs from easily accessible and less invasive tissues, like blood. We have recently reported the efficient generation of human iPSCs from adult fibroblasts using a combination of plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53. We herein report a modified protocol enabling efficient iPSC induction from CD34+ cord blood cells and from peripheral blood isolated from healthy donors using these plasmid vectors. The original plasmid mixture could induce iPSCs; however, the efficiency was low. The addition of EBNA1, an essential factor for episomal amplification of the vectors, by an extra plasmid greatly increased the efficiency of iPSC induction, especially when the induction was performed from αβT cells. This improvement enabled the establishment of blood-derived iPSCs from seven healthy donors ranging in age from their 20s to their 60s. This induction method will be useful for the derivation of patient-specific integration-free iPSCs and would also be applicable to the generation of clinical-grade iPSCs in the future.

Title: Adipose stem cells originate from perivascular cells. Content: Recent research has shown that adipose tissues contain abundant MSCs (mesenchymal stem cells). The origin and location of the adipose stem cells, however, remain unknown, presenting an obstacle to the further purification and study of these cells. In the present study, we aimed at investigating the origins of adipose stem cells. α-SMA (α-smooth muscle actin) is one of the markers of pericytes. We harvested ASCs (adipose stromal cells) from α-SMA-GFP (green fluorescent protein) transgenic mice and sorted them into GFP-positive and GFP-negative cells by FACS. Multilineage differentiation tests were applied to examine the pluripotent ability of the α-SMA-GFP-positive and -negative cells. Immunofluorescent staining for α-SMA and PDGF-Rβ (platelet-derived growth factor receptor β) were applied to identify the α-SMA-GFP-positive cells. Then α-SMA-GFP-positive cells were loaded on a collagen-fibronectin gel with endothelial cells to test their vascularization ability both in vitro and in vivo. Results show that, in adipose tissue, all of the α-SMA-GFP-positive cells congregate around the blood vessels. Only the α-SMA-GFP-positive cells have multilineage differentiation ability, while the α-SMA-GFP-negative cells can only differentiate in an adipogenic direction. The α-SMA-GFP-positive cells maintained expression of α-SMA during multilineage differentiation. The α-SMA-GFP-positive cells can promote the vascularization of endothelial cells in three-dimensional culture both in vitro and in vivo. We conclude that the adipose stem cells originate from perivascular cells and congregate around blood vessels.

Title: In Vitro–expanded Antigen-specific Regulatory T Cells Suppress Autoimmune Diabetes Content: The low number of CD4+ CD25+ regulatory T cells (Tregs), their anergic phenotype, and diverse antigen specificity present major challenges to harnessing this potent tolerogenic population to treat autoimmunity and transplant rejection. In this study, we describe a robust method to expand antigen-specific Tregs from autoimmune-prone nonobese diabetic mice. Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2. The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.

Title: IL-18 induction of osteopontin mediates cardiac fibrosis and diastolic dysfunction in mice. Content: Osteopontin (OPN), a key component of the extracellular matrix, is associated with the fibrotic process during tissue remodeling. OPN and the cytokine interleukin (IL)-18 have been shown to be overexpressed in an array of human cardiac pathologies. In the present study, we determined the role of IL-18 in the regulation of cardiac OPN expression and the subsequent interstitial fibrosis and diastolic dysfunction. We demonstrated parallel increases in IL-18, OPN expression, and interstitial fibrosis in murine models of left ventricular pressure and volume overload. Exogenous recombinant (r)IL-18 administered for 2 wk increased cardiac OPN expression, interstitial fibrosis, and diastolic dysfunction. Stimulation of the T helper (Th)1 lymphocyte phenotype with a selective toll-like receptor (TLR)9 agonist induced cardiac IL-18 and OPN expression, which was associated with increased cardiac fibrillar collagen concentrations and interstitial fibrosis resulting in diastolic dysfunction. rIL-18 induced OPN expression and protein levels in primary of cardiac fibroblast cultures. Conditioned media from TLR9-stimulated T lymphocyte cultures induced IL-18 and OPN expression in cardiac fibroblasts, while blockade of the IL-18 receptor with a neutralizing antibody abolished the increase in OPN expression. Furthermore, a mutation in the transcriptional factor interferon regulatory factor (IRF)1 or IRF1 small interfering RNA (siRNA) resulted in the decreased expression of IL-18 and OPN in cardiac fibroblasts. With pressure overload, IRF1-mutant mice showed downregulation of IL-18 and OPN expression in cardiac tissue, reduced cardiac fibrotic development, and increased left ventricular function compared with wild type. These results provide direct evidence that the induction of IL-18 regulates OPN-mediated cardiac fibrosis and diastolic dysfunction.

Title: Lethal autoimmune myocarditis in interferon-gamma receptor-deficient mice: enhanced disease severity by impaired inducible nitric oxide synthase induction. Content: BACKGROUND Interferon-gamma (IFN-gamma) is an essential cytokine in the regulation of inflammatory responses in autoimmune diseases. Little is known about its role in inflammatory heart disease. METHODS AND RESULTS We showed that IFN-gamma receptor-deficient mice (IFN-gammaR(-/-)) on a BALB/c background immunized with a peptide derived from cardiac alpha-myosin heavy chain develop severe myocarditis with high mortality. Although myocarditis subsided in wild-type mice after 3 weeks, IFN-gammaR(-/-) mice showed persistent disease. The persistent inflammation was accompanied by vigorous in vitro CD4 T-cell responses and impaired inducible nitric oxide synthase expression, together with evidence of impaired nitric oxide production in IFN-gammaR(-/-) hearts. Treatment of wild-type mice with the nitric oxide synthetase inhibitor N:-nitro-l-arginine-methyl-ester enhanced in vitro CD4 T-cell proliferation and prevented healing of myocarditis. CONCLUSIONS Our data provide evidence that IFN-gamma protects mice from lethal autoimmune myocarditis by inducing the expression of inducible nitric oxide synthase followed by the downregulation of T-cell responses.

