the situation seems to be inversed in hESCs, where the G1/S checkpoint has been suggested to be functional [59], while the S-phase checkpoint appears to be inefficient [60]. This difference can be explained as possible differences in the molecular circuits that regulate pluripotency between mouse and human stem cells. Absence of a G1/S checkpoint in mESCs was originally suggested to be due to inefficient p53 function [57]. However, it has been shown that p53 can transactivate target genes in these cells [53, 56, 61]. The molecular grounds of inefficient G1/S checkpoint in mESCs have been more recently explained by the presence of high levels of the CDC25A phosphatase [58] due to its stabilization by the ubiquitin hydrolase DUB3 which is expressed at high levels in mESCs [53]. This results in constitutive dephosphorylation of the CDK2 kinase, which pushes cells into S-phase even in the presence of DNA damage, similar to the phenotype observed in cancer cells overexpressing CDC25A [62]. Interestingly, DUB3 has been shown to regulate the ubiquitination of both H2AX and γH2AX in somatic cells [63]. If this is also the case in mESCs, then it may explain why these cells repair inefficiently DSBs [64], aside from expressing low levels of DNA–PK*cs* [65]. Suppression of the G1/S checkpoint is untimely linked to pluripotency. The expression of the DUB3 gene in mESCs [53] and that of the CDC25A gene in hESCs [66] are under control of pluripotency factors. Indeed, down‐ regulation of CDC25A induces a G1/S delay upon DNA damage and cells spontaneously differentiate [53]. Consistent with this observation, DUB3 is more rapidly downregulated than OCT4 upon onset of differentiation (starting from day 1), making this gene a novel and highly specific marker of pluripotency in mESCs. Another work has shown that the contraction of the G1 phase is crucial to suppress differentiation of mESCs [67]. Collectively these observations

124 Pluripotent Stem Cells - From the Bench to the Clinic

suggest that cell cycle contraction is an essential feature of pluripotency in mESCs.

to CHK2 activation independently of ATM.

mESCs exhibit spontaneous formation of γH2AX, RPA, and Rad51 foci but do not appear to display DSBs accumulation consistent with the absence of 53BP1 foci [68, 69], although activation of downstream DDR transducers (CHK1/2, CDC25A) does not seem to be affected [53]. It is possible to envisage the presence of multiple levels of regulation of the S-phase checkpoint by various factors, such as effectors of signaling pathways, unique to stem cells. One example is provided by the observation that the CHK2 kinase appears to be sequestered at the centrosome in mESCs so that it is not activated following induction of DSBs [70]. New evidence suggests that H2AX phosphorylation in cultured ESCs is neither DNA–PK*cs*- nor ATM-dependent but is in part ATR-dependent. This is associated with ssDNA gaps accumu‐ lation, reduced fork speed, and frequent fork reversal. All these features are lost upon onset of differentiation [71]. Why is ATR spontaneously activated in mESCs? Ahuja et al. [71] show that hypoxia, DNA methylation, and transcription do not seem to be the main cause of RS in mESCs. RS appears to be linked to the maintenance of self-renewal of embryonic stem cells. Turinetto et al. [68] demonstrated that γH2AX level decrease during mESCs differentiation, while it increases upon treatment with self-renewal-enhancing small molecules such as GSK and MEK inhibitor, which correlates with increased OCT4 and NANOG expression. Further, a pluripotent state-specific gene, named FILIA, has been recently shown to be important for genomic stability in mESCs [72]. This protein is constitutively localized to the centrosomes, is recruited to DNA damage sites, where it stimulates PARP1 enzymatic activity, and contributes Somatic cells expressing defined pluripotency factors can be reprogrammed into iPSCs [76]. These cells share several similarities with ESCs such as a similar contracted cell cycle [77], the ability to undergo self-renewal and differentiation, as well as expression of pluripotency markers such as NANOG, OCT4, SOX2, and SSE-4 amongst others. Reprogramming increases γH2AX levels [78–80] and induces accumulation of genomic aberrancies ranging from whole chromosome aneuploidies, CNVs to point mutations [81], as well as epigenetic abnormalities [82]. The mutation frequency of iPSCs is also increased and has been estimated to be 10 times higher than that of ESCs [83–84]. This is in some way not surprising since the reprogramming protocol involves overexpression of oncogenes, such as *c-myc*, that introduces RS. Decreased genomic instability can be achieved by overexpression of the CHK1 kinase or by nucleosides supplementation during reprogramming [80]. ATM is also important for reprogramming. It has been reported that iPSCs deleted of ATM reprogram less efficiently and have increased genomic instability. Interestingly, these cells display gene expression profiles similar to wildtype ESCs and maintain the ability to differentiate into all three germ layers [85]. In line with this data, iPSCs exhibit G2/M cell cycle arrest and efficient DSB repair if ATM-dependent checkpoint activation signaling cascade is activated by ionizing radiation. iPSCs arrest the cell cycle in G2-phase and repair DSB by HR probably by overexpressing DNA repair genes [86]. Altogether these observations point out to a general requirement for the DNA damage checkpoint in sustaining reprogramming, suggesting that forced induction of proliferation induces RS and cells need a functional DDR to cope with this.

