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**11**

*Iran*

Mohaddeseh Behjati

*Isfahan University of Medical Sciences*

**Pursuing Candidate Stem Cells for**

**Optimal Cardiac Regeneration in Patients**

**Suffered from Acute Coronary Syndrome**

It has been estimated that cardiovascular diseases will increase to 23.3 million in 2030 [Mathers & Loncar, 2006]. Acute myocardial infarction (AMI) typically occurs as a result of death of millions of myocytes, replaced by non-contractile scar tissue, which imposes a great load burden on surviving myocytes [Segers & Lee, 2010]. Despite of the fundamental progress in the treatment of cardiovascular diseases, a substantial limitation is still present. Current reperfusion strategies afford a great myocardial salvage but limited regenerative capacity of the human heart is a barrier for complete myocardial recovery after necrotic events. Thus, the heart responds to injury by scar formation and persistence muscle damage. Massive cell death and replacement of fibrotic tissue lead to cavitary dilatation and negative left ventricular (LV) remodeling [Ren et al., 2005]. The aftermath event is contractile dysfunction and terminal failure. Thus, regenerating the infracted heart should be adjunct to the therapeutic strategies capable to restore the blood supply to the territory of the infarct related artery. These needs can be met using a cluster of cells with self-renewal capacity, clonal expansion and ability to differentiate into multiple cell lineages. Pluripotent embryonic stem cells (ESCs) or multipotent adult stem cells (ASCs) showed remarkable capacity in heart regeneration. But it needs to be emphasized that heart is not just a pump and orchestrated temporospatial activities consisting sequential electrical stimulation and mechanical contraction are highly demanded. By now, our knowledge about the genetic bases and natural underlying events of cardiovascular disease precede the advancement of therapeutic strategies. A candidate therapeutic strategy should improve cardiac remodeling and function through formation of new blood vessels and inducing reconstitution of functional myocardium. Thus, the aim of this chapter is to focus on the different aspects of

Human ESCs (hESCs) isolated from the inner cell mass of the blastocyst stage of human embryo [Ding et al., 2011]. These cells have a unique ability to differentiate into various derivatives of three germ layers and construct ~ 220 diverse cell types of adult human body [Mingxia et al., 2011, Ding et al., 2011]. Application of clinically unsuitable or

stem cell therapy as a growing field for cell-based strategies.

**1. Introduction**

**2. Stem cells**

## **Pursuing Candidate Stem Cells for Optimal Cardiac Regeneration in Patients Suffered from Acute Coronary Syndrome**

Mohaddeseh Behjati *Isfahan University of Medical Sciences Iran*

## **1. Introduction**

224 Coronary Artery Diseases

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2011, 123:779-797

It has been estimated that cardiovascular diseases will increase to 23.3 million in 2030 [Mathers & Loncar, 2006]. Acute myocardial infarction (AMI) typically occurs as a result of death of millions of myocytes, replaced by non-contractile scar tissue, which imposes a great load burden on surviving myocytes [Segers & Lee, 2010]. Despite of the fundamental progress in the treatment of cardiovascular diseases, a substantial limitation is still present. Current reperfusion strategies afford a great myocardial salvage but limited regenerative capacity of the human heart is a barrier for complete myocardial recovery after necrotic events. Thus, the heart responds to injury by scar formation and persistence muscle damage. Massive cell death and replacement of fibrotic tissue lead to cavitary dilatation and negative left ventricular (LV) remodeling [Ren et al., 2005]. The aftermath event is contractile dysfunction and terminal failure. Thus, regenerating the infracted heart should be adjunct to the therapeutic strategies capable to restore the blood supply to the territory of the infarct related artery. These needs can be met using a cluster of cells with self-renewal capacity, clonal expansion and ability to differentiate into multiple cell lineages. Pluripotent embryonic stem cells (ESCs) or multipotent adult stem cells (ASCs) showed remarkable capacity in heart regeneration. But it needs to be emphasized that heart is not just a pump and orchestrated temporospatial activities consisting sequential electrical stimulation and mechanical contraction are highly demanded. By now, our knowledge about the genetic bases and natural underlying events of cardiovascular disease precede the advancement of therapeutic strategies. A candidate therapeutic strategy should improve cardiac remodeling and function through formation of new blood vessels and inducing reconstitution of functional myocardium. Thus, the aim of this chapter is to focus on the different aspects of stem cell therapy as a growing field for cell-based strategies.

## **2. Stem cells**

Human ESCs (hESCs) isolated from the inner cell mass of the blastocyst stage of human embryo [Ding et al., 2011]. These cells have a unique ability to differentiate into various derivatives of three germ layers and construct ~ 220 diverse cell types of adult human body [Mingxia et al., 2011, Ding et al., 2011]. Application of clinically unsuitable or

Pursuing Candidate Stem Cells for Optimal

molecular imaging are warranted.

beneficial than BM-MSCs [In 't Anker et al., 2010].

Cardiac Regeneration in Patients Suffered from Acute Coronary Syndrome 227

criterion for pluripotency is demonstration of the cell lineage's ability to reconstitute tissue composed of three layers by creating chimeras, tetraploid complementation or teratoma formation tests [Swelstad & Kerr, 2009]. Teratoma assay in immunodeficient SCID mice is currently used to test pluripotency in vivo for human iPSCs [Tan et al., 2008]. Teratoma formation (mature or immature) with differentiated ESC- or –iPSC-derived cells is attributed to the insufficient purity and remnant undifferentiated cell population within the transplanted cells [Kooreman & Wu, 2010]. Teratoma formation with injected mouse ESC (mESC)-derived beating embryoid bodies and undifferentiated mESCs is seen in experimental studies [Lin et al., 2010]. Transplantation of pure hESC-derived cardiomyocytes (82.6±6.6%) into immunodeficient rats was not associated with teratoma formation [Laflamme et al., 2007]. For ultimate translation of pluripotent stem cells into clinical benefits, highly purified cells and early detection of teratoma using novel non-invasive tracking strategies and advanced

Despite of the substantial progresses made in the field of reprogramming, low reprogramming efficiencies (0.01-0.1% of input cells), slow kinetics of process, partial reprogramming and genetic instability of the manipulated cells hamper clinical application of iPSCs [Utikal et al., 2009, Kanawaty & Henderson, 2009, Stadtfeld et al., 2008]. The type of original somatic cell used for iPSCs, its cycle status and genetic/epigenetic background affect the functional/molecular characteristic of the derived cells [Polo et al., 2010]. These factors, in addition to the "epigenetic memory" of hiPSCs affect the total reprogramming efficiency [Polo et al., 2010]. Thus, alternative promising stem cell source with remarkable plasticity as easily extracted adult stem cells (ASCs), hematopoietic stem cells (HSCs), adipose-derived stem cells and derived MSCs seem useful surrogates [Lodi et al., 2011]. ASCs showed wide range of paracrine effects as cytoprotection, enhanced angiogenesis, recruitment of hematopoietic stem cells and activation of resident cardiac stem cells for endogenous repair [Gnecchi et al., 2008]. Umbilical cord blood (UCB) containing hematopoietic (UC-HS) and mesenchymal stem cells (UC-MS) with higher immunological tolerance are another cell source [Mihu et al., 2008]. Generally, UC-MS grafts are more

The activated vs. silenced pluripotency gene cluster is needed for proper programming. Mitotic errors, mutation occurrence and karyotypic changes have been observed in hESCs cultured over long passages [Kooreman & Wu, 2010]. Alterations in imprinted region on chromosome 12, location of pluripotency marker Nanog gene, have been proposed in the tumorigenicity of pluripotent cells [Draper et al., 2004]. Suppressed p53 signaling necessary for reprogramming brings tumorigenicity to the derived stem cells [Hong et al., 2009]. Tumorigenicity is an inherent property of pluripotent cells which is reduced upon differentiation. Thus, decreased tumorigenicity of the pluripotent cells means parallel decrease in their pluripotency and self-renewal potentials. Despite of the presence of intact spindle assembly checkpoints (SAC), mitotic failure-induced polyploidy has been observed in ESCs without occurrence of apoptosis [Kooreman & Wu, 2010]. In contrast with phenotypically resistant ESCs to DNA-damaging agents, embryoid bodies (EB) undergo caspase-3-induced apoptosis by these agents [Kooreman & Wu, 2010]. Human EB aggregates could be propagated from embryonic germ (EG) cells with multi-lineage differentiation potential and limited proliferation [Wobus & Löser, 2011]. It has been speculated that hEG cells might be an alternative to hESCs in future for therapeutic

developmentally arrested embryos can overcome the ethical problems related to embryo manipulation. Obtaining cells from single blastomeres of human embryo hampers the need for embryo destruction. Induced pluripotent stem cells (iPSCs) using cellular reprogramming via forced expression of certain stimulating factors essential for maintenance of stemness, brought the ultimate solution without the need to human embryo [David et al., 2011]. By transferring the nuclear materials of somatic cells into the oocytes conferred pluripotency or totipotency of somatic cells became possible [Gurdon & Wilmut, 2011]. Despite of some epigenetic variations between iPSCs (mainly factor-free) and ESCs, these cells are similar in terms of proliferation, morphology, differentiation potential, imprinting, chromatin profiles and global gene/protein expression signature [Nordin et al., 2011]. This technology bypasses the need for the embryo or the desired tissue as heart. Thus, it is ethically accepted for both therapeutic applications and diagnostic measures like patient's disease modeling in vitro in order to find a treatment. Disease-specific iPS cells are also of paramount importance for achievement of this goal. In this way, tissue matching for organ transplantation is not a matter more. These goals are achievable using exogenous expression of two pluripotency transcription factors (e.g. Nanong, Oct4 and Sox2) and two proto-oncogenes (e.g. c-Myc and Klf4) [Nordin et al., 2011]. Induction of these programming factors is possible through application of retroviral and lentiviral vectors. Both of these vectors act for a period of time and then get silenced state once the endogenous genes had taken over the management of pluripotency [Stadtfeld & Hochedlinger, 2010]. The larger insert capacity of defective lentiviral vectors let them to deliver all of the programming factors without the need for separate individual vectors [Sinn et al., 2005]. In contrast with retroviruses, lentiviral vectors potently infect both dividing and non-dividing cells [Škalamera et al., 2011]. Viral vectors without integration into the genomic material of the target cells, like Adenovirus, have been used but their extremely low efficiency has faded their wide application [Okita & Yamanaka, 2011, Stadtfeld et al., 2008]. In addition to genetic manipulation, cell preconditioning and reprogramming could be performed thorough chemical and pharmacological cell manipulation. The advent of virus-free induction methods seems a revolutionary step in stem cell biotechnology. Despite of their low efficiency, Transposons, protein and mRNA-based induction methods seem advantageous due to their transgene-free nature [Si-Tayeb, 2010]. Small molecules as chromatin-modifying agents like Valporic acid (VPA) are also promising options for transgene-free cell reprogramming with replacement of potentially oncogenic-reprogramming factors [Medvedev et al., 2011]. In addition to the induction methods, knowledge about the through interplay between reprogramming factors will help in identification of more powerful reprogramming strategies. In this context, the effect of c-Myc in augmentation of the Oct4, Sox2 and Klf4 has been consequently associated with enhanced proliferation and differentiation arrest [Takahashi. et al., 2007]. Fully programmed cells raised safety concerns due to induced tumorigenicity by applied preconditioning factors as c-Myc [Kooreman & Wu, 2010]. Recently it has been suggested that creation of iPSCs using L-Myc instead of c-Myc brings less tumorigenicity [Nakagawa et al., 2010]. iPSCs are characterized by the expression of the above mentioned transcription factors and cell surface molecules (e.g. SSEA-3/4, Tra-1-60 and Tra-1-81) [Swelstad & Kerr, 2009]. High alkaline phosphatase and telomerase activity, rapid proliferation, lack of contact inhibition and high nucleus to cytoplasmic ratio with prominent nuclear growing in flat colonies are also further confirmatory indices of successful iPSC achievement [Kooreman & Wu, 2010]. The major