Title: Strong association between respiratory viral infection early after hematopoietic stem cell transplantation and the development of life-threatening acute and chronic alloimmune lung syndromes. Content: Alloimmune lung syndromes (allo-LS), including idiopathic pneumonia syndrome, bronchiolitis obliterans syndrome, and bronchiolitis obliterans organizing pneumonia, are severe complications after hematopoietic stem cell transplantation (HSCT). In our cohort of 110 pediatric patients, 30 had allo-LS (27.3%), 18 with idiopathic pneumonia syndrome and 12 with bronchiolitis obliterans syndrome. Multivariate analysis showed that respiratory viral infection early after HSCT is an important predictor for the development of allo-LS (P <.0001). This was true for all viruses tested. In multivariate analysis, allo-LS was the only predictor for higher mortality (P = .04). Paradoxically, prolonged administration of immunosuppressive agents because of acute graft-versus-host disease had a protective effect on the development of allo-LS (P = .004). We hypothesize that early infection of the respiratory tract with a common cold virus makes the lungs a target for alloimmunity.

Title: Long-term immune deficiency after allogeneic stem cell transplantation: B-cell deficiency is associated with late infections. Content: Immune reconstitution was analyzed in 140 consecutive patients who were 2-year disease-free and who underwent myeloablative allogeneic transplantation. A CD4 and CD8 defect was observed involving naive, terminally differentiated, memory and competent cells and above limits values for activated subsets. Natural killer cells normalize at six months while we observed expansion of CD19(+)/CD5(+) B cells after three months and a persisting defect of memory B cells. Chronic graft-versus-host disease did not influence significantly those parameters for CD8 subsets while the naïve and competent CD4 subsets were strongly affected. But the most profound impact of chronic graft-versus-host disease was on B-cell subsets, especially on the memory B population. The cumulative incidence of late severe infections was low (14% at four years). Using Cox's models, only low B-cell counts at 12 (P=0.02) and 24 (P=0.001) months were associated with the hazard of developing late infection, in particular if patients did not develop chronic graft-versus-host disease.

Title: PU.1-mediated upregulation of CSF1R is crucial for leukemia stem cell potential induced by MOZ-TIF2 Content: Leukemias and other cancers possess self-renewing stem cells that help to maintain the cancer. Cancer stem cell eradication is thought to be crucial for successful anticancer therapy. Using an acute myeloid leukemia (AML) model induced by the leukemia-associated monocytic leukemia zinc finger (MOZ)-TIF2 fusion protein, we show here that AML can be cured by the ablation of leukemia stem cells. The MOZ fusion proteins MOZ-TIF2 and MOZ-CBP interacted with the transcription factor PU.1 to stimulate the expression of macrophage colony–stimulating factor receptor (CSF1R, also known as M-CSFR, c-FMS or CD115). Studies using PU.1-deficient mice showed that PU.1 is essential for the ability of MOZ-TIF2 to establish and maintain AML stem cells. Cells expressing high amounts of CSF1R (CSF1Rhigh cells), but not those expressing low amounts of CSF1R (CSF1Rlow cells), showed potent leukemia-initiating activity. Using transgenic mice expressing a drug-inducible suicide gene controlled by the CSF1R promoter, we cured AML by ablation of CSF1Rhigh cells. Moreover, induction of AML was suppressed in CSF1R-deficient mice and CSF1R inhibitors slowed the progression of MOZ-TIF2–induced leukemia. Thus, in this subtype of AML, leukemia stem cells are contained within the CSF1Rhigh cell population, and we suggest that targeting of PU.1-mediated upregulation of CSF1R expression might be a useful therapeutic approach.

Title: Organ-resident, nonlymphoid cells suppress proliferation of autoimmune T-helper lymphocytes. Content: Local presentation of autoantigen by organ-resident cells inappropriately expressing Ia determinants has been implicated in organ-specific autoimmunity. Experimental autoimmune uveoretinitis, induced in rats by immunization with retinal soluble antigen, is used as a model of organ-specific autoimmunity. In an in vitro system derived from this model, uveitogenic rat T-helper lymphocytes specific to the retinal soluble antigen, or control T-helper lymphocytes reactive to the purified protein derivative of tuberculin, were cocultured with Ia-expressing syngeneic retinal glial cells (Müller cells) in the presence of specific antigen. Antigen presentation was not apparent under ordinary culture conditions, and the Müller cells profoundly suppressed the proliferative response of primed T-helper lymphocytes to antigen presented on conventional antigen-presenting cells, as well as their subsequent interleukin-2 (IL-2)-dependent expansion. Suppression of proliferation was accompanied by inhibition of IL-2 production in response to antigen, as well as by reduction in high-affinity IL-2 receptor expression, and proceeded via a contact-dependent mechanism. These results suggest a role for locally acting suppression mechanisms in immune regulation and maintenance of tissue homeostasis.