#### **3.3. ASCs**

ASCs are characterized by a narrower differentiation potential than ESCs. These cells selfrenew to preserve both specific tissue and organ homeostasis throughout the life of an individual. Although ASCs show much less signs of genomic instability than ESCs, they deteriorate with age [87]. It is likely that the accumulation of lesions and mutations observed during ageing of stem cells is caused by acquired defects in DNA repair pathways that reduce stem cell potential. Interestingly, defective DNA repair is tightly linked to regeneration failure in certain tissues. Fanconi anemia patients, who are deficient in ICL repair, are characterized by a premature failure of bone marrow hematopoiesis. This event is triggered by the accumu‐ lation of DNA lesions leading to excessive DDR activation in hematopoietic stem cells (HSCs) and their progenitors [88]. In addition, NER is required for the maintenance of HSCs and prevention of premature ageing [89]. NHEJ is critical for the maintenance of skeletal muscle and muscle stem cells, since decreased Ku80 expression (a subunit of the heterodimeric complex made of Ku70 and 80 proteins that that functions with DNA–PK*cs* in NHEJ) causes accelerated exhaustion of stem cell pool and ageing [90]. In HSCs and their progenitors, ROS accumulation can be provoked by loss of ATM, affecting cell cycle progression. Conditional depletion of ATR or its downstream effector CHK1 is responsible for premature ageing phenotypes in skin, bones, small intestine, and the hematopoietic system [91, 92], resulting in apoptosis and cell cycle arrest because of rapid accumulation of DNA lesions [93, 94].

Interestingly, it has been reported that aging HSCs have a higher rate of genomic instability than young HSCs, fuelled by a high level of RS generated by the reduced expression of components of the MCM2-7 replicative helicase [95]. Reduced expression of the MCM3 gene was also recently shown to be sufficient to impair hematopoietic progenitor cells due to RS [94]. A recently identified protein, NUCLEOSTEMIN, rules a primary function in maintaining the genomic stability of neural stem cells. This protein promotes recruitment of RAD51 to replication-induced DNA damage foci and activates growth arrest independently of p53 [96]. Analysis of the transcriptional program of ESCs compared to ASCs (i.e., neural and hemato‐ poietic) showed unexpected high similarities of gene expression profiles and identified a core set of about 200 genes expressed in all three cell types, accordingly coined as the "*stemness*" factors [97].

**Table 1.** Summary of main causes of genomic instability of pluripotent stem cells.

The cell cycle of ASCs is remarkably different from that of ESCs. ASCs are mostly quiescent (being mostly in the G0 state) and display a very slow cell cycle. For example, about 75% of HSCs reside in G0 [98], whereas ESCs grown in culture display less than 20% of cells in G1. It has been suggested that HSCs may divide once every 145 days (about five times during a mouse lifetime [99]), while ESCs divide every 11–12 h. These features make ASCs able to activate checkpoints and allow efficient repair. Since NHEJ can also act during G0/G1 because of its template independency, HSCs make an attempt to avoid DNA lesions by maintaining a hypoxic status [100] and decreasing the generation of ROS. In these conditions, ATP is generated mainly through glycolysis rather than mitochondrial respiration. Since this latter is activated only following cell cycle entry [101, 102], it explains why the first process is mainly employed by HSCs that are usually quiescent (**Table 1**).

by a premature failure of bone marrow hematopoiesis. This event is triggered by the accumu‐ lation of DNA lesions leading to excessive DDR activation in hematopoietic stem cells (HSCs) and their progenitors [88]. In addition, NER is required for the maintenance of HSCs and prevention of premature ageing [89]. NHEJ is critical for the maintenance of skeletal muscle and muscle stem cells, since decreased Ku80 expression (a subunit of the heterodimeric complex made of Ku70 and 80 proteins that that functions with DNA–PK*cs* in NHEJ) causes accelerated exhaustion of stem cell pool and ageing [90]. In HSCs and their progenitors, ROS accumulation can be provoked by loss of ATM, affecting cell cycle progression. Conditional depletion of ATR or its downstream effector CHK1 is responsible for premature ageing phenotypes in skin, bones, small intestine, and the hematopoietic system [91, 92], resulting in

apoptosis and cell cycle arrest because of rapid accumulation of DNA lesions [93, 94].

factors [97].

iPSCs ASCs

\*

Observed in HSCs [100].