developmentally arrested embryos can overcome the ethical problems related to embryo manipulation. Obtaining cells from single blastomeres of human embryo hampers the need for embryo destruction. Induced pluripotent stem cells (iPSCs) using cellular reprogramming via forced expression of certain stimulating factors essential for maintenance of stemness, brought the ultimate solution without the need to human embryo [David et al., 2011]. By transferring the nuclear materials of somatic cells into the oocytes conferred pluripotency or totipotency of somatic cells became possible [Gurdon & Wilmut, 2011]. Despite of some epigenetic variations between iPSCs (mainly factor-free) and ESCs, these cells are similar in terms of proliferation, morphology, differentiation potential, imprinting, chromatin profiles and global gene/protein expression signature [Nordin et al., 2011]. This technology bypasses the need for the embryo or the desired tissue as heart. Thus, it is ethically accepted for both therapeutic applications and diagnostic measures like patient's disease modeling in vitro in order to find a treatment. Disease-specific iPS cells are also of paramount importance for achievement of this goal. In this way, tissue matching for organ transplantation is not a matter more. These goals are achievable using exogenous expression of two pluripotency transcription factors (e.g. Nanong, Oct4 and Sox2) and two proto-oncogenes (e.g. c-Myc and Klf4) [Nordin et al., 2011]. Induction of these programming factors is possible through application of retroviral and lentiviral vectors. Both of these vectors act for a period of time and then get silenced state once the endogenous genes had taken over the management of pluripotency [Stadtfeld & Hochedlinger, 2010]. The larger insert capacity of defective lentiviral vectors let them to deliver all of the programming factors without the need for separate individual vectors [Sinn et al., 2005]. In contrast with retroviruses, lentiviral vectors potently infect both dividing and non-dividing cells [Škalamera et al., 2011]. Viral vectors without integration into the genomic material of the target cells, like Adenovirus, have been used but their extremely low efficiency has faded their wide application [Okita & Yamanaka, 2011, Stadtfeld et al., 2008]. In addition to genetic manipulation, cell preconditioning and reprogramming could be performed thorough chemical and pharmacological cell manipulation. The advent of virus-free induction methods seems a revolutionary step in stem cell biotechnology. Despite of their low efficiency, Transposons, protein and mRNA-based induction methods seem advantageous due to their transgene-free nature [Si-Tayeb, 2010]. Small molecules as chromatin-modifying agents like Valporic acid (VPA) are also promising options for transgene-free cell reprogramming with replacement of potentially oncogenic-reprogramming factors [Medvedev et al., 2011]. In addition to the induction methods, knowledge about the through interplay between reprogramming factors will help in identification of more powerful reprogramming strategies. In this context, the effect of c-Myc in augmentation of the Oct4, Sox2 and Klf4 has been consequently associated with enhanced proliferation and differentiation arrest [Takahashi. et al., 2007]. Fully programmed cells raised safety concerns due to induced tumorigenicity by applied preconditioning factors as c-Myc [Kooreman & Wu, 2010]. Recently it has been suggested that creation of iPSCs using L-Myc instead of c-Myc brings less tumorigenicity [Nakagawa et al., 2010]. iPSCs are characterized by the expression of the above mentioned transcription factors and cell surface molecules (e.g. SSEA-3/4, Tra-1-60 and Tra-1-81) [Swelstad & Kerr, 2009]. High alkaline phosphatase and telomerase activity, rapid proliferation, lack of contact inhibition and high nucleus to cytoplasmic ratio with prominent nuclear growing in flat colonies are also further confirmatory indices of successful iPSC achievement [Kooreman & Wu, 2010]. The major criterion for pluripotency is demonstration of the cell lineage's ability to reconstitute tissue composed of three layers by creating chimeras, tetraploid complementation or teratoma formation tests [Swelstad & Kerr, 2009]. Teratoma assay in immunodeficient SCID mice is currently used to test pluripotency in vivo for human iPSCs [Tan et al., 2008]. Teratoma formation (mature or immature) with differentiated ESC- or –iPSC-derived cells is attributed to the insufficient purity and remnant undifferentiated cell population within the transplanted cells [Kooreman & Wu, 2010]. Teratoma formation with injected mouse ESC (mESC)-derived beating embryoid bodies and undifferentiated mESCs is seen in experimental studies [Lin et al., 2010]. Transplantation of pure hESC-derived cardiomyocytes (82.6±6.6%) into immunodeficient rats was not associated with teratoma formation [Laflamme et al., 2007]. For ultimate translation of pluripotent stem cells into clinical benefits, highly purified cells and early detection of teratoma using novel non-invasive tracking strategies and advanced molecular imaging are warranted.

Despite of the substantial progresses made in the field of reprogramming, low reprogramming efficiencies (0.01-0.1% of input cells), slow kinetics of process, partial reprogramming and genetic instability of the manipulated cells hamper clinical application of iPSCs [Utikal et al., 2009, Kanawaty & Henderson, 2009, Stadtfeld et al., 2008]. The type of original somatic cell used for iPSCs, its cycle status and genetic/epigenetic background affect the functional/molecular characteristic of the derived cells [Polo et al., 2010]. These factors, in addition to the "epigenetic memory" of hiPSCs affect the total reprogramming efficiency [Polo et al., 2010]. Thus, alternative promising stem cell source with remarkable plasticity as easily extracted adult stem cells (ASCs), hematopoietic stem cells (HSCs), adipose-derived stem cells and derived MSCs seem useful surrogates [Lodi et al., 2011]. ASCs showed wide range of paracrine effects as cytoprotection, enhanced angiogenesis, recruitment of hematopoietic stem cells and activation of resident cardiac stem cells for endogenous repair [Gnecchi et al., 2008]. Umbilical cord blood (UCB) containing hematopoietic (UC-HS) and mesenchymal stem cells (UC-MS) with higher immunological tolerance are another cell source [Mihu et al., 2008]. Generally, UC-MS grafts are more beneficial than BM-MSCs [In 't Anker et al., 2010].

The activated vs. silenced pluripotency gene cluster is needed for proper programming. Mitotic errors, mutation occurrence and karyotypic changes have been observed in hESCs cultured over long passages [Kooreman & Wu, 2010]. Alterations in imprinted region on chromosome 12, location of pluripotency marker Nanog gene, have been proposed in the tumorigenicity of pluripotent cells [Draper et al., 2004]. Suppressed p53 signaling necessary for reprogramming brings tumorigenicity to the derived stem cells [Hong et al., 2009]. Tumorigenicity is an inherent property of pluripotent cells which is reduced upon differentiation. Thus, decreased tumorigenicity of the pluripotent cells means parallel decrease in their pluripotency and self-renewal potentials. Despite of the presence of intact spindle assembly checkpoints (SAC), mitotic failure-induced polyploidy has been observed in ESCs without occurrence of apoptosis [Kooreman & Wu, 2010]. In contrast with phenotypically resistant ESCs to DNA-damaging agents, embryoid bodies (EB) undergo caspase-3-induced apoptosis by these agents [Kooreman & Wu, 2010]. Human EB aggregates could be propagated from embryonic germ (EG) cells with multi-lineage differentiation potential and limited proliferation [Wobus & Löser, 2011]. It has been speculated that hEG cells might be an alternative to hESCs in future for therapeutic

Pursuing Candidate Stem Cells for Optimal

occurred after trans-differentiation into myocardium.