Title: Advances in graft-versus-host disease biology and therapy Content: Allogeneic haematopoietic stem cell transplantation is used to treat a variety of disorders, but its efficacy is limited by the occurrence of graft-versus-host disease (GVHD). The past decade has brought impressive advances in our understanding of the role of stimulatory and suppressive elements of the adaptive and innate immune systems from both the donor and the host in GVHD pathogenesis. New insights from basic immunology, preclinical models and clinical studies have led to novel approaches for prevention and treatment. This Review highlights the recent advances in understanding the pathophysiology of GVHD and its treatment, with a focus on manipulations of the immune system that are amenable to clinical application.

Title: CD200R/CD200 Inhibits Osteoclastogenesis: New Mechanism of Osteoclast Control by Mesenchymal Stem Cells in Human Content: Bone homeostasis is maintained by the balance between bone-forming osteoblasts and bone-degrading osteoclasts. Osteoblasts have a mesenchymal origin whereas osteoclasts belong to the myeloid lineage. Osteoclast and osteoblast communication occurs through soluble factors secretion, cell-bone interaction and cell-cell contact, which modulate their activities. CD200 is an immunoglobulin superfamilly member expressed on various types of cells including mesenchymal stem cells (MSCs). CD200 receptor (CD200R) is expressed on myeloid cells such as monocytes/macrophages. We assume that CD200 could be a new molecule involved in the control of osteoclastogenesis and could play a role in MSC-osteoclast communication in humans. In this study, we demonstrated that soluble CD200 inhibited the differentiation of osteoclast precursors as well as their maturation in bone-resorbing cells in vitro. Soluble CD200 did not modify the monocyte phenotype but inhibited the receptor activator of nuclear factor kappa-B ligand (RANKL) signaling pathway as well as the gene expression of osteoclast markers such as osteoclast-associated receptor (OSCAR) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Moreover, MSCs inhibited osteoclast formation, which depended on cell-cell contact and was associated with CD200 expression on the MSC surface. Our results clearly demonstrate that MSCs, through the expression of CD200, play a major role in the regulation of bone resorption and bone physiology and that the CD200-CD200R couple could be a new target to control bone diseases.

Title: Matrix Metalloproteinase 1 Is Necessary for the Migration of Human Bone Marrow-Derived Mesenchymal Stem Cells Toward Human Glioma Content: Human mesenchymal stem cells (MSCs) have increasingly been used as cellular vectors for the delivery of therapeutic genes to tumors. However, the precise mechanism of mobilization remains poorly defined. In this study, MSCs that expressed similar cell surface markers and exhibited multilineage differentiation potentials were isolated from various donors. Interestingly, different MSC isolates displayed differential migration ability toward human glioma cells. We hypothesized that distinct molecular signals may be involved in the varied tumor tropisms exhibited by different MSC isolates. To test this hypothesis, gene expression profiles of tumor-trophic MSCs were compared with those of non-tumor-trophic MSCs. Among the various differentially regulated genes, matrix metalloproteinase one (MMP1) gene expression and its protein activities were enhanced by 27-fold and 21-fold, respectively, in highly migrating MSCs compared with poorly migrating MSCs. By contrast, there was no change in the transcriptional levels of other MMPs. Functional inactivation of MMP1 abrogated the migratory potential of MSCs toward glioma-conditioned medium. Conversely, the nonmigratory phenotype of poorly migrating MSC could be rescued in the presence of either recombinant MMP1 or conditioned medium from the highly migrating MSCs. Ectopic expression of MMP1 in these poorly migrating cells also rendered the cells responsive to the signaling cues from the glioma cells in vivo. However, blocking the interaction of MMP1 and its cognate receptor PAR1 effectively diminished the migratory ability of MSCs. Taken together, this study provides, for the first time, supporting evidence that MMP1 is critically involved in the migration capacity of MSCs, acting through the MMP1/PAR1 axis.

Title: Induced pluripotent stem cells in Alzheimer’s disease: applications for disease modeling and cell-replacement therapy Content: Alzheimer's disease (AD) is the most common cause of dementia in those over the age of 65. While a numerous of disease-causing genes and risk factors have been identified, the exact etiological mechanisms of AD are not yet completely understood, due to the inability to test theoretical hypotheses on non-postmortem and patient-specific research systems. The use of recently developed and optimized induced pluripotent stem cells (iPSCs) technology may provide a promising platform to create reliable models, not only for better understanding the etiopathological process of AD, but also for efficient anti-AD drugs screening. More importantly, human-sourced iPSCs may also provide a beneficial tool for cell-replacement therapy against AD. Although considerable progress has been achieved, a number of key challenges still require to be addressed in iPSCs research, including the identification of robust disease phenotypes in AD modeling and the clinical availabilities of iPSCs-based cell-replacement therapy in human. In this review, we highlight recent progresses of iPSCs research and discuss the translational challenges of AD patients-derived iPSCs in disease modeling and cell-replacement therapy.

Title: Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer Prrx1. Content: The epithelial-mesenchymal transition (EMT) is required in the embryo for the formation of tissues for which cells originate far from their final destination. Carcinoma cells hijack this program for tumor dissemination. The relevance of the EMT in cancer is still debated because it is unclear how these migratory cells colonize distant tissues to form macrometastases. We show that the homeobox factor Prrx1 is an EMT inducer conferring migratory and invasive properties. The loss of Prrx1 is required for cancer cells to metastasize in vivo, which revert to the epithelial phenotype concomitant with the acquisition of stem cell properties. Thus, unlike the classical EMT transcription factors, Prrx1 uncouples EMT and stemness, and is a biomarker associated with patient survival and lack of metastasis.