ESCs Short cell cycle

RS

126 Pluripotent Stem Cells - From the Bench to the Clinic

RS

**Cell type Causes of genomic instability of stem cells**

Inefficient checkpoints

Inefficient checkpoints

Mutations carry over High mutation frequency

Aging-induced RS\*

Reprogramming-induced DNA damage

**Table 1.** Summary of main causes of genomic instability of pluripotent stem cells.

Short cell cycle

Interestingly, it has been reported that aging HSCs have a higher rate of genomic instability than young HSCs, fuelled by a high level of RS generated by the reduced expression of components of the MCM2-7 replicative helicase [95]. Reduced expression of the MCM3 gene was also recently shown to be sufficient to impair hematopoietic progenitor cells due to RS [94]. A recently identified protein, NUCLEOSTEMIN, rules a primary function in maintaining the genomic stability of neural stem cells. This protein promotes recruitment of RAD51 to replication-induced DNA damage foci and activates growth arrest independently of p53 [96]. Analysis of the transcriptional program of ESCs compared to ASCs (i.e., neural and hemato‐ poietic) showed unexpected high similarities of gene expression profiles and identified a core set of about 200 genes expressed in all three cell types, accordingly coined as the "*stemness*"

## **4. Implications of PSCs genomic instability in regenerative medicine**

PSCs are of great interest for their use in cell-based therapy. Current protocols involve PSCs differentiation into a specific cell type and then injection into an organ in the aim of replacing existing faulty cells. From a clinical point of view, this is a major concern due to the threat of transplanting immature cells with instable genome. Indeed, when injected in mice, immature PSCs induce teratoma. Hence, a greater understanding of the factors that regulate genomic stability in PSCs is critical to address this issue.

#### **4.1. Hurdles in translating iPSCs technology into the clinic: problems and perspectives**

Genetic instability and a high mutation rate constitute the dark side of iPSCs when taken into the clinic [4]. Hence, current efforts are made to generate iPSCs with reduced mutation load and having more stable genomes. Ji et al. [103] have shown that antioxydants reduce the level of γH2AX and *de novo* formation of CNVs in iPSCs suggesting that excessive ROS production in iPSCs increases their genetic instability. Indeed, a very recent report that analyzed the "metabolome" of naïve ESCs compared to that of primed ESCs show significant differences between these two cell types, which in turn may impinge on the level of ROS [104]. Further, recent work suggests that the use of non-integrative vectors to induce reprogramming significantly reduces the number of CNVs in the resulting iPSCs [105, 106]. Furthermore, a recent report that analyzed the mutational load of three distinct pluripotency induction methods shows that a non-integrative approach results in lower mutation load than either retrovirus or Sendai virus-based reprogramming methods [104]. Because integrative vectors induce DNA damage by generating DSBs, this suggests that the manipulation of the DDR can be a useful tool to reduce the genetic instability of iPSCs. It is then conceivable to think that DNA damage generated during reprogramming may be not well taken care, one reason being that iPSCs have inefficient checkpoints [86]. Indeed, a recent report indicates that manipulating the DDR can decrease the genomic instability of iPSCs [80]. This work shows that increasing the cellular levels of the CHK1 protein kinase decreases the level of γH2AX in these cells. In sum, in order to reduce undesired genetic burden arising during reprogramming of somatic cells, supplementing medium with both antioxidants and nucleoside should be combined to significantly reduce RS and CNVs in iPSCs.