**4. Culture conditions**

**5. Special considerations**

Cardiac Regeneration in Patients Suffered from Acute Coronary Syndrome 229

teratoma formation [van der Bogt et al**.,** 2006, Lee et al., 2009]. Early detected teratomas can be ablated by its targeting using reporter-suicide gene construct [Cao et al., 2007]. But limited signal penetration in larger animals and lack of provided spatial three-dimensional data hurdles clinical application of BLI using Fluc reporter gene [Kooreman & Wu, 2010]. Higher spatial resolution of PET and MRI made them good candidate for clinical application but their substantial low detection threshold remained an obstacle [Kooreman & Wu, 2010]. Thus, combining image modalities is of crucial importance especially for clinical insights about identification of the safe limit of stem cell numbers without teratoma formation. Using BLI, Lee et al found safe limit of undifferentiated hESCs for cardiac transplantation into SCID mice to be 1×104 [Lee et al., 2009]. Progress in tracking strategies should be in parallel with identification of the appropriate markers for tracking of both stem cell homing and cardiac differentiation. Markers of undifferentiated cells as Oct4, hTert (human telomerase reverse transcriptase) and Dusp6 (dual specifity phosphatase 6) have been shown to be decreased during cardiac trans-differentiation [Wobus & Löser, 2011]. Thus, markers of mesoderm and early cardiogenesis as GATA-4 and Brachyury were found to be suitable for tracking cardiac differentiation [Wobus & Löser, 2011]. Precise imaging technologies should solve uncertainties real cardiomyocyte trans-differentiation vs. cell fusion. Cell fusion as an overlooked phenomenon occurs due to the autoflorescence problems regarding label transfer to neighboring cells or fusion of donor and recipient cells [Reinecke et al., 2008]. The former is avoidable using cell lineage markers as genetic materials [Reinecke et al., 2008]. This process gives rise to bi-nucleated, mono-nucleated cells with tetraploid synkaryon or cells with normal karyotype during division [Wang et al., 2003]. This phenomenon might be

The first ESC was cultured on mouse embryonic fibroblast- feeder layer cells (MEF-FL) [Wobus, 2010]. Growing of stem cells in suspension as aggregates or removing of feeder fibroblasts promotes differentiation of ESCs [Dambrot et al., 2011]. Derivatives of three germ layers like mesoderm (cardiomyocytes, blood and vascular endothelial cells) were derived by this method [Dambrot et al., 2011]. By advent of novel culture media stem cells could be kept in undifferentiated state even in the absence of feeder cells. Commercial culture media as mTeSR**®**1 and TeSR™2 (STEMCELL technologies) in combination with matrix containing a mixture of human collagen IV, fibronectin, vitronectin and laminin are beneficial in this context [Dambrot et al., 2011]. The goal of these culture conditions is to promote stem cell scale up while keeping karyotypic stability through successive enzymatic passages or

suspension cultures. This field of stem cell technology needs to be promoted further.

Surveys to find the candidate stem cell should be parallel with search to find the candidate animal model. By now, mice are the most common used animal models due to the feasibility of the mutation induction and targeted deletion in them. Despite of these advantages, there are some fundamental differences between mouse and human heart. The predominantly expressed isoform of Myosin heavy chain (MHC) in fetal and adult mice are βMHC and αMHC, respectively [Dambrot et al., 2011]. The inverse pattern is seen in humans. The higher beating rate of mice (500 bpm) is surprisingly different from human heart (70 bpm)

applications [Wobus & Löser, 2011]. Embryonic-like stem cells as Spermatogonial stem cells (SGSCs), parthenogenetic stem cells (PSCs) and male germline stem cells in pre-menopausal women can also give rise into fully active cardiomyocytes [Guan et al., 2007, Zimmermann, 2011].

## **3. Image platform**

Molecular imaging for in vivo tracking the proliferating and viable stem cells made a substantial help in the field of bench to bedside application of stem cells. Pre-transplantation labeling through cell inoculation with nanoparticles or reporter gene is helpful [Kooreman & Wu, 2010]. Semiconductor quantum dots capable to emit different light wavelengths show photostable bright image signals but their aggregation inside the cytosol made the process of cell delivery difficult [Kooreman & Wu, 2010]. Non-specific binding is another issue. Mesenchymal stem cells have been tracked by MRI after labeling with Ferumoxides [Kraitchman et al.**,** 2003, Amado et al**.,** 2005, Arai et al**.,** 2006]. MRI signals elicited by changes in T2 relaxation are induced due to the endocytosis of the iron oxide particles (SPIOs) or ultrasmall superparamagnetic iron oxide particles (USPIOs) [Kooreman & Wu, 2010]. MRI signals are detectable for a period between three weeks up to two months but these signals can already exist in the presence of dead stem cells due to engulfed iron particles in scavenging macrophages [Lee et al., 2009]. Alternatively, direct stem cell labeling applying radionucleotides has been used for circulating-progenitor cells successfully [Hofmann et al., 2005]. The radionucleotide-bound cells could be detected using SPECT, PET, gamma camera and cardiac magnetic resonance tracing [Kooreman & Wu. 2010]. Ultimately, the tracking duration of the radionucleotide-bound cells depends greatly on the individual half-lives of the applied radionucleotide [Kooreman & Wu, 2010]. The enhanced false-positive rate, attributed to the radionucleotide leakage into the non-target cells is still a remained limitation for this highly valuable labeling technique [Kooreman & Wu, 2010].

Reporter gene imagining using intracellular enzyme, cell surface receptor, transmembrane protein and intracellular storage protein probes can provoke detectable signals after interaction with the used exogenous reporters [Cao et al., 2006, MacLaren et al., 1999, Miyagawa et al., 2005, Liu et al., 2009]. Facilitated evaluation of survival and proliferation of the mother stem cells is possible through transferring stably integrated reporter genes [Kooreman & Wu, 2010]. However, concerns persist with regards to the altered cellular behaviors due to the inserted gene [Kooreman & Wu. 2010]. Assurance can be achieved using safer site-specific integration approaches [Keravala et al., 2009]. Double fusion construct containing firefly luciferase (Fluc) which interact with the reporter probe Dluciferin and enhanced green florescence protein (eGFP) can be used for cell tracking in small animals [van der Bogt et al., 2006]. Low-energy photons (2-3 ev) made by Fluc suitable for high-throughput bioluminescence imaging (BLI) and signals of eGFP can be detected using ultrasensitive CCD camera and postmortem histology experiments, respectively [Kooreman & Wu, 2010]. In vivo monitoring of survival, proliferation and migration of the injected intramyocardial mESCs were performed using triple-fusion construct composed of Fluc, monomeric red fluorescent protein (mRFP) and Herpes simplex virus truncated thymidine kinase (HSVttk) [Cao et al., 2006]. PET is preferred to BLI due to the greater anatomical details and applicability in humans [Yaghoubi et al., 2009]. Indeed, easily performed and sensitive imaging modalities like BLI are able to detect early stages of teratoma formation [van der Bogt et al**.,** 2006, Lee et al., 2009]. Early detected teratomas can be ablated by its targeting using reporter-suicide gene construct [Cao et al., 2007]. But limited signal penetration in larger animals and lack of provided spatial three-dimensional data hurdles clinical application of BLI using Fluc reporter gene [Kooreman & Wu, 2010]. Higher spatial resolution of PET and MRI made them good candidate for clinical application but their substantial low detection threshold remained an obstacle [Kooreman & Wu, 2010]. Thus, combining image modalities is of crucial importance especially for clinical insights about identification of the safe limit of stem cell numbers without teratoma formation. Using BLI, Lee et al found safe limit of undifferentiated hESCs for cardiac transplantation into SCID mice to be 1×104 [Lee et al., 2009]. Progress in tracking strategies should be in parallel with identification of the appropriate markers for tracking of both stem cell homing and cardiac differentiation. Markers of undifferentiated cells as Oct4, hTert (human telomerase reverse transcriptase) and Dusp6 (dual specifity phosphatase 6) have been shown to be decreased during cardiac trans-differentiation [Wobus & Löser, 2011]. Thus, markers of mesoderm and early cardiogenesis as GATA-4 and Brachyury were found to be suitable for tracking cardiac differentiation [Wobus & Löser, 2011]. Precise imaging technologies should solve uncertainties real cardiomyocyte trans-differentiation vs. cell fusion. Cell fusion as an overlooked phenomenon occurs due to the autoflorescence problems regarding label transfer to neighboring cells or fusion of donor and recipient cells [Reinecke et al., 2008]. The

former is avoidable using cell lineage markers as genetic materials [Reinecke et al., 2008]. This process gives rise to bi-nucleated, mono-nucleated cells with tetraploid synkaryon or cells with normal karyotype during division [Wang et al., 2003]. This phenomenon might be occurred after trans-differentiation into myocardium.

## **4. Culture conditions**

228 Coronary Artery Diseases

applications [Wobus & Löser, 2011]. Embryonic-like stem cells as Spermatogonial stem cells (SGSCs), parthenogenetic stem cells (PSCs) and male germline stem cells in pre-menopausal women can also give rise into fully active cardiomyocytes [Guan et al., 2007, Zimmermann,

Molecular imaging for in vivo tracking the proliferating and viable stem cells made a substantial help in the field of bench to bedside application of stem cells. Pre-transplantation labeling through cell inoculation with nanoparticles or reporter gene is helpful [Kooreman & Wu, 2010]. Semiconductor quantum dots capable to emit different light wavelengths show photostable bright image signals but their aggregation inside the cytosol made the process of cell delivery difficult [Kooreman & Wu, 2010]. Non-specific binding is another issue. Mesenchymal stem cells have been tracked by MRI after labeling with Ferumoxides [Kraitchman et al.**,** 2003, Amado et al**.,** 2005, Arai et al**.,** 2006]. MRI signals elicited by changes in T2 relaxation are induced due to the endocytosis of the iron oxide particles (SPIOs) or ultrasmall superparamagnetic iron oxide particles (USPIOs) [Kooreman & Wu, 2010]. MRI signals are detectable for a period between three weeks up to two months but these signals can already exist in the presence of dead stem cells due to engulfed iron particles in scavenging macrophages [Lee et al., 2009]. Alternatively, direct stem cell labeling applying radionucleotides has been used for circulating-progenitor cells successfully [Hofmann et al., 2005]. The radionucleotide-bound cells could be detected using SPECT, PET, gamma camera and cardiac magnetic resonance tracing [Kooreman & Wu. 2010]. Ultimately, the tracking duration of the radionucleotide-bound cells depends greatly on the individual half-lives of the applied radionucleotide [Kooreman & Wu, 2010]. The enhanced false-positive rate, attributed to the radionucleotide leakage into the non-target cells is still a remained limitation for this highly valuable labeling technique [Kooreman & Wu, 2010].

Reporter gene imagining using intracellular enzyme, cell surface receptor, transmembrane protein and intracellular storage protein probes can provoke detectable signals after interaction with the used exogenous reporters [Cao et al., 2006, MacLaren et al., 1999, Miyagawa et al., 2005, Liu et al., 2009]. Facilitated evaluation of survival and proliferation of the mother stem cells is possible through transferring stably integrated reporter genes [Kooreman & Wu, 2010]. However, concerns persist with regards to the altered cellular behaviors due to the inserted gene [Kooreman & Wu. 2010]. Assurance can be achieved using safer site-specific integration approaches [Keravala et al., 2009]. Double fusion construct containing firefly luciferase (Fluc) which interact with the reporter probe Dluciferin and enhanced green florescence protein (eGFP) can be used for cell tracking in small animals [van der Bogt et al., 2006]. Low-energy photons (2-3 ev) made by Fluc suitable for high-throughput bioluminescence imaging (BLI) and signals of eGFP can be detected using ultrasensitive CCD camera and postmortem histology experiments, respectively [Kooreman & Wu, 2010]. In vivo monitoring of survival, proliferation and migration of the injected intramyocardial mESCs were performed using triple-fusion construct composed of Fluc, monomeric red fluorescent protein (mRFP) and Herpes simplex virus truncated thymidine kinase (HSVttk) [Cao et al., 2006]. PET is preferred to BLI due to the greater anatomical details and applicability in humans [Yaghoubi et al., 2009]. Indeed, easily performed and sensitive imaging modalities like BLI are able to detect early stages of

2011].