Title: Long‐Term Follow‐up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia Content: Background CD19‐specific chimeric antigen receptor (CAR) T cells induce high rates of initial response among patients with relapsed B‐cell acute lymphoblastic leukemia (ALL) and long‐term remissions in a subgroup of patients. Methods We conducted a phase 1 trial involving adults with relapsed B‐cell ALL who received an infusion of autologous T cells expressing the 19‐28z CAR at the Memorial Sloan Kettering Cancer Center (MSKCC). Safety and long‐term outcomes were assessed, as were their associations with demographic, clinical, and disease characteristics. Results A total of 53 adults received 19‐28z CAR T cells that were manufactured at MSKCC. After infusion, severe cytokine release syndrome occurred in 14 of 53 patients (26%; 95% confidence interval [CI], 15 to 40); 1 patient died. Complete remission was observed in 83% of the patients. At a median follow‐up of 29 months (range, 1 to 65), the median event‐free survival was 6.1 months (95% CI, 5.0 to 11.5), and the median overall survival was 12.9 months (95% CI, 8.7 to 23.4). Patients with a low disease burden (<5% bone marrow blasts) before treatment had markedly enhanced remission duration and survival, with a median event‐free survival of 10.6 months (95% CI, 5.9 to not reached) and a median overall survival of 20.1 months (95% CI, 8.7 to not reached). Patients with a higher burden of disease (≥5% bone marrow blasts or extramedullary disease) had a greater incidence of the cytokine release syndrome and neurotoxic events and shorter long‐term survival than did patients with a low disease burden. Conclusions In the entire cohort, the median overall survival was 12.9 months. Among patients with a low disease burden, the median overall survival was 20.1 months and was accompanied by a markedly lower incidence of the cytokine release syndrome and neurotoxic events after 19‐28z CAR T‐cell infusion than was observed among patients with a higher disease burden. (Funded by the Commonwealth Foundation for Cancer Research and others; ClinicalTrials.gov number, NCT01044069.)

Title: Atg5-independent autophagy regulates mitochondrial clearance and is essential for iPSC reprogramming Content: Successful generation of induced pluripotent stem cells entails a major metabolic switch from mitochondrial oxidative phosphorylation to glycolysis during the reprogramming process. The mechanism of this metabolic reprogramming, however, remains elusive. Here, our results suggest that an Atg5-independent autophagic process mediates mitochondrial clearance, a characteristic event involved in the metabolic switch. We found that blocking such autophagy, but not canonical autophagy, inhibits mitochondrial clearance, in turn, preventing iPSC induction. Furthermore, AMPK seems to be upstream of this autophagic pathway and can be targeted by small molecules to modulate mitochondrial clearance during metabolic reprogramming. Our work not only reveals that the Atg5-independent autophagy is crucial for establishing pluripotency, but it also suggests that iPSC generation and tumorigenesis share a similar metabolic switch.

Title: Dorsomorphin, a Selective Small Molecule Inhibitor of BMP Signaling, Promotes Cardiomyogenesis in Embryonic Stem Cells Content: BACKGROUND Pluripotent embryonic stem (ES) cells, which have the capacity to give rise to all tissue types in the body, show great promise as a versatile source of cells for regenerative therapy. However, the basic mechanisms of lineage specification of pluripotent stem cells are largely unknown, and generating sufficient quantities of desired cell types remains a formidable challenge. Small molecules, particularly those that modulate key developmental pathways like the bone morphogenetic protein (BMP) signaling cascade, hold promise as tools to study in vitro lineage specification and to direct differentiation of stem cells toward particular cell types. METHODOLOGY/ PRINCIPAL FINDINGS We describe the use of dorsomorphin, a selective small molecule inhibitor of BMP signaling, to induce myocardial differentiation in mouse ES cells. Cardiac induction is very robust, increasing the yield of spontaneously beating cardiomyocytes by at least 20 fold. Dorsomorphin, unlike the endogenous BMP antagonist Noggin, robustly induces cardiomyogenesis when treatment is limited to the initial 24-hours of ES cell differentiation. Quantitative-PCR analyses of differentiating ES cells indicate that pharmacological inhibition of BMP signaling during the early critical stage promotes the development of the cardiomyocyte lineage, but reduces the differentiation of endothelial, smooth muscle, and hematopoietic cells. CONCLUSIONS/ SIGNIFICANCE Administration of a selective small molecule BMP inhibitor during the initial stages of ES cell differentiation substantially promotes the differentiation of primitive pluripotent cells toward the cardiomyocytic lineage, apparently at the expense of other mesodermal lineages. Small molecule modulators of developmental pathways like dorsomorphin could become versatile pharmacological tools for stem cell research and regenerative medicine.

Title: The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function Content: Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin(-) MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin(+) cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP(+) Pdgfrα(-) cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation.

Title: FGF-2 enhances intestinal stem cell survival and its expression is induced after radiation injury. Content: Fibroblast growth factors (FGFs) have mitogenic activity toward a wide variety of cells of mesenchymal, neuronal, and epithelial origin and regulate events in normal embryonic development, angiogenesis, wound repair, and neoplasia. FGF-2 is expressed in many normal adult tissues and can regulate migration and replication of intestinal epithelial cells in culture. However, little is known about the effects of FGF-2 on intestinal epithelial stem cells during either normal epithelial renewal or regeneration of a functional epithelium after injury. In this study, we investigated the expression of FGF-2 in the mouse small intestine after irradiation and determined the effect of exogenous FGF-2 on crypt stem cell survival after radiation injury. Expression of FGF-2 mRNA and protein began to increase at 12 h after gamma-irradiation, and peak levels were observed from 48 to 120 h after irradiation. At all times after irradiation, the higher molecular mass isoform ( approximately 24 kDa) of FGF-2 was the predominant form expressed in the small intestine. Immunohistochemical analysis of FGF-2 expression after radiation injury demonstrated that FGF-2 was predominantly found in the mesenchyme surrounding regenerating crypts. Exogenous recombinant human FGF-2 (rhFGF-2) markedly enhanced crypt stem cell survival when given before irradiation. We conclude that expression of FGF-2 is induced by radiation injury and that rhFGF-2 can enhance crypt stem cell survival after subsequent injury.