In an effort to reduce genetic manipulation and consequent DNA damage, it has been shown that several transcription factors needed for iPSCs generation (except OCT4) can be replaced with a cocktail of chemical compounds [107]. More recently, generating iPSCs with a mix of small molecules inhibitors that can also replace OCT4 appears to strongly suppress the level of γH2AX, suggesting a reduction in spontaneous DNA damage, while keeping these cells pluripotent [108, 109]. As an example for the downside of genetic manipulation, NANOG expression has been shown to be cell cycle-regulated in human and mouse ESCs [53, 66, 110], whereas during reprogramming, this transcription factor is under constitutive expression. Altogether these data suggest that the use of an optimized set of chemical compounds may not alter natural gene expression during reprogramming and therefore would likely reduce unwanted off-target effects otherwise generated using genetic manipulation for reprogram‐ ming. Hence, chemical reprogramming remains a potentially more appropriate method since standardization of the approach is foreseeable and paves a new way of keeping genomic instability of iPSCs under control using pharmacological inhibitors.

Interestingly, mutation in the cell surface protein Glypican4 (Gpc4), implicated in the control of the Wnt/ß-catenin signaling pathway, has been reported to strongly reduce formation of teratoma upon implantation of mESCs in nude mice without affecting pluripotency [111]. Gpc4 mutant ESCs appear to be able to differentiate in all three embryonic layers when injected into developing blastocysts, although with faster kinetics compared to wild-type ESCs. These data propose Gpc4 as a promising target to modulate the teratogenic potential of ESCs. Indeed, more recent data show that ESCs bearing a hypomorphic Gpc4 allele improve recovery of motoneuron defects in a rat model for Parkinson disease without generating teratoma [112]. It is not yet known whether Gpc4 mutations have a similar effect also on human ESCs or iPSCs, and whether spontaneous DNA damage and/or genomic instability are affected.

## **5. Conclusions and perspectives**

Differentiation of iPSCs has been successfully achieved to generate hematopoietic cells, neurons, pancreatic β-islet, and cardiomyocytes; however, production of other cell types is still challenging. One major hurdle is the efficiency of differentiation that still remains very low. In addition, PSCs show several signs of genetic instability, not only in culture but also *in vivo* [71, 106], yet embryos manage to keep this instability under control by generating viable and healthy organisms. Hence, the question arises of how this control is achieved. First, cells with unstable genomes can be eliminated by apoptosis during differentiation, which is actually what it is observed during *in vitro* differentiation. However, γH2AX detection in blastocysts shows that most of the cells stain positive for this marker [71, 106], which makes unlikely that most of them bear indeed highly unstable genomes. Another possibility is that the γH2AX observed in these cells is not only a mark of genetic instability but perhaps also a marker of other DNA transactions, including chromatin remodeling. Chromatin remodeling is known to change dramatically during differentiation; hence, the decrease of γH2AX observed upon differentiation onset may be also due to changes in chromatin structure. If this is the case, the chromatin structure and epigenetic marks responsible for constitutive γH2AX in ESCs remain to be discovered. Furthermore, recent data show the presence of a high proportion of ssDNA gaps in mESCs; however, the link between these gaps and the level of γH2AX is unclear. Despite the high level of genetic instability, a highly contracted cell cycle and an inefficient G1/S checkpoint, the mutation rate of mESCs has been surprisingly reported to be lower than that of isogenic somatic cells. The significance of this discrepancy needs to be further under‐ stood.

How our current knowledge on PSCs can be translated into improving their genetic stability so to foster the development of PSCs with lower mutation load that can be used with success in regenerative medicine? In principle, identifying the molecular basis of genomic instability of PSCs opens the perspective of manipulating the genes implicated, in the aim to decrease their tendency to introduce mutations and so doing, reduce their teratogenicity. For instance, being able to manipulate the structure of the cell cycle of PSCs so to decrease RS and still maintain pluripotency may be of value. Further work in this direction is expected to generate novel insights and hopes into this rather difficult though exciting task. Clinically-compatible quantitative methods to comprehensively analyze the genetic stability of iPSCs would greatly facilitate the selection process of most appropriate iPSCs clones. Recent efforts have shifted the practice and proposed pathway signaling as readout to compare for functionality [113]. Ease of application and selection would guarantee large-scale testing in clinics.