**3. Image platform**

The first ESC was cultured on mouse embryonic fibroblast- feeder layer cells (MEF-FL) [Wobus, 2010]. Growing of stem cells in suspension as aggregates or removing of feeder fibroblasts promotes differentiation of ESCs [Dambrot et al., 2011]. Derivatives of three germ layers like mesoderm (cardiomyocytes, blood and vascular endothelial cells) were derived by this method [Dambrot et al., 2011]. By advent of novel culture media stem cells could be kept in undifferentiated state even in the absence of feeder cells. Commercial culture media as mTeSR**®**1 and TeSR™2 (STEMCELL technologies) in combination with matrix containing a mixture of human collagen IV, fibronectin, vitronectin and laminin are beneficial in this context [Dambrot et al., 2011]. The goal of these culture conditions is to promote stem cell scale up while keeping karyotypic stability through successive enzymatic passages or suspension cultures. This field of stem cell technology needs to be promoted further.

## **5. Special considerations**

Surveys to find the candidate stem cell should be parallel with search to find the candidate animal model. By now, mice are the most common used animal models due to the feasibility of the mutation induction and targeted deletion in them. Despite of these advantages, there are some fundamental differences between mouse and human heart. The predominantly expressed isoform of Myosin heavy chain (MHC) in fetal and adult mice are βMHC and αMHC, respectively [Dambrot et al., 2011]. The inverse pattern is seen in humans. The higher beating rate of mice (500 bpm) is surprisingly different from human heart (70 bpm)

Pursuing Candidate Stem Cells for Optimal

**6. Cardiac differentiation**

Cardiac Regeneration in Patients Suffered from Acute Coronary Syndrome 231

angiogenesis and myogenesis in injured heart. Finally, it should be beer in mind that the goal of stem cell differentiation methods should be achievement of functional myocardium.

By depletion of differentiation-repressing factors or growing cells as EBs, hESCs are easily committed to the target lineage [Dambrot et al., 2011]. EB-based directed differentiation occurs on specific matrixes in the presence of multiple inducers as growth factors, differentiation repressors or small molecules [Mohr et al., 2010, Boheler et al., 2002, Zwi et al., 2009, Mummery et al., 2007, Passier et al., 2006]. This method showed more success with mESCs rather than hESCs [Huangfu et al., 2008]. Spin EBs created from exactly defined cell numbers and centrifugated in V-shaped wells enhanced directed differentiation down the cardiac lineage [Ng et al., 2005, Ng et al., 2008]. Its yield is comparable with cardiomyocyte achievement of less than 5% of all cells using "hanging drop" EBs [Yoon et al., 2006]. Spontaneously beating cardiac clusters in the EB-outgrowths, varying in number from 8 to 70%, will be stable for up to three months [He et al., 2003, Kehat et al., 2001, Xu et al., 2002]. Contracting EB depends on the applied growth factors, cell line used and size of EB [Burridge et al., 2007, Pal & Khanna, 2007, Mikkola et al., 2006, Niebruegge et al., 2009, Mohr et al., 2010]. Moreover, crucial additions of major regulators of cardiac development as fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), bone morphogenic protein (BMP), activin, vascular endothelial growth factor (VEGF), stem cell factor (SCF), ascorbic acid and members of Wnt family added the yield of this technique [Dambrot et al., 2011]. Some inhibitors like Wnt-inhibitor DKK1 (added to culture media at late stages), mitogen-activated protein kinase inhibitor (p38 MAPK) and glycogen synthase kinase 3 (GSK3) inhibitor added more to this yield [Dambrot et al., 2011]. Co-cultures with visceral endoderm-like cell lines (END) in serum-free media supplanted with Insulin or their conditioned medium is an alternative approach [Passier et al., 2005, Freund et al., 2008]. This method, applies mechanical rather than enzymatic passage of undifferentiated cells [Dambrot et al., 2011]. Co-culture with END-2 cell line led a more homogenous ventricular cardiomyocyte population [Mummery et al., 2003]. Laflamme et al achieved cardiomyocytes more efficiently than EB-derived cells using high-density monolayer model in serum-free medium in the presence of BMP4 and activin A [Laflamme et al., 2007]. Achievement of homogenous mature cardiac cells as homogenous atrial, ventricular, conduction fibers or a mixture of them is the main goal of directed cardiomyocyte differentiation [Dambrot et al., 2011]. Currently applied technique yield a heterogeneous cell population with cardiomyocytes ranging from 1% to ~50% of the total cell mass [Dambrot et al., 2011]. Indeed, the premature phenotype of induced cells is an important issue which needs maturation induction using cell re-plating or END-2 co-culture methods followed by limited three-dimensional culturing [Otsuji et al., 2010]. Cyclic stretches or forcing alignments might enhance tissue maturity further. Gradient centrifugation method isolates largely-sized cardiomyocytes, physically [Xu et al., 2006]. Cardiomyocyte harvesting based on the cell surface receptors like protein fetal liver kinase 1 [Flk1; also known as VEGF-receptor or kinase insert domain-containing receptor (KDR)] has been demonstrated [Yang et al., 2008]. This method will provide a mixed population of cells like endothelial progenitor cells (EPCs), endothelial cells, smooth muscle cells and some other undifferentiated cells [Dambrot et al., 2011]. An easy and reversible method of isolation has been introduced using reversible mitochondria labeling by tetramethylrhodamine methyl ester perchlorate

[Dambrot et al., 2011]. Some inherent properties of mESCs and hESCs are noteworthy but their clinical significance is still unknown. mESCs and hESCs differ in expression of surface markers and culture requirements, mainly attributed to the more naïve state of mESCs [Dambrot et al., 2011]. Regarding culture requirements for maintenance of undifferentiated state, mESCs are leukemia inhibitory factor (LIF)-dependent but humans are dependent to basic fibroblast growth factor (bFGF) and Activin/Nodal-controlled signaling pathways [Tesar et al., 2007, Xu et al., 2008]. Indeed, mESCs express SSEA-1 surface molecule rather than SSEA-3/4 in hESCs [Wobus & Löser, 2011]. These basic differences make scientist to seek for a more comprehensively matched research model for regenerative purposes and diagnostic applications. In vitro drug screening, drug geneotoxicity/mutagenecity, chemical safety assessment, predictive toxicology and cardiac safety pharmacology are other avenues for beneficial application of stem cell technology. Due to species-specific pharmacotoxicological effects, animal models are not representative for human beings. Indeed, high number of animals needed for in vitro compound screening and toxicology tests [Wobus & Löser, 2011]. In vitro human cellular tests overcame the limitations of inadequate standardized animal-based tests [Wobus & Löser, 2011]. These species specific toxicology tests using immortalized human cell cultures were not real representative of normal cell types and mortal primary human cells loose their tissue-specific functions in cultures [Wobus & Löser, 2011]. Thus, stem cells can provide a good source of cells without the need for immortalization measures; facilitate human-specific cardiac pharmaco-toxicology test systems. These stem-cell based compound screening, is of paramount importance for drugs synthesized for treatment of acute ischemic events. Stem cells can be potentially used for preimplantation genetic diagnosis (PGD) and –screening (PGS) of cases with genetic predisposition to cardiac ischemic events, but this aspect of stem cell technology needs to be wrought further: extended EST (Embryonic Stem Cell Test).

Creating predictive in vitro human models of acute coronary events may be possible using cardiac stem cells. Stem cell-based models might be helpful both diagnostically and therapeutically. In terms of diagnosis and treatment, induction of gain-of function (selective turn-on) and loss-of-function (selective turn-off) mutations allows selective genetic manipulation of stem cells serving as vehicles. In addition, these assays are complementary to understand the effects of constitutively expressed genes in cell function and during the differentiation process. Loss-of-function mutations will potentially serve in identifying the cardiac lethality and survival genes. Generally, non-homologous joint recombination, homologous recombination, site-specific double-strand breaks and transpositional recombination are used strategies for genomic manipulation of stem cells [Dambrot et al., 2011]. Direct stem cell reprogramming using three cardiac transcription factors, mouse fibroblasts can be differentiated into the cardiomyocytes [Ieda et al., 2010]. Examination of a cocktail of genes, introduced Gata4, Mef2c and Tbx5 as "master regulator genes" for rapid and stable direct reprogramming of fibroblasts into cardiomyocytes [Dambrot et al., 2011]. This method showed superior efficiency to iPSC technology by eliminating concerns regarding the presence of residual undifferentiated cells [Ieda et al., 2010]. Accelerated delivery of cells to the patients with lower costs is other benefit of this method [Dambrot et al., 2011]. But the inability of these emerged cardiomyocytes to expand in vitro is its major limitation [Dambrot et al., 2011]. Adipose-derived stem cells are also an attractive easily accessible source of stem cells for clinical application. These cells safely improve both angiogenesis and myogenesis in injured heart. Finally, it should be beer in mind that the goal of stem cell differentiation methods should be achievement of functional myocardium.