Title: HIV–1 Infects Multipotent Progenitor Cells Causing Cell Death and Establishing Latent Cellular Reservoirs Content: HIV causes a chronic infection characterized by depletion of CD4(+) T lymphocytes and the development of opportunistic infections. Despite drugs that inhibit viral spread, HIV infection has been difficult to cure because of uncharacterized reservoirs of infected cells that are resistant to highly active antiretroviral therapy (HAART) and the immune response. Here we used CD34(+) cells from infected people as well as in vitro studies of wild-type HIV to show infection and killing of CD34(+) multipotent hematopoietic progenitor cells (HPCs). In some HPCs, we detected latent infection that stably persisted in cell culture until viral gene expression was activated by differentiation factors. A unique reporter HIV that directly detects latently infected cells in vitro confirmed the presence of distinct populations of active and latently infected HPCs. These findings have major implications for understanding HIV bone marrow pathology and the mechanisms by which HIV causes persistent infection.

Title: TCR stimulation with modified anti-CD3 mAb expands CD8+ T cell population and induces CD8+CD25+ Tregs. Content: Modified anti-CD3 mAbs are emerging as a possible means of inducing immunologic tolerance in settings including transplantation and autoimmunity such as in type 1 diabetes. In a trial of a modified anti-CD3 mAb [hOKT3gamma1(Ala-Ala)] in patients with type 1 diabetes, we identified clinical responders by an increase in the number of peripheral blood CD8+ cells following treatment with the mAb. Here we show that the anti-CD3 mAb caused activation of CD8+ T cells that was similar in vitro and in vivo and induced regulatory CD8+CD25+ T cells. These cells inhibited the responses of CD4+ cells to the mAb itself and to antigen. The regulatory CD8+CD25+ cells were CTLA4 and Foxp3 and required contact for inhibition. Foxp3 was also induced on CD8+ T cells in patients during mAb treatment, which suggests a potential mechanism of the anti-CD3 mAb immune modulatory effects involving induction of a subset of regulatory CD8+ T cells.

Title: Transcriptional Signature and Memory Retention of Human-Induced Pluripotent Stem Cells Content: Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However, it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here, we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions, revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions, pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover, the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research.

Title: Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors. Content: Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human-induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt-NES cell-derived neurons displayed neuronal properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, for example, such as epilepsy.

Title: Geometric cues for directing the differentiation of mesenchymal stem cells. Content: Significant efforts have been directed to understanding the factors that influence the lineage commitment of stem cells. This paper demonstrates that cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature-and with each occupying the same area-display different adipogenesis and osteogenesis profiles. The results reveal that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. Taken together, this work points to the role that geometric shape cues can play in orchestrating the mechanochemical signals and paracrine/autocrine factors that can direct MSCs to appropriate fates.

Title: Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population Content: The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1+ (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDRlow/C-KIT(CD117)neg population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDRlow/C-KITneg cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDRlow/C-KITneg fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.

Title: A graphene-based platform for induced pluripotent stem cells culture and differentiation. Content: Induced pluripotent stem cells (iPSCs) hold great promise as a cell source for regenerative medicine yet its culture, maintenance of pluripotency and induction of differentiation remain challenging. Conversely, graphene (G) and graphene oxide (GO) have captured tremendous interests in the fields of materials science, physics, chemistry and nanotechnology. Here we report on that G and GO can support the mouse iPSCs culture and allow for spontaneous differentiation. Intriguingly, G and GO surfaces led to distinct cell proliferation and differentiation characteristics. In comparison with the glass surface, iPSCs cultured on the G surface exhibited similar degrees of cell adhesion and proliferation while iPSCs on the GO surface adhered and proliferated at a faster rate. Moreover, G favorably maintained the iPSCs in the undifferentiated state while GO expedited the differentiation. The iPSCs cultured on both G and GO surfaces spontaneously differentiated into ectodermal and mesodermal lineages without significant disparity, but G suppressed the iPSCs differentiation towards the endodermal lineage whereas GO augmented the endodermal differentiation. These data collectively demonstrated that the different surface properties of G and GO governed the iPSCs behavior and implicate the potentials of graphene-based materials as a platform for iPSCs culture and diverse applications.

Title: Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4. Content: Embryonic stem (ES) cells homozygous for a Shp-2 mutation (Shp-2(Delta46-110)) demonstrate leukemia inhibitory factor (LIF) hypersensitivity and increased LIF-stimulated phosphorylation of signal transducer and activator of transcription (STAT3). We hypothesized that LIF-responsive genes in Shp-2(Delta46-110) cells would represent potential candidates for molecules vital for ES cell self-renewal. Using microarray analysis, we detected 41 genes whose expression was modified by LIF in Shp-2(Delta46-110) ES cells. Induction of 2 significantly up-regulated genes, suppressor of cytokine signaling-3 (SOCS-3) and Kruppel-like factor 4 (Klf4), was verified using Northern blotting. ES cells overexpressing SOCS-3 had an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ES cells overexpressing Klf4 had a greater capacity to self-renew based on secondary embryoid body (EB) formation. Klf4-transduced d6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ES cell self-renewal. These findings verify the negative role of SOCS-3 on LIF signaling and provide a novel role for Klf4 in ES cell function.