## **Author details**

cells, supplementing medium with both antioxidants and nucleoside should be combined to

In an effort to reduce genetic manipulation and consequent DNA damage, it has been shown that several transcription factors needed for iPSCs generation (except OCT4) can be replaced with a cocktail of chemical compounds [107]. More recently, generating iPSCs with a mix of small molecules inhibitors that can also replace OCT4 appears to strongly suppress the level of γH2AX, suggesting a reduction in spontaneous DNA damage, while keeping these cells pluripotent [108, 109]. As an example for the downside of genetic manipulation, NANOG expression has been shown to be cell cycle-regulated in human and mouse ESCs [53, 66, 110], whereas during reprogramming, this transcription factor is under constitutive expression. Altogether these data suggest that the use of an optimized set of chemical compounds may not alter natural gene expression during reprogramming and therefore would likely reduce unwanted off-target effects otherwise generated using genetic manipulation for reprogram‐ ming. Hence, chemical reprogramming remains a potentially more appropriate method since standardization of the approach is foreseeable and paves a new way of keeping genomic

Interestingly, mutation in the cell surface protein Glypican4 (Gpc4), implicated in the control of the Wnt/ß-catenin signaling pathway, has been reported to strongly reduce formation of teratoma upon implantation of mESCs in nude mice without affecting pluripotency [111]. Gpc4 mutant ESCs appear to be able to differentiate in all three embryonic layers when injected into developing blastocysts, although with faster kinetics compared to wild-type ESCs. These data propose Gpc4 as a promising target to modulate the teratogenic potential of ESCs. Indeed, more recent data show that ESCs bearing a hypomorphic Gpc4 allele improve recovery of motoneuron defects in a rat model for Parkinson disease without generating teratoma [112]. It is not yet known whether Gpc4 mutations have a similar effect also on human ESCs or iPSCs,

Differentiation of iPSCs has been successfully achieved to generate hematopoietic cells, neurons, pancreatic β-islet, and cardiomyocytes; however, production of other cell types is still challenging. One major hurdle is the efficiency of differentiation that still remains very low. In addition, PSCs show several signs of genetic instability, not only in culture but also *in vivo* [71, 106], yet embryos manage to keep this instability under control by generating viable and healthy organisms. Hence, the question arises of how this control is achieved. First, cells with unstable genomes can be eliminated by apoptosis during differentiation, which is actually what it is observed during *in vitro* differentiation. However, γH2AX detection in blastocysts shows that most of the cells stain positive for this marker [71, 106], which makes unlikely that most of them bear indeed highly unstable genomes. Another possibility is that the γH2AX observed in these cells is not only a mark of genetic instability but perhaps also a marker of other DNA transactions, including chromatin remodeling. Chromatin remodeling is known

and whether spontaneous DNA damage and/or genomic instability are affected.

instability of iPSCs under control using pharmacological inhibitors.

significantly reduce RS and CNVs in iPSCs.

128 Pluripotent Stem Cells - From the Bench to the Clinic

**5. Conclusions and perspectives**

Elena Lo Furno1 , Siem van der Laan2 and Domenico Maiorano1\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Institute of Human Genetics, CNRS-UPR1142, Genome Surveillance and Stability Laboratory, Department of Molecular Bases of Human Diseases, University of Montpellier, Montpellier, France

2 CNRS FRE3690, Parc Euromedicine Cap Delta, Montpellier, France

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136 Pluripotent Stem Cells - From the Bench to the Clinic

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**Section 3**

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138 Pluripotent Stem Cells - From the Bench to the Clinic

## **Moving toward Xeno-free Culture of Human Pluripotent Stem Cells**

## Kamthorn Pruksananonda and Ruttachuk Rungsiwiwut

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62770

#### **Abstract**

Human pluripotent stem cells (hPSCs) were conventionally cultured on feeder cells that are isolated from mouse embryonic fibroblast (MEF). However, these culture compo‐ nents could contaminate the hPSCs and can limit the application of hPSCs for clinical use. On the other hand, we demonstrated that exogenous basic fibroblast growth factor (bFGF) could be omitted from the hPSC culture media if we used the suitable feeder cells. We also showed that although hPSCs can proliferate on the feeder-free culture system, however, genetic instability of hPSCs has been reported in such environment. Feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS) prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. We proposed the use of human cord blood-derived serum (hUCS) and showed a positive effect on culture of mesenchymal stem cells. The results showed that human foreskin fibroblasts (HFFs) cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, short population doubling times, high proliferation rates, and normal karyotypes after prolonged culture. These studies of hPSC xeno-free culture have been growing in both basic research and clinical trial. The data regarding the current clinical trials of using hPSCs convince the researchers not only about the possibility of application of hPSCs for cell-based therapy, but also the quality of established hPSC lines. Most of the hPSC lines that were published in the literature and registered in the National Institute of Health (NIH), hPSCreg of the European Union are not Good Manufacturing Practice (GMP) grade cell lines. Since one of the goals of using hPSCs is therapeutic purpose, GMP for derivation, cultivation, and handling the hPSCs are required. This chapter also reviews the state-of-the-art xeno-free culture system of hPSCs in the respect of future clinical applications.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Keywords:** xeno-free, pluripotent stem cells, cell culture, cell therapy, regenerative medicine