## **6. Cardiac differentiation**

230 Coronary Artery Diseases

[Dambrot et al., 2011]. Some inherent properties of mESCs and hESCs are noteworthy but their clinical significance is still unknown. mESCs and hESCs differ in expression of surface markers and culture requirements, mainly attributed to the more naïve state of mESCs [Dambrot et al., 2011]. Regarding culture requirements for maintenance of undifferentiated state, mESCs are leukemia inhibitory factor (LIF)-dependent but humans are dependent to basic fibroblast growth factor (bFGF) and Activin/Nodal-controlled signaling pathways [Tesar et al., 2007, Xu et al., 2008]. Indeed, mESCs express SSEA-1 surface molecule rather than SSEA-3/4 in hESCs [Wobus & Löser, 2011]. These basic differences make scientist to seek for a more comprehensively matched research model for regenerative purposes and diagnostic applications. In vitro drug screening, drug geneotoxicity/mutagenecity, chemical safety assessment, predictive toxicology and cardiac safety pharmacology are other avenues for beneficial application of stem cell technology. Due to species-specific pharmacotoxicological effects, animal models are not representative for human beings. Indeed, high number of animals needed for in vitro compound screening and toxicology tests [Wobus & Löser, 2011]. In vitro human cellular tests overcame the limitations of inadequate standardized animal-based tests [Wobus & Löser, 2011]. These species specific toxicology tests using immortalized human cell cultures were not real representative of normal cell types and mortal primary human cells loose their tissue-specific functions in cultures [Wobus & Löser, 2011]. Thus, stem cells can provide a good source of cells without the need for immortalization measures; facilitate human-specific cardiac pharmaco-toxicology test systems. These stem-cell based compound screening, is of paramount importance for drugs synthesized for treatment of acute ischemic events. Stem cells can be potentially used for preimplantation genetic diagnosis (PGD) and –screening (PGS) of cases with genetic predisposition to cardiac ischemic events, but this aspect of stem cell technology needs to be

Creating predictive in vitro human models of acute coronary events may be possible using cardiac stem cells. Stem cell-based models might be helpful both diagnostically and therapeutically. In terms of diagnosis and treatment, induction of gain-of function (selective turn-on) and loss-of-function (selective turn-off) mutations allows selective genetic manipulation of stem cells serving as vehicles. In addition, these assays are complementary to understand the effects of constitutively expressed genes in cell function and during the differentiation process. Loss-of-function mutations will potentially serve in identifying the cardiac lethality and survival genes. Generally, non-homologous joint recombination, homologous recombination, site-specific double-strand breaks and transpositional recombination are used strategies for genomic manipulation of stem cells [Dambrot et al., 2011]. Direct stem cell reprogramming using three cardiac transcription factors, mouse fibroblasts can be differentiated into the cardiomyocytes [Ieda et al., 2010]. Examination of a cocktail of genes, introduced Gata4, Mef2c and Tbx5 as "master regulator genes" for rapid and stable direct reprogramming of fibroblasts into cardiomyocytes [Dambrot et al., 2011]. This method showed superior efficiency to iPSC technology by eliminating concerns regarding the presence of residual undifferentiated cells [Ieda et al., 2010]. Accelerated delivery of cells to the patients with lower costs is other benefit of this method [Dambrot et al., 2011]. But the inability of these emerged cardiomyocytes to expand in vitro is its major limitation [Dambrot et al., 2011]. Adipose-derived stem cells are also an attractive easily accessible source of stem cells for clinical application. These cells safely improve both

wrought further: extended EST (Embryonic Stem Cell Test).

By depletion of differentiation-repressing factors or growing cells as EBs, hESCs are easily committed to the target lineage [Dambrot et al., 2011]. EB-based directed differentiation occurs on specific matrixes in the presence of multiple inducers as growth factors, differentiation repressors or small molecules [Mohr et al., 2010, Boheler et al., 2002, Zwi et al., 2009, Mummery et al., 2007, Passier et al., 2006]. This method showed more success with mESCs rather than hESCs [Huangfu et al., 2008]. Spin EBs created from exactly defined cell numbers and centrifugated in V-shaped wells enhanced directed differentiation down the cardiac lineage [Ng et al., 2005, Ng et al., 2008]. Its yield is comparable with cardiomyocyte achievement of less than 5% of all cells using "hanging drop" EBs [Yoon et al., 2006]. Spontaneously beating cardiac clusters in the EB-outgrowths, varying in number from 8 to 70%, will be stable for up to three months [He et al., 2003, Kehat et al., 2001, Xu et al., 2002]. Contracting EB depends on the applied growth factors, cell line used and size of EB [Burridge et al., 2007, Pal & Khanna, 2007, Mikkola et al., 2006, Niebruegge et al., 2009, Mohr et al., 2010]. Moreover, crucial additions of major regulators of cardiac development as fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), bone morphogenic protein (BMP), activin, vascular endothelial growth factor (VEGF), stem cell factor (SCF), ascorbic acid and members of Wnt family added the yield of this technique [Dambrot et al., 2011]. Some inhibitors like Wnt-inhibitor DKK1 (added to culture media at late stages), mitogen-activated protein kinase inhibitor (p38 MAPK) and glycogen synthase kinase 3 (GSK3) inhibitor added more to this yield [Dambrot et al., 2011]. Co-cultures with visceral endoderm-like cell lines (END) in serum-free media supplanted with Insulin or their conditioned medium is an alternative approach [Passier et al., 2005, Freund et al., 2008]. This method, applies mechanical rather than enzymatic passage of undifferentiated cells [Dambrot et al., 2011]. Co-culture with END-2 cell line led a more homogenous ventricular cardiomyocyte population [Mummery et al., 2003]. Laflamme et al achieved cardiomyocytes more efficiently than EB-derived cells using high-density monolayer model in serum-free medium in the presence of BMP4 and activin A [Laflamme et al., 2007]. Achievement of homogenous mature cardiac cells as homogenous atrial, ventricular, conduction fibers or a mixture of them is the main goal of directed cardiomyocyte differentiation [Dambrot et al., 2011]. Currently applied technique yield a heterogeneous cell population with cardiomyocytes ranging from 1% to ~50% of the total cell mass [Dambrot et al., 2011]. Indeed, the premature phenotype of induced cells is an important issue which needs maturation induction using cell re-plating or END-2 co-culture methods followed by limited three-dimensional culturing [Otsuji et al., 2010]. Cyclic stretches or forcing alignments might enhance tissue maturity further. Gradient centrifugation method isolates largely-sized cardiomyocytes, physically [Xu et al., 2006]. Cardiomyocyte harvesting based on the cell surface receptors like protein fetal liver kinase 1 [Flk1; also known as VEGF-receptor or kinase insert domain-containing receptor (KDR)] has been demonstrated [Yang et al., 2008]. This method will provide a mixed population of cells like endothelial progenitor cells (EPCs), endothelial cells, smooth muscle cells and some other undifferentiated cells [Dambrot et al., 2011]. An easy and reversible method of isolation has been introduced using reversible mitochondria labeling by tetramethylrhodamine methyl ester perchlorate

Pursuing Candidate Stem Cells for Optimal

application is not yet well clarified.

still in its infancy.

**8. Stem cell-based therapies for ACS**

**9. Pre-administration perquisites**

history respective to these arrhythmias.

Cardiac Regeneration in Patients Suffered from Acute Coronary Syndrome 233

heart, improved LV function and attenuated pathologic remodeling mostly through an indirect paracrine pathway [Winter et al., 2007]. The clinical efficacy of these cells for human

Inadequate cardiac regeneration and cell death with subsequent progressive remodeling following acute ischemic insults make a vicious cycle toward further degeneration: degeneration begets degeneration. Measures should be performed to break this vicious cycle at earlier reversible stages. Despite of the potential role of stem cells in the regeneration of advanced stages of disease spectrum, it will bring battery of influential effects if used at the acute phase of ischemic event. Final goal should be replacement of the damaged and necrotic regions with alive and regenerative cells. Stem cell nomenclatures based on their function, phenotype, special characteristics and practical applications for heart regeneration seem helpful. Stem cell-based cardiac repair put forth new therapeutic paradigms for treatment of relentless progression of heart diseases after acute myocardial insults. But it is

Prior planning for stem cell-based therapies, issues regarding their safety and feasibility should be determined. Ethical considerations for stem cell-based therapies should be identified based on the district rules. The risks and benefits of the proposed procedure, the superiority of this procedure over other approaches and its probable durability and reproducibility should be reviewed with patient. Signed informed consents are perquisite for stem cell-based therapies. Pre-hospital issues to potentiate outcomes of the cases intended for this treatment modality are unknown. In clinically indicated cases, electrolyte abnormalities should be corrected before stem cell implantation. Patients should be evaluated for arrhythmogenic clinical grounds. It is not yet determined if patients at high risk for development of life-threatening arrhythmia are eligible for stem cell-based therapies. By virtue of the predisposition to arrhythmia in ischemic myocardium, the amplified arrhythmia risk might not be clinically favored. The benefits should be weighted in patients with currently under treatment of fatal arrhythmia or cases with remarkable past

After all, it is noteworthy that each stem cell-based therapy should be administrated in equipped hospitals. Isolated stem cells should be transferred to intervention room easily. Perhaps, the best condition is performance of stem cell-based therapies in hospitals equipped with stem cell laboratories. This minimizes troubles and cautions related to cell transferring. Moreover, time wasting would be minimized. For this purpose, standardized cell isolation protocols and scale-up procedures should be emerged before wide clinical applications. Expanded stem cells should be characterized prior to clinical application. Of note, pre-administration evaluation of cell sterility, quality and functionality both in vitro (migratory and colony forming abilities) and in vivo (ability to reperfuse blood flow to ischemic district) should be performed. Technical challenges must be met thoroughly before clinical stem cell application. The impact of quality of cell processing and purity of the final cell on the final outcomes has been previously clarified. In an equipped hospital, a trained assembled team composed of basic stem-cell researcher, cardiologist and nurses is

(TMRM) in the mixed cell population [Hattori et al., 2010]. This labeling yields three cell fractions as follow: cardiomyocyte with high fluorescent fraction, intermediate fraction of non-cardiomyocyte viable cells and dead low fraction or blood cells [Hattori et al., 2010]. In this way, cardiomyocytes are maintained more than 50 days in cultures [Hattori et al., 2010]. Alternation in culturing protocols might insight to valuable information about alternative approaches for obtaining cardiomyocytes. A yield up to 50% cardiomyocytes was achieved using feeder-dependent enzymatic passage of hESCs in knockout serum replacement (KOSR) followed by spin EBs and addition of BMP4 and activin A with subsequent replating [Ng et al., 2008]. As a complication of acute coronary syndrome, conduction defects are notable which calls attention for the importance of nodal cell achievement besides cardiomyocytes. For this purpose, inhibition of neuregulin (NRG)-1β/ErbB pathway has been shown to enhance nodal-like cell achievement, in vitro [Zhu et al., 2010].