Title: Serious infections after unrelated donor transplantation in 136 children: impact of stem cell source. Content: How the infection risks compare after umbilical cord blood (UCB) and bone marrow (BM) transplantation is not known. Therefore, we compared serious infections in the 2 years after pediatric myeloablative unrelated donor transplantation with unmanipulated BM (n = 52), T cell-depleted (TCD) BM (n = 24), or UCB (n = 60) for the treatment of hematologic malignancy. Overall, the cumulative incidence of 1 or more serious infections was comparable between groups (BM, 81%; TCD, 83%; UCB, 90%; P = .12). Furthermore, by taking all serious infections into account and using multivariate techniques with unmanipulated BM as the reference, there were also no significant differences between groups (TCD relative risk [RR], 1.6; P = .10; UCB RR, 1.0; P = .84). Within the time periods days 0 to 42, days 43 to 100, and days 101 to 180, the only difference was a greater risk of viral infections from days 0 to 42 in TCD recipients (RR, 3.5; P = .02). Notably, after day 180, TCD recipients had a significantly increased infection risk (RR, 3.1; P = .03), whereas the risk in UCB recipients (RR, 0.5; P = .23) was comparable to that in BM recipients. Other factors associated with an increased infection risk in the 2 years after transplantation were age > or = 8 years, graft failure, and severe acute graft-versus-host disease. These data suggest that the risk of serious infection after pediatric UCB transplantation is comparable to that with unmanipulated BM.

Title: Embryonic-like stem cell derived from adult bone marrow: immature morphology, cell surface markers, ultramicrostructure and differentiation into multinucleated fibers in vitro. Content: Embryonic-like stem cell (ELSC), expressing part of surface markers of human embryonic stem cells, may be a better candidate for cell therapy of degenerative muscular disease than mesenchymal stem cell (MSC). We isolated ELSC and MSC from bone marrow, respectively, and compared their differences in the characteristics and the capacity of myogenic differentiation. Results showed that ELSC could be isolated successfully from 3 adult bone marrow samples by using serum-free medium with 10ng/ml basic fibroblast growth factor (bFGF). At the same cell density, MSC could also be isolated from the same samples by using DMEM/F12 medium containing 10% new cattle serum. However, ELSC appeared as small, morphologically slenderer, upregulated expression of SSEA-4 and ultramicroscopically more immature than MSC derived from the same samples. Immunofluorescent staining and RT-PCR analysis showed ELSC weakly expressed Oct-4, Nanog-3 and Sox-2. Moreover, ELSC and MSC could be induced into long, multinucleated fibers expressing myogenin and myosin heavy chain (MHC) in myogenic differentiation medium, but by day 10, proportion of multinucleated fibers positive for MHC was respectively 25.0%+/-6.9% and 13.8%+/-7.6% in ELSC and MSC culture. These data suggest that bone marrow derived ELSC represent an ideal candidate for cell therapy of degenerative muscular disease.

Title: Programmed death-1–induced interleukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection Content: Viral replication and microbial translocation from the gut to the blood during HIV infection lead to hyperimmune activation, which contributes to the decline in CD4+ T cell numbers during HIV infection. Programmed death-1 (PD-1) and interleukin-10 (IL-10) are both upregulated during HIV infection. Blocking interactions between PD-1 and programmed death ligand-1 (PD-L1) and between IL-10 and IL-10 receptor (IL-10R) results in viral clearance and improves T cell function in animal models of chronic viral infections. Here we show that high amounts of microbial products and inflammatory cytokines in the plasma of HIV-infected subjects lead to upregulation of PD-1 expression on monocytes that correlates with high plasma concentrations of IL-10. Triggering of PD-1 expressed on monocytes by PD-L1 expressed on various cell types induced IL-10 production and led to reversible CD4+ T cell dysfunction. We describe a new function for PD-1 whereby microbial products inhibit T cell expansion and function by upregulating PD-1 levels and IL-10 production by monocytes after binding of PD-1 by PD-L1.

Title: Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity Content: The midbody is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. Midbodies persist in cells long after division as midbody derivatives (MBds), but their fate is unclear. Here we show that MBds are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells, induced pluripotent stem cells and potential cancer ‘stem cells’ in vivo and in vitro. MBd loss accompanies stem-cell differentiation, and involves autophagic degradation mediated by binding of the autophagic receptor NBR1 to the midbody protein CEP55. Differentiating cells and normal dividing cells do not accumulate MBds and possess high autophagic activity. Stem cells and cancer cells accumulate MBds by evading autophagosome encapsulation and exhibit low autophagic activity. MBd enrichment enhances reprogramming to induced pluripotent stem cells and increases the in vitro tumorigenicity of cancer cells. These results indicate unexpected roles for MBds in stem cells and cancer ‘stem cells’.

Title: Serum immunoglobulin A from patients with celiac disease inhibits human T84 intestinal crypt epithelial cell differentiation. Content: BACKGROUND & AIMS Celiac disease is characterized by disturbed jejunal crypt-villus axis biology with immunoglobulin (Ig) A deposits underlining the epithelium. The aim of this study was to test whether celiac disease serum IgA (reticulin/endomysial autoantibodies) interferes with the mesenchymal-epithelial cell cross-talk. METHODS Differentiation of T84 epithelial cells was induced with IMR-90 fibroblasts or transforming growth factor beta in three-dimensional collagen gel cultures. The effects of purified celiac IgA and monoclonal tissue transglutaminase antibodies (CUB7402) were studied by adding the antibodies to the cocultures. RESULTS Active celiac disease IgA, reactive for tissue transglutaminase, significantly inhibited T84 epithelial cell differentiation (P < 0.001) and increased epithelial cell proliferation (P = 0.024). Similar effects were obtained with antibodies against tissue transglutaminase. CONCLUSIONS Celiac disease-associated IgA class antibodies disturb transforming growth factor beta-mediated fibroblast-epithelial cell cross-talk in this in vitro crypt-villus axis model. This primary finding indicates that celiac disease-specific autoantibodies may also contribute to the formation of the gluten-triggered jejunal mucosal lesion in celiac disease.