## **1. Introduction**

The ultimate goal of pluripotent stem cell research is to improve quality of life and patient treatment. The human pluripotent stem cells (hPSCs) can be classified as human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs). hESCs can be derived from the inner cell mass (ICM) of the preimplantation embryos [1]. hiPSCs can be generated by reprogramming the somatic cells using the exogenous transcriptional factors and resulting in the pluripotent cells [2]. The successful derivation of hESCs and hiPSCs holds the great hope of treatment of incurable diseases, and the hPSC-related studies have been growing with regard to the potential of hPSCs.

hPSCs was firstly cultured in the medium containing the fetal bovine serum (FBS) and cocultured with mouse embryonic fibroblast (MEF) feeders [1, 2]. However, such conditions might introduce the contamination of animal pathogens to the hPSCs and make hPSCs unsuitable for clinical applications. Thus, elimination of all animal products during the derivation, long-term culture as well as differentiation of hPSCs is necessary prior to applica‐ tion of hPSCs in clinical cell therapy. Because the ultimate aim of pluripotent stem cell research is to improve quality of life and patient outcome, the more defined culture systems of hPSCs were progressively developed. The researchers firstly developed xeno-free culture system that consisted of a growth medium containing Knockout serum replacement (KO-SR) and basic fibroblast growth factor (bFGF) instead of FBS [3–5]. MEF feeder cells were replaced by the humanized feeder cells derived from human tissues. Later, the researchers have developed various xeno-free culture media, and xeno-free extracellular matrices which support the growth and pluripotency of hPSCs [6–10]. Recently, commercial xeno-free, defined culture media and extracellular matrix has been developed and available for the researchers.

## **2. Human pluripotent stem cells properties**

hPSCs are widely accepted as the valuable source for cell-based therapy due to their ability of differentiation toward three embryonic germ layers and process cell division indefinitely [11]. Although the origin of hESCs and hiPSCs is different, they share similarities in the morpho‐ logical appearances and the molecular levels. The unique morphological appearances of hPSCs can be identified by the tightly pack colonies with defined borders. The clear cytoplasm and distinctive nuclei of the individual cells can be observed. hPSCs expressed the pluripotentrelated transcriptional factors including OCT-4, Nanog, Sox2, Nodal, hTERT, Rex1 as well as positive for stage-specific-embryonic antigen (SSEA)-3, SSEA −4, tumor-recognition antigen (TRA)-1-60 and −1-81, expressed high level of alkaline phosphatase and telomerase activity [1– 10]. Mellon et al. 12] and Guenther et al. [13] reported that there were no significant gene expression differences between hESCs and hiPSCs. However, some studies reported the differences in the methylation profile between hESCs and hiPSCs [14–16]. This can reflect the genetic and epigenetic abnormalities during the reprogramming process or even the differ‐ ences of cell culture condition in different laboratories [17]. Although the similarities and differences between hESCs and hiPSCs are contradictory, the researchers around the world tried to standardize the culture conditions and characterization of hPSCs with the ultimate goal to achieve the development of the hPSC culture system for the future clinical application [18, 19].

## **3. Good Manufacturing Practice for human Pluripotent Stem Cells**

Since one of the goals of using hPSCs is therapeutic purpose, Good Manufacturing Practice (GMP) for derivation, cultivation, and handling the hPSCs are required. Most of the hPSC lines that were registered in the National Institute of Health (NIH), hPSCreg of the European Union as well as the data published in the literature are not GMP grade cell lines. Non-GMP grade cell lines are usually contacts to the animal products especially through the feeder cells, serum, and extracellular matrix [20, 21]. The researchers found that culture of hESCs on the feeder cells derived from the mouse embryonic tissues may contaminate nonhuman sialic acid (Neu5Gc) molecules, which can stimulate the immune rejection after transplantation [22]. Moreover, FBS that was supplemented in the culture medium of feeder cells might contain bovine pathogens such as bovine spongiform encephalitis (BSE) and limits the use of hPSCs for clinical applications [23]. Therefore, the materials for hPSC culture system that compliant for clinical application should be verified that the materials are GMP-grade and animal-free products. Importantly, the clinical-grade hPSCs should be generated under the ethical consideration guidelines that strictly followed the national law, from the traceable and healthy donors and the culture system should follow the standard operating procedures (SOPs) of GMP.