## **7. Resident cardiac stem cells**

Mammalian cardiomyocytes are not totally terminally differentiated post-mitotic cells and cardiomyocyte turn-over has been observed in adult hearts [Walsh et al., 2010]. In aggregate, CSCs seem more efficient and natural for cardiogenesis than other non-heart origin stem cells. Resident CSCs restore the dead myocardium by proliferation and differentiation into newly mechanically effective myocardium [Dergilev et al., 2011]. These cells are tissuespecific, mostly pre-committed to cardiac lineage fate [Limana et al., 2011]. Thus, activation of the few resident cardiac stem cells (rCSCs) available in the heart via exogenous factors might exert beneficial effects [Leri et al., 2005]. But their insufficient numbers limit the benefits derived from their activation. Some resident cardiac progenitor cells are explained here.

Side-population (SP) cells are some resident which trans-differentiate into mature cardiomyocytes by co-culture with mature ventricular myocytes. SP cells express Abcg2 transporter and exporters of Hoechst dye [Balbuena et al., 2011]. The immediate replacement of SP cell by bone marrow cells after AMI suggests the presence of homing mechanism and phenotypic conversion [Guan & Hasenfuss, 2007]. This inspires the possibility of SP cell hunting from peripheral blood and avoiding their extraction by surgery and cardiac biopsy. Among SP cells the maximum potency for cardiac differentiation belongs to the Sca-1+, CD31- cells [Pfister et al., 2005]. Cardiac SP-derived Cardiospheres, self-adherent clusters derived from mild enzymatic digestion of cardiomyocytes, express both endothelial and stem cell markers [Chamuleau et al., 2009, Reinecke et al., 2008]. These cells belong to CSCs and contain firm cardiac stemness phenotypes [Guan & Hasenfuss, 2007]. Cardiosphere-derived stem cells as well as C-Kit+ cells are able to differentiate into the major cardiac vascular and muscular specialized cells [Guan & Hasenfuss, 2007].

Skeletal myoblasts were the first relevant cells used clinically [Guan & Hasenfuss, 2007]. Upon transplantation into the infracted myocardium, these cells were clonally expanded, propagated and differentiated into myotubes clustering in specific foci and improved cardiac contractility [Guan & Hasenfuss, 2007, Taylor et al., 1998, Scorsin et al., 2000]. However, there is some controversies regarding the arrhythmogenicity of these cells [Moreno et al., 2010]. Another cardiac stem cell capable to contribute to approximately all cellular elements of the cardiac interstitium and coronary vasculature is referred as epicardium-derived cell (EPDC) [Lie-Venema et al., 2007]. EPDCs transplanted into mouse heart, improved LV function and attenuated pathologic remodeling mostly through an indirect paracrine pathway [Winter et al., 2007]. The clinical efficacy of these cells for human application is not yet well clarified.

## **8. Stem cell-based therapies for ACS**

232 Coronary Artery Diseases

(TMRM) in the mixed cell population [Hattori et al., 2010]. This labeling yields three cell fractions as follow: cardiomyocyte with high fluorescent fraction, intermediate fraction of non-cardiomyocyte viable cells and dead low fraction or blood cells [Hattori et al., 2010]. In this way, cardiomyocytes are maintained more than 50 days in cultures [Hattori et al., 2010]. Alternation in culturing protocols might insight to valuable information about alternative approaches for obtaining cardiomyocytes. A yield up to 50% cardiomyocytes was achieved using feeder-dependent enzymatic passage of hESCs in knockout serum replacement (KOSR) followed by spin EBs and addition of BMP4 and activin A with subsequent replating [Ng et al., 2008]. As a complication of acute coronary syndrome, conduction defects are notable which calls attention for the importance of nodal cell achievement besides cardiomyocytes. For this purpose, inhibition of neuregulin (NRG)-1β/ErbB pathway has

Mammalian cardiomyocytes are not totally terminally differentiated post-mitotic cells and cardiomyocyte turn-over has been observed in adult hearts [Walsh et al., 2010]. In aggregate, CSCs seem more efficient and natural for cardiogenesis than other non-heart origin stem cells. Resident CSCs restore the dead myocardium by proliferation and differentiation into newly mechanically effective myocardium [Dergilev et al., 2011]. These cells are tissuespecific, mostly pre-committed to cardiac lineage fate [Limana et al., 2011]. Thus, activation of the few resident cardiac stem cells (rCSCs) available in the heart via exogenous factors might exert beneficial effects [Leri et al., 2005]. But their insufficient numbers limit the benefits derived from their activation. Some resident cardiac progenitor cells are explained

Side-population (SP) cells are some resident which trans-differentiate into mature cardiomyocytes by co-culture with mature ventricular myocytes. SP cells express Abcg2 transporter and exporters of Hoechst dye [Balbuena et al., 2011]. The immediate replacement of SP cell by bone marrow cells after AMI suggests the presence of homing mechanism and phenotypic conversion [Guan & Hasenfuss, 2007]. This inspires the possibility of SP cell hunting from peripheral blood and avoiding their extraction by surgery and cardiac biopsy. Among SP cells the maximum potency for cardiac differentiation belongs to the Sca-1+, CD31- cells [Pfister et al., 2005]. Cardiac SP-derived Cardiospheres, self-adherent clusters derived from mild enzymatic digestion of cardiomyocytes, express both endothelial and stem cell markers [Chamuleau et al., 2009, Reinecke et al., 2008]. These cells belong to CSCs and contain firm cardiac stemness phenotypes [Guan & Hasenfuss, 2007]. Cardiosphere-derived stem cells as well as C-Kit+ cells are able to differentiate into

the major cardiac vascular and muscular specialized cells [Guan & Hasenfuss, 2007].

Skeletal myoblasts were the first relevant cells used clinically [Guan & Hasenfuss, 2007]. Upon transplantation into the infracted myocardium, these cells were clonally expanded, propagated and differentiated into myotubes clustering in specific foci and improved cardiac contractility [Guan & Hasenfuss, 2007, Taylor et al., 1998, Scorsin et al., 2000]. However, there is some controversies regarding the arrhythmogenicity of these cells [Moreno et al., 2010]. Another cardiac stem cell capable to contribute to approximately all cellular elements of the cardiac interstitium and coronary vasculature is referred as epicardium-derived cell (EPDC) [Lie-Venema et al., 2007]. EPDCs transplanted into mouse

been shown to enhance nodal-like cell achievement, in vitro [Zhu et al., 2010].

**7. Resident cardiac stem cells**

here.

Inadequate cardiac regeneration and cell death with subsequent progressive remodeling following acute ischemic insults make a vicious cycle toward further degeneration: degeneration begets degeneration. Measures should be performed to break this vicious cycle at earlier reversible stages. Despite of the potential role of stem cells in the regeneration of advanced stages of disease spectrum, it will bring battery of influential effects if used at the acute phase of ischemic event. Final goal should be replacement of the damaged and necrotic regions with alive and regenerative cells. Stem cell nomenclatures based on their function, phenotype, special characteristics and practical applications for heart regeneration seem helpful. Stem cell-based cardiac repair put forth new therapeutic paradigms for treatment of relentless progression of heart diseases after acute myocardial insults. But it is still in its infancy.

## **9. Pre-administration perquisites**

Prior planning for stem cell-based therapies, issues regarding their safety and feasibility should be determined. Ethical considerations for stem cell-based therapies should be identified based on the district rules. The risks and benefits of the proposed procedure, the superiority of this procedure over other approaches and its probable durability and reproducibility should be reviewed with patient. Signed informed consents are perquisite for stem cell-based therapies. Pre-hospital issues to potentiate outcomes of the cases intended for this treatment modality are unknown. In clinically indicated cases, electrolyte abnormalities should be corrected before stem cell implantation. Patients should be evaluated for arrhythmogenic clinical grounds. It is not yet determined if patients at high risk for development of life-threatening arrhythmia are eligible for stem cell-based therapies. By virtue of the predisposition to arrhythmia in ischemic myocardium, the amplified arrhythmia risk might not be clinically favored. The benefits should be weighted in patients with currently under treatment of fatal arrhythmia or cases with remarkable past history respective to these arrhythmias.

After all, it is noteworthy that each stem cell-based therapy should be administrated in equipped hospitals. Isolated stem cells should be transferred to intervention room easily. Perhaps, the best condition is performance of stem cell-based therapies in hospitals equipped with stem cell laboratories. This minimizes troubles and cautions related to cell transferring. Moreover, time wasting would be minimized. For this purpose, standardized cell isolation protocols and scale-up procedures should be emerged before wide clinical applications. Expanded stem cells should be characterized prior to clinical application. Of note, pre-administration evaluation of cell sterility, quality and functionality both in vitro (migratory and colony forming abilities) and in vivo (ability to reperfuse blood flow to ischemic district) should be performed. Technical challenges must be met thoroughly before clinical stem cell application. The impact of quality of cell processing and purity of the final cell on the final outcomes has been previously clarified. In an equipped hospital, a trained assembled team composed of basic stem-cell researcher, cardiologist and nurses is

Pursuing Candidate Stem Cells for Optimal

Cardiac Regeneration in Patients Suffered from Acute Coronary Syndrome 235

The goal of catheter based needle intramyocardial cell injection should be promoted cell dispersion with limited immediate cell washout. Since formation of cell clusters rather than cell dispersion is proposed as a mechanism of arrhythmia-induction following stem cell application, dispersed-delivery techniques should be sought. Strategies which enhance homogenous and aligned cell integration with host tissue are more desired. But the most

Myocyte-specific strategies prevent tumor formation and growth in other tissues and allow safe systemic delivery bypassing complicated local delivery approaches. Factors specific for cardiomyocytes in the contracting walls bordering the infract zone should be identified. This strategy might be potentially with pronounced efficacy without imposing side effects like hypertrophy on remote resident cardiomyocytes. Implantation of tissue-engineered autologous myoblast sheets showed promising results in rat, canine and porcine ischemic models [Sawa et al., 2010]. Sheets would cover larger area with fewer arrhythmogenic potential. Since stem cell therapy is not just based on the administration of crude stem cells,

Stem cell application might be possible through seeding of stem cells on appropriate scaffolds and cell delivery at the site of damage. A step further back, might be approaches based on the enhanced homing of stem cells via promoted endogenous or exogenous stimuli with high specifity for stem cells involved in cardiac regeneration. Providing an accommodation for stem cells released after acute ischemic insult into the circulation, will be another alternative. These approaches will minimize the untoward effects of the exogenously delivered stem cells. A combination of exogenous stem cell administration and activation of endogenous stem cells using endogenous or exogenous stimulating factors might be attractive. Last, integrated and multi-disciplinary stem cell therapy for ACS needs fusing basic and clinical researches to narrow the gaps. Moreover, identification of key proteins involved in cardiac regeneration and cell differentiation opened the field of "Protein therapeutics". Proteins should be modified in the way to limit immunogenicity and rapid degradation in plasma and tissues. Delivered proteins exert paracrine effects on neighboring myocardium. Regardless of the type of regenerative agent, each candidate