Title: SIRT1 is required for long-term growth of human mesenchymal stem cells Content: Human mesenchymal stem cells (MSCs) have therapeutic potential because of their ability to self-renew and differentiate into multiple tissues. However, senescence often occurs in MSCs when they are cultured in vitro and the molecular mechanisms underlying this effect remain unclear. In this study, we found that NAD-dependent protein deacetylase SIRT1 is differentially expressed in both human bone marrow-derived MSCs (B-MSCs) and adipose tissue-derived MSCs after increasing passages of cell culture. Using lentiviral shRNA we demonstrated that selective knockdown of SIRT1 in human MSCs at early passage slows down cell growth and accelerates cellular senescence. Conversely, overexpression of SIRT1 delays senescence in B-MSCs that have undergone prolonged in vitro culturing and the cells do not lose adipogenic and osteogenic potential. In addition, we found that the delayed accumulation of the protein p16 is involved in the effect of SIRT1. However, resveratrol, which has been used as an activator of SIRT1 deacetylase activity, only transiently promotes proliferation of B-MSCs. Our findings will help us understand the role of SIRT1 in the aging of normal diploid cells and may contribute to the prevention of human MSCs senescence thus benefiting MSCs-based tissue engineering and therapies.

Title: Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin Content: Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, postnatally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.

Title: Induction of circadian gene expression in human subcutaneous adipose-derived stem cells. Content: OBJECTIVE Genes encoding the circadian transcriptional apparatus exhibit robust oscillatory expression in murine adipose tissues. This study tests the hypothesis that human subcutaneous adipose-derived stem cells (ASCs) provide an in vitro model in which to monitor the activity of the core circadian transcriptional apparatus. RESEARCH METHODS AND PROCEDURES Primary cultures of undifferentiated or adipocyte-differentiated ASCs were treated with dexamethasone, rosiglitazone, or 30% fetal bovine serum. The response of undifferentiated ASCs to dexamethasone was further evaluated in the presence of lithium chloride. Lithium inhibits glycogen synthase kinase 3, a key component of the circadian apparatus. Total RNA was harvested at 4-hour intervals over 48 hours and examined by real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS Adipocyte-differentiated cells responded more rapidly to treatments than their donor-matched undifferentiated controls; however, the period of the circadian gene oscillation was longer in the adipocyte-differentiated cells. Dexamethasone generated circadian gene expression patterns with mean periods of 25.4 and 26.7 hours in undifferentiated and adipocyte-differentiated ASCs, respectively. Both rosiglitazone and serum shock generated a significantly longer period in adipocyte-differentiated ASCs relative to undifferentiated ASCs. The Bmal1 profile was phase-shifted by approximately 8 to 12 hours relative to Per1, Per3, and Cry2, consistent with their expression in vivo. Lithium chloride inhibited adipogenesis and significantly lengthened the period of Per3 and Rev-erbalpha gene expression profiles by >5 hours in dexamethasone-activated undifferentiated ASCs. DISCUSSION These results support the initial hypothesis and validate ASCs as an in vitro model for the analysis of circadian biology in human adipose tissue.

Title: Treatment and management of graft-versus-host disease: improving response and survival. Content: Graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following allogenic haematopoietic stem-cell transplantation and thus the focus of much ongoing research. Despite considerable advances in our understanding of the pathophysiology, diagnosis and predisposing factors for both acute and chronic forms of the disease, a standardised therapeutic strategy is still lacking. There is good evidence for initial treatment of both acute and chronic forms of the disease with corticosteroid therapy. However, the most effective approach to steroid-refractory disease remains controversial, with current practice based mainly on smaller studies and varying considerably between local institutions. Timely diagnosis, multidisciplinary working and good supportive care, including infection prophylaxis, are clearly important in optimizing response and survival in such patients. It is hoped that in the future systematic research strategies and the identification of novel therapeutic targets may improve outcome further. The following review aims to outline some of the existing options for the treatment and management of acute and chronic GVHD.

Title: Adult T-cell leukemia/lymphoma in London: clinical experience of 21 cases. Content: Adult T-cell leukemia/lymphoma (ATLL) is uncommon in the United Kingdom and has so far been restricted to people of Afro-Caribbean extraction. Between 1981 and 1995, 21 cases presented to 2 inner London teaching hospitals where 17% of the population are of Afro-Caribbean origin. Clinical presentations were similar to those of the disease in HTLV-I-endemic areas. Major responses (CR + PR) were obtained in 10/16 assessable patients (63%) treated with combination chemotherapy. However, median survival was only 5.5 months. Disease progression and opportunistic infection were the major causes of treatment failure and death. Three patients (14%) relapsed in the central nervous system (CNS). Our cases confirm the profound immunosuppression in ATLL. The poor prognosis of acute and lymphoma types of ATLL highlight the need for new approaches to treatment such as zidovudine and alpha-interferon, incorporating prophylaxis against CNS disease and opportunistic infections.