## **4. Derivation of human pluripotent stem cells**

**Keywords:** xeno-free, pluripotent stem cells, cell culture, cell therapy, regenerative

The ultimate goal of pluripotent stem cell research is to improve quality of life and patient treatment. The human pluripotent stem cells (hPSCs) can be classified as human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs). hESCs can be derived from the inner cell mass (ICM) of the preimplantation embryos [1]. hiPSCs can be generated by reprogramming the somatic cells using the exogenous transcriptional factors and resulting in the pluripotent cells [2]. The successful derivation of hESCs and hiPSCs holds the great hope of treatment of incurable diseases, and the hPSC-related studies have been growing with regard

hPSCs was firstly cultured in the medium containing the fetal bovine serum (FBS) and cocultured with mouse embryonic fibroblast (MEF) feeders [1, 2]. However, such conditions might introduce the contamination of animal pathogens to the hPSCs and make hPSCs unsuitable for clinical applications. Thus, elimination of all animal products during the derivation, long-term culture as well as differentiation of hPSCs is necessary prior to applica‐ tion of hPSCs in clinical cell therapy. Because the ultimate aim of pluripotent stem cell research is to improve quality of life and patient outcome, the more defined culture systems of hPSCs were progressively developed. The researchers firstly developed xeno-free culture system that consisted of a growth medium containing Knockout serum replacement (KO-SR) and basic fibroblast growth factor (bFGF) instead of FBS [3–5]. MEF feeder cells were replaced by the humanized feeder cells derived from human tissues. Later, the researchers have developed various xeno-free culture media, and xeno-free extracellular matrices which support the growth and pluripotency of hPSCs [6–10]. Recently, commercial xeno-free, defined culture

media and extracellular matrix has been developed and available for the researchers.

hPSCs are widely accepted as the valuable source for cell-based therapy due to their ability of differentiation toward three embryonic germ layers and process cell division indefinitely [11]. Although the origin of hESCs and hiPSCs is different, they share similarities in the morpho‐ logical appearances and the molecular levels. The unique morphological appearances of hPSCs can be identified by the tightly pack colonies with defined borders. The clear cytoplasm and distinctive nuclei of the individual cells can be observed. hPSCs expressed the pluripotentrelated transcriptional factors including OCT-4, Nanog, Sox2, Nodal, hTERT, Rex1 as well as positive for stage-specific-embryonic antigen (SSEA)-3, SSEA −4, tumor-recognition antigen (TRA)-1-60 and −1-81, expressed high level of alkaline phosphatase and telomerase activity [1–

**2. Human pluripotent stem cells properties**

medicine

142 Pluripotent Stem Cells - From the Bench to the Clinic

**1. Introduction**

to the potential of hPSCs.

The recent advances in GMP derivation of hPSC lines are using xeno-free reagents/materials and replacing the mouse feeders with GMP-qualified human feeders or recombinant human proteins [24–28]. Regarding the xeno-free hPSC derivation, there are several steps that should be considered in order to meet the GMP or criteria for clinical grade hPSCs.

For hESC derivation, the zona pellucida of the blastocyst embryos has to be removed prior to isolation of the ICM. The zona pellucida could be digested by the acid Tyrode's solution or pronase [3–5, 29]. To avoid the destructive effect of the acid or enzyme, the laser-assisted zona pellucida removal and the mechanical cut using surgical blade or needle were successfully applied [30–32]. Enzymatic removal, laser assisted, and mechanical dissection of zona pellucida are the xeno-free procedures due to these methods has no contact to the animal materials. However, further step for derivation of hESCs is separation of the ICM from the trophectoderm (TE) which the embryo or ICM might contact to the animal products. Tradi‐ tionally, the TE is separated from the ICM by the procedure called immunosurgery. The immunosurgery technique involves the selective lyses of the TE but not the ICM, resulting to the TE-free ICM. The use of anti-human serum combined with complement mediated lysis using guinea pig serum made the immunosurgery technique unsuitable for derivation of xenofree hESCs. Therefore, other means such as microdissection of blastocysts using fine needle, laser-assisted biopsy, and whole blastocyst culture are the promising techniques for xeno-free ICM isolation [30–32]. The isolated ICM is subsequently plated and cultured for generation of hESC lines. The following procedures including culture conditions, cell propagation, and cryopreservation have to manage under the xeno-free conditions.