Irrespective of the applied method for myocardial cell delivery, cells should be engrafted in suitable place. Viable myocardial segments are most desired sites for cell delivery. Patients who suffered from ACS might have chronic scar tissues rather than freshly made scar due to the acute event. Obviously, cell grafting at the sites of chronic scars would be of no benefit.  Cell loss during and

Targeting the ideal site of cell deployment is of paramount value. This site reflects the mentioned regenerative focus which can send out constructive signals. Thus, numerous 3-D intracardiac navigation systems as electromechanical mapping techniques have been developed for correct cell seeding [Banovic et al., 2011]. Interrogation using intracardiac echocardiography might yield more. Mapping catheters integrated with injection ports conjunct with manipulated guiding catheters might help in direct endomyocardial injection mapping for targeted cell delivery. Optimal imaging techniques should be applied to both guide characterization of the cell-delivery site and monitoring the functionality and efficacy

approaches for delivery of other regenerative agents should also be discussed here.

method should elicit a durable effect in a significant number of myocytes.

after transplantation lowers the efficacy of stem cell therapy.

important item is delivery of healthy cells, not crushed or squeezed cells.

necessary. Caring nurses must be able to recognize and deal with the challenges specified to stem cells recipients. Cardiac surgeon should also be attendant if surgical cell delivery is intended. Thus, those qualified centers should have on-site surgical back-up. Both team and hospital should have certificates for these operations by maintenance of good tissue practice (GTP) and GMP (good manufacturing practice). Periodic quality control should not be missed. Minimal stem-cell based procedures per year for the team and hospital need to be defined by experts. High-volume operation centers might offer less risk to the patient compared with low-volume ones. Poor clinical attainments might be reflection of technical failure. But, to date there is no absolute definition for primary and late procedural success and failure. Of course, clinical failure attributed to time delay to reperfusion and major adverse cardiac events due complications of angioplasty should be discriminated from pure cell-related outcomes.

## **10. Administration of regenerative agents**

By now the maximized cell migration and adhesion through percutaneous delivery of stem cells is done with stop-flow balloon catheter to achieve total flow occlusion within three minutes followed by stem cell infusion and reflow through deflation [Nuri & Hafeez, 2011]. In the case of extensive MI and multi-vessel disease, the eligible vessel should be identified. The superiority of antegrade vs. retrograde and proximal vs. distal stem cell delivery is not yet elucidated. Inflamed necrotic myocardium makes a hot microenvironment for delivered stem cells. The impact of this even slightly higher temperature on the efficacy of stem cells should undergo exploration. If any, cooling devices assembled with mechanical cell delivery instruments seem attractive. Perhaps stem cell eluting stents containing stem cell seeding on stem cell-friendly biomaterials without the problem of much scaffolding become available in future. If so, combination of drug-release plus stem cell-eluting stents and other combination might become revolutionary. Cells delivered directly through intracoronary route, need migration out of the vessel walls into the adjacent myocardium. This method brings the risk of coronary artery obstruction due to the plagued stem cells and consequently leads to further myocardial damage [Grieve et al., 2010]. The diameter of the target vessel and number of delivered cells seem detrimental with respect to cell stasis and vessel obstruction. The inherent risk of embolic events "cell embolism" is another limitation for intracoronary administration of stem cells [Zhang et al., 2007]. The underperfused myocardium potentially makes an unfavored environment for stable graft survival.

Intravenous stem cell injection has been shown to be safe for allogenic MSCs [Vassalli & Moccetti, 2011]. By intravenous cell administration, the majority of infused cells were shown to be harbored in kings and this cell trapping consequently limit the efficacy of this approach [Wang. et al., 2011]. Poor cell survival and drastic safety outcomes due to the extensive cell redistribution throughout the body limits this delivery approach. Alternatively, direct intramyocardial cell delivery is possible through transendocardial (percutaneous) or transepicardial (surgical) cell injection into the LV walls [Nuri & Hafeez, 2011]. Percutaneous route or "interventional cardiomyoplasty" requires retrograde passage of specially designed injection catheters into the left ventricle via femoral or arterial access [Psaltis et al., 2010]. Direct cell injection into the scar tissue or hibernated myocardium can be performed during open heart surgery or minimally invasive thoracostomy. Traumatic myocardial perforation especially at the site of freshly infracted tissue is a major side effect of this method.

necessary. Caring nurses must be able to recognize and deal with the challenges specified to stem cells recipients. Cardiac surgeon should also be attendant if surgical cell delivery is intended. Thus, those qualified centers should have on-site surgical back-up. Both team and hospital should have certificates for these operations by maintenance of good tissue practice (GTP) and GMP (good manufacturing practice). Periodic quality control should not be missed. Minimal stem-cell based procedures per year for the team and hospital need to be defined by experts. High-volume operation centers might offer less risk to the patient compared with low-volume ones. Poor clinical attainments might be reflection of technical failure. But, to date there is no absolute definition for primary and late procedural success and failure. Of course, clinical failure attributed to time delay to reperfusion and major adverse cardiac events due complications of angioplasty should be discriminated from pure

By now the maximized cell migration and adhesion through percutaneous delivery of stem cells is done with stop-flow balloon catheter to achieve total flow occlusion within three minutes followed by stem cell infusion and reflow through deflation [Nuri & Hafeez, 2011]. In the case of extensive MI and multi-vessel disease, the eligible vessel should be identified. The superiority of antegrade vs. retrograde and proximal vs. distal stem cell delivery is not yet elucidated. Inflamed necrotic myocardium makes a hot microenvironment for delivered stem cells. The impact of this even slightly higher temperature on the efficacy of stem cells should undergo exploration. If any, cooling devices assembled with mechanical cell delivery instruments seem attractive. Perhaps stem cell eluting stents containing stem cell seeding on stem cell-friendly biomaterials without the problem of much scaffolding become available in future. If so, combination of drug-release plus stem cell-eluting stents and other combination might become revolutionary. Cells delivered directly through intracoronary route, need migration out of the vessel walls into the adjacent myocardium. This method brings the risk of coronary artery obstruction due to the plagued stem cells and consequently leads to further myocardial damage [Grieve et al., 2010]. The diameter of the target vessel and number of delivered cells seem detrimental with respect to cell stasis and vessel obstruction. The inherent risk of embolic events "cell embolism" is another limitation for intracoronary administration of stem cells [Zhang et al., 2007]. The underperfused

myocardium potentially makes an unfavored environment for stable graft survival.

Intravenous stem cell injection has been shown to be safe for allogenic MSCs [Vassalli & Moccetti, 2011]. By intravenous cell administration, the majority of infused cells were shown to be harbored in kings and this cell trapping consequently limit the efficacy of this approach [Wang. et al., 2011]. Poor cell survival and drastic safety outcomes due to the extensive cell redistribution throughout the body limits this delivery approach. Alternatively, direct intramyocardial cell delivery is possible through transendocardial (percutaneous) or transepicardial (surgical) cell injection into the LV walls [Nuri & Hafeez, 2011]. Percutaneous route or "interventional cardiomyoplasty" requires retrograde passage of specially designed injection catheters into the left ventricle via femoral or arterial access [Psaltis et al., 2010]. Direct cell injection into the scar tissue or hibernated myocardium can be performed during open heart surgery or minimally invasive thoracostomy. Traumatic myocardial perforation especially at the site of freshly infracted tissue is a major side effect

cell-related outcomes.

of this method.

**10. Administration of regenerative agents**

The goal of catheter based needle intramyocardial cell injection should be promoted cell dispersion with limited immediate cell washout. Since formation of cell clusters rather than cell dispersion is proposed as a mechanism of arrhythmia-induction following stem cell application, dispersed-delivery techniques should be sought. Strategies which enhance homogenous and aligned cell integration with host tissue are more desired. But the most important item is delivery of healthy cells, not crushed or squeezed cells.

Myocyte-specific strategies prevent tumor formation and growth in other tissues and allow safe systemic delivery bypassing complicated local delivery approaches. Factors specific for cardiomyocytes in the contracting walls bordering the infract zone should be identified. This strategy might be potentially with pronounced efficacy without imposing side effects like hypertrophy on remote resident cardiomyocytes. Implantation of tissue-engineered autologous myoblast sheets showed promising results in rat, canine and porcine ischemic models [Sawa et al., 2010]. Sheets would cover larger area with fewer arrhythmogenic potential. Since stem cell therapy is not just based on the administration of crude stem cells, approaches for delivery of other regenerative agents should also be discussed here.

Stem cell application might be possible through seeding of stem cells on appropriate scaffolds and cell delivery at the site of damage. A step further back, might be approaches based on the enhanced homing of stem cells via promoted endogenous or exogenous stimuli with high specifity for stem cells involved in cardiac regeneration. Providing an accommodation for stem cells released after acute ischemic insult into the circulation, will be another alternative. These approaches will minimize the untoward effects of the exogenously delivered stem cells. A combination of exogenous stem cell administration and activation of endogenous stem cells using endogenous or exogenous stimulating factors might be attractive. Last, integrated and multi-disciplinary stem cell therapy for ACS needs fusing basic and clinical researches to narrow the gaps. Moreover, identification of key proteins involved in cardiac regeneration and cell differentiation opened the field of "Protein therapeutics". Proteins should be modified in the way to limit immunogenicity and rapid degradation in plasma and tissues. Delivered proteins exert paracrine effects on neighboring myocardium. Regardless of the type of regenerative agent, each candidate method should elicit a durable effect in a significant number of myocytes.

Irrespective of the applied method for myocardial cell delivery, cells should be engrafted in suitable place. Viable myocardial segments are most desired sites for cell delivery. Patients who suffered from ACS might have chronic scar tissues rather than freshly made scar due to the acute event. Obviously, cell grafting at the sites of chronic scars would be of no benefit.  Cell loss during and after transplantation lowers the efficacy of stem cell therapy.