Title: LIN28B enhanced tumorigenesis in an autochthonous KRASG12V-driven lung carcinoma mouse model Content: LIN28B is a RNA-binding protein regulating predominantly let-7 microRNAs with essential functions in inflammation, wound healing, embryonic stem cells, and cancer. LIN28B expression is associated with tumor initiation, progression, resistance, and poor outcome in several solid cancers, including lung cancer. However, the functional role of LIN28B, especially in non-small cell lung adenocarcinomas, remains elusive. Here, we investigated the effects of LIN28B expression on lung tumorigenesis using LIN28B transgenic overexpression in an autochthonous KRASG12V-driven mouse model. We found that LIN28B overexpression significantly increased the number of CD44+/CD326+ tumor cells, upregulated VEGF-A and miR-21 and promoted tumor angiogenesis and epithelial-to-mesenchymal transition (EMT) accompanied by enhanced AKT phosphorylation and nuclear translocation of c-MYC. Moreover, LIN28B accelerated tumor initiation and enhanced proliferation which led to a shortened overall survival. In addition, we analyzed lung adenocarcinomas of the Cancer Genome Atlas (TCGA) and found LIN28B expression in 24% of KRAS-mutated cases, which underscore the relevance of our model.

Title: The developmental potential of iPSCs is greatly influenced by reprogramming factor selection. Content: Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.

Title: Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma. Content: PURPOSE Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Title: Requirement of endogenous stem cell factor and granulocyte-colony-stimulating factor for IL-17-mediated granulopoiesis. Content: IL-17 is a novel, CD4+ T cell-restricted cytokine. In vivo, it stimulates hematopoiesis and causes neutrophilia consisting of mature granulocytes. In this study, we show that IL-17-mediated granulopoiesis requires G-CSF release and the presence or induction of the transmembrane form of stem cell factor (SCF) for optimal granulopoiesis. However, IL-17 also protects mice from G-CSF neutralization-induced neutropenia. G-CSF neutralization completely reversed IL-17-induced BM progenitor expansion, whereas splenic CFU-GM/CFU-granulocyte-erythrocyte-megakaryocyte-monocyte was only reduced by 50% in both Sl/Sld and littermate control mice. Thus, there remained a significant SCF/G-CSF-independent effect of IL-17 on splenic granulopoiesis, resulting in a preservation of mature circulating granulocytes. IL-17 is a cytokine that potentially interconnects lymphocytic and myeloid host defense and may have potential for therapeutic development.

Title: Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Content: Studies of the identity and physiological function of mesenchymal stromal cells (MSCs) have been hampered by a lack of markers that permit both prospective identification and fate mapping in vivo. We found that Leptin Receptor (LepR) is a marker that highly enriches bone marrow MSCs. Approximately 0.3% of bone marrow cells were LepR(+) , 10% of which were CFU-Fs, accounting for 94% of bone marrow CFU-Fs. LepR(+) cells formed bone, cartilage, and adipocytes in culture and upon transplantation in vivo. LepR(+) cells were Scf-GFP(+), Cxcl12-DsRed(high), and Nestin-GFP(low), markers which also highly enriched CFU-Fs, but negative for Nestin-CreER and NG2-CreER, markers which were unlikely to be found in CFU-Fs. Fate-mapping showed that LepR(+) cells arose postnatally and gave rise to most bone and adipocytes formed in adult bone marrow, including bone regenerated after irradiation or fracture. LepR(+) cells were quiescent, but they proliferated after injury. Therefore, LepR(+) cells are the major source of bone and adipocytes in adult bone marrow.

Title: Axicabtagene Ciloleucel CAR T‐Cell Therapy in Refractory Large B‐Cell Lymphoma Content: Background In a phase 1 trial, axicabtagene ciloleucel (axi‐cel), an autologous anti‐CD19 chimeric antigen receptor (CAR) T‐cell therapy, showed efficacy in patients with refractory large B‐cell lymphoma after the failure of conventional therapy. Methods In this multicenter, phase 2 trial, we enrolled 111 patients with diffuse large B‐cell lymphoma, primary mediastinal B‐cell lymphoma, or transformed follicular lymphoma who had refractory disease despite undergoing recommended prior therapy. Patients received a target dose of 2×106 anti‐CD19 CAR T cells per kilogram of body weight after receiving a conditioning regimen of low‐dose cyclophosphamide and fludarabine. The primary end point was the rate of objective response (calculated as the combined rates of complete response and partial response). Secondary end points included overall survival, safety, and biomarker assessments. Results Among the 111 patients who were enrolled, axi‐cel was successfully manufactured for 110 (99%) and administered to 101 (91%). The objective response rate was 82%, and the complete response rate was 54%.With a median follow‐up of 15.4 months, 42% of the patients continued to have a response, with 40% continuing to have a complete response. The overall rate of survival at 18 months was 52%. The most common adverse events of grade 3 or higher during treatment were neutropenia (in 78% of the patients), anemia (in 43%), and thrombocytopenia (in 38%). Grade 3 or higher cytokine release syndrome and neurologic events occurred in 13% and 28% of the patients, respectively. Three of the patients died during treatment. Higher CAR T‐cell levels in blood were associated with response. Conclusions In this multicenter study, patients with refractory large B‐cell lymphoma who received CAR T‐cell therapy with axi‐cel had high levels of durable response, with a safety profile that included myelosuppression, the cytokine release syndrome, and neurologic events. (Funded by Kite Pharma and the Leukemia and Lymphoma Society Therapy Acceleration Program; ZUMA‐1 ClinicalTrials.gov number, NCT02348216.)