Targeting the ideal site of cell deployment is of paramount value. This site reflects the mentioned regenerative focus which can send out constructive signals. Thus, numerous 3-D intracardiac navigation systems as electromechanical mapping techniques have been developed for correct cell seeding [Banovic et al., 2011]. Interrogation using intracardiac echocardiography might yield more. Mapping catheters integrated with injection ports conjunct with manipulated guiding catheters might help in direct endomyocardial injection mapping for targeted cell delivery. Optimal imaging techniques should be applied to both guide characterization of the cell-delivery site and monitoring the functionality and efficacy

Pursuing Candidate Stem Cells for Optimal

**12. Clinical trials**

Cardiac Regeneration in Patients Suffered from Acute Coronary Syndrome 237

The first clinical attempt of cell therapy was performed using BMC (bone-marrow derived cells). Rapid transfer of BMCs from bench to bedside without the need for ex-vivo expansion facilitates their clinical applications. BMCs showed modest and reproducible improvement in cardiac function by enhanced angiogenesis, augmented myocardial perfusion [Segers & Lee, 2008, Sun et al., 2009]. Reduced end-systolic volume by these cells reflects reduced negative remodeling [Orlic et al., 2001]. Un-fractionated BMCs encompass heterogeneous stem cell population including stromal cells, circulating progenitor cells (CPCs), EPC, MSC and HSC [Soejitno et al., 2010]. These cells release biologically active factors in favor of healing of the infracted myocardium [Henning, 2011]. Meta-analysis demonstrated a mean absolute increase 3-4% in ejection fraction by intracoronary infusion of patient's own reconstituted BMC aspirate [Rangappa et al., 2010]. Despite of the patient's pain relief and improved systolic and diastolic cardiac performance, the ultimate long-term effects were limited [Passier et al., 2008, Mummery et al., 2010, van Ramshorst et al., 2009]. Practically, there is no proved superiority between BMCs and CPCs in terms of clinical utilities and both are readily aspirated and administrated in contrary with difficult expansion of competent cardiac stem cells (CSCs) [Soejitno et al., 2010]. Ex-vivo expanded BMCs and unfractionated CPCs were infused post AMI via intracoronary rout in Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI) trial [Schächinger et al., 2004]. It showed improved regional and global wall motion up to 9 and 1.2 percentage points from baseline during 4-month follow-up [Soejitno et al., 2010]. Significant enhancement was found in coronary blood flow reserve, cardiac geometry and myocardial viability [Schächinger et al., 2004]. EPCs augment tissue perfusion through proneovasculogenic functions and show very low efficient cardiac trans-differentiation in coculture with mature cardiomyocytes [Kawamoto & Asahara, 2007, Badorff et al., 2003]. These cells increase perfusion of the ischemic tissue [Murohara et al., 2000, Kawamoto et al., 2001, Kalka et al., 2000]. In bone marrow transfer to enhance ST-elevation infarct regeneration (BOOST) trial, Mononucleated BMCs applied days after post-MI coronary intervention, increased regional and global cardiac performance during 6-month follow-up [Wollert et al., 2004]. Similar to TOPCARE-AMI trial, most improvement was seen in infarct border zone. But unexplained statistically insignificant decline in LVEF after 18 and 61 months later occurred in treatment arm [Meyer et al., 2006, Meyer et al., 2009]. Sustained improvements in LV function after 12-month of follow-up has been underlined through post-PCI BMC application in Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial [Schächinger et al., 2006]. There are some discouraging data from Autologous Stem Cell Transplantation in Acute Myocardial Infarction trial **(**ASTAMI) and Multicenter, randomized trial of intracoronary infusion of autologous mononuclear bone marrow cells or peripheral mononuclear blood cells after primary PCI (HEBE) trial which failed to demonstrate clinical benefits of intracoronary transfusion of BMC and BMC or CPC, respectively [van der Laan et al., 2008, Lunde et al., 2006]. Similar negative results with BMC were achieved by Leuven AMI trial with intracoronary application of BMCs 24 hrs after reperfusion in AMI cases [Janssens et al., 2006]. These patients showed improvement in regional function of only infracted segment [Janssens et al., 2006]. The same, Regeneration by Intracoronary Infusion of Selected Population of Stem Cells in Acute Myocardial Infarction (REGENT) trail failed to show promising outcomes of intracoronary infusion of bone marrow-derived selected CD34+CXCR4+ cells and non-selected mononuclear cells on LVEF or volumes [Tendera et

of the transplanted cells. Stem cell scintigraphy, lineage tracing and intravital imaging protocols might be helpful. Since the heterogeneity of the grafted stem cells with native myocytes is the principal cause of arrhythmia induction, one potential application of imaging modalities might be detection of patients with greatest uncoupling between grafted and native cells. In this way, patents at high risk for development of arrhythmia presumably could be identified earlier. Patients at high risk of arrhythmogenicity can receive prophylactic measures before development of life-threatening arrhythmias. Whether if routine stem cell tracking after application of stem cell-based therapies is valuable or not should be determined clinically. Labeled grafts facilitate following of applied cells, but need advent of non-toxic and or timely degrades labels. Non-invasive objectifying of myogenic cell grafts and assessment of the fate and bio-distribution of applied cells might be valuable in certain patient populations at risk of early stem cell failure. If so, such at risk patients should be characterized by risk assessment algorithms. Indeed, the importance of early detection of residual ischemia in patients seems valuable. Residual ischemia might limit the potential benefits of applied stem cell-based therapies as soon as the time of application. All of the deteriorating underlying conditions like anemia and poor glycemic control, in recipients of stem-cell based therapies should be approached similar to the patients receiving standard medical cares.

## **11. Post-administration perquisites**

Induction of malignant arrhythmia by transplanted stem cells has been demonstrated in several studies. This increased incidence has been attributed to the non-synchronized contraction and electromechanical non-incorporation of novel myocytes with background cells [Song et al., 2011]. Other mechanisms as Anisotropy, scar-implanted cell interaction and the presence of immature cardiomyocytes with intrinsic pacemaker activities are also speculated [Dambrot et al., 2011]. Recipients should be monitored in-hospital with more intense attention to electrocardiographic evidences of arrhythmia. Electrolyte abnormalities in favor of arrhythmogenicity should be identified and eliminated earlier. Routine electrolyte evaluation might be remarkable after stem cell implantation for reduction of the risk of arrhythmia. In the case of life-threatening arrhythmia, prophylactic and therapeutic anti-arrhythmic approaches should be provided for the patient. At least in-hospital monitoring interval before discharge should be determined. Indeed, the optimal hospitalization place for the recipient should also be evaluated. When the recipient should be sent to the step-down wards out of CCU? Certainly, the observation unit for the recipient should be equipped with central monitoring systems for the occurrence of fatal and nonfatal arrhythmias. Really, the interval with the greatest risk of arrhythmia induction should be determined. The impact of traditional risk factors and multiple comorbidities on either exogenous and mobilized endogenous stem cell outcomes or organ toxicities should be determined clearly. Irrelative to the type of applied therapies, ongoing risk factor modification and life-style modification should be started at early hospitalization in patients suffered from acute coronary syndrome. These strategies impact the general efficiency of applied therapies as stem cell-based treatments in the recipient patients. After stem cell transplantation, recipient should be followed-up for functional performance, clinical complications and mortality. Follow-up modalities and intervals should be determined. In this context, stem cell-specific comorbidity index (CI) charts derived from long-term patient follow-ups are helpful.

## **12. Clinical trials**

236 Coronary Artery Diseases

of the transplanted cells. Stem cell scintigraphy, lineage tracing and intravital imaging protocols might be helpful. Since the heterogeneity of the grafted stem cells with native myocytes is the principal cause of arrhythmia induction, one potential application of imaging modalities might be detection of patients with greatest uncoupling between grafted and native cells. In this way, patents at high risk for development of arrhythmia presumably could be identified earlier. Patients at high risk of arrhythmogenicity can receive prophylactic measures before development of life-threatening arrhythmias. Whether if routine stem cell tracking after application of stem cell-based therapies is valuable or not should be determined clinically. Labeled grafts facilitate following of applied cells, but need advent of non-toxic and or timely degrades labels. Non-invasive objectifying of myogenic cell grafts and assessment of the fate and bio-distribution of applied cells might be valuable in certain patient populations at risk of early stem cell failure. If so, such at risk patients should be characterized by risk assessment algorithms. Indeed, the importance of early detection of residual ischemia in patients seems valuable. Residual ischemia might limit the potential benefits of applied stem cell-based therapies as soon as the time of application. All of the deteriorating underlying conditions like anemia and poor glycemic control, in recipients of stem-cell based therapies should be approached similar to the patients

Induction of malignant arrhythmia by transplanted stem cells has been demonstrated in several studies. This increased incidence has been attributed to the non-synchronized contraction and electromechanical non-incorporation of novel myocytes with background cells [Song et al., 2011]. Other mechanisms as Anisotropy, scar-implanted cell interaction and the presence of immature cardiomyocytes with intrinsic pacemaker activities are also speculated [Dambrot et al., 2011]. Recipients should be monitored in-hospital with more intense attention to electrocardiographic evidences of arrhythmia. Electrolyte abnormalities in favor of arrhythmogenicity should be identified and eliminated earlier. Routine electrolyte evaluation might be remarkable after stem cell implantation for reduction of the risk of arrhythmia. In the case of life-threatening arrhythmia, prophylactic and therapeutic anti-arrhythmic approaches should be provided for the patient. At least in-hospital monitoring interval before discharge should be determined. Indeed, the optimal hospitalization place for the recipient should also be evaluated. When the recipient should be sent to the step-down wards out of CCU? Certainly, the observation unit for the recipient should be equipped with central monitoring systems for the occurrence of fatal and nonfatal arrhythmias. Really, the interval with the greatest risk of arrhythmia induction should be determined. The impact of traditional risk factors and multiple comorbidities on either exogenous and mobilized endogenous stem cell outcomes or organ toxicities should be determined clearly. Irrelative to the type of applied therapies, ongoing risk factor modification and life-style modification should be started at early hospitalization in patients suffered from acute coronary syndrome. These strategies impact the general efficiency of applied therapies as stem cell-based treatments in the recipient patients. After stem cell transplantation, recipient should be followed-up for functional performance, clinical complications and mortality. Follow-up modalities and intervals should be determined. In this context, stem cell-specific comorbidity index (CI) charts derived from long-term patient

receiving standard medical cares.

follow-ups are helpful.

**11. Post-administration perquisites**