For hPSC derivation, the early step for derivation of xeno-free hPSCs is isolation and culture of the somatic cells prior to reprogramming. The somatic cells should be isolated under the aseptic condition with the minimal invasive technique. It has been reported that hPSCs can be generated by using several somatic cell types such as skin fibroblasts, peripheral blood mononuclear cells and even the cells isolated from urine [2, 33, 34]. Replacement of the animal products by using human grade or recombinant synthetic products during the isolation and culture of somatic cells is necessary. For example, human dermal fibroblasts can be isolated and propagated in the culture medium supplemented with human serum. These fibroblast cells can multiply under the xeno-free condition and suitable for using as the starting cells for reprogramming. We proposed the use of human cord blood-derived serum (hUCS) and showed a positive effect on culture of mesenchymal stem cells. The results showed that human foreskin fibroblasts (HFFs) cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and transforming growth factor beta-1 (TGFβ1), which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSC culture system.

However, one of the bottle-neck of derivation of xeno-free hPSCs is the method for reprog‐ ramming of the somatic cells. Originally, the exogenous genes were delivered to the somatic cells by means of virus such as lentivirus or retrovirus [2, 35]. Because the integration of lentiviral- or retroviral genome to the host (somatic cells) genome might occur during the reprogramming process, thus the hPSCs generated by lentivirus- or retrovirus are not the ideal hPSCs for the clinical use. The clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through GMP. Recently, reprogramming by protein, microRNA, episomal vectors, or Sendai virus, a nonintegrating virus is considered suitable for delivery of the exogenous genes into the somatic cells [36]. We demonstrated recently that the transgene-free hPSCs can be generated by the temperature-sensitive strain of Sendai virus and the viral particles were clearly eliminated from the established hPSCs by the heat treatment [37]. Thus, derivation of transgene-free hPSCs is a very important step for generation of xeno-free hPSCs that match the future clinical applications.

## **5. Culture of human pluripotent stem cells**

materials. However, further step for derivation of hESCs is separation of the ICM from the trophectoderm (TE) which the embryo or ICM might contact to the animal products. Tradi‐ tionally, the TE is separated from the ICM by the procedure called immunosurgery. The immunosurgery technique involves the selective lyses of the TE but not the ICM, resulting to the TE-free ICM. The use of anti-human serum combined with complement mediated lysis using guinea pig serum made the immunosurgery technique unsuitable for derivation of xenofree hESCs. Therefore, other means such as microdissection of blastocysts using fine needle, laser-assisted biopsy, and whole blastocyst culture are the promising techniques for xeno-free ICM isolation [30–32]. The isolated ICM is subsequently plated and cultured for generation of hESC lines. The following procedures including culture conditions, cell propagation, and

For hPSC derivation, the early step for derivation of xeno-free hPSCs is isolation and culture of the somatic cells prior to reprogramming. The somatic cells should be isolated under the aseptic condition with the minimal invasive technique. It has been reported that hPSCs can be generated by using several somatic cell types such as skin fibroblasts, peripheral blood mononuclear cells and even the cells isolated from urine [2, 33, 34]. Replacement of the animal products by using human grade or recombinant synthetic products during the isolation and culture of somatic cells is necessary. For example, human dermal fibroblasts can be isolated and propagated in the culture medium supplemented with human serum. These fibroblast cells can multiply under the xeno-free condition and suitable for using as the starting cells for reprogramming. We proposed the use of human cord blood-derived serum (hUCS) and showed a positive effect on culture of mesenchymal stem cells. The results showed that human foreskin fibroblasts (HFFs) cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and transforming growth factor beta-1 (TGFβ1), which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit

However, one of the bottle-neck of derivation of xeno-free hPSCs is the method for reprog‐ ramming of the somatic cells. Originally, the exogenous genes were delivered to the somatic cells by means of virus such as lentivirus or retrovirus [2, 35]. Because the integration of lentiviral- or retroviral genome to the host (somatic cells) genome might occur during the reprogramming process, thus the hPSCs generated by lentivirus- or retrovirus are not the ideal hPSCs for the clinical use. The clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through GMP. Recently, reprogramming by protein, microRNA, episomal vectors, or Sendai virus, a nonintegrating virus is considered suitable for delivery of the exogenous genes into the somatic cells [36]. We demonstrated recently that the transgene-free hPSCs can be generated by the temperature-sensitive strain of Sendai virus and the viral particles were clearly eliminated from the established hPSCs by the

cryopreservation have to manage under the xeno-free conditions.

of hUCS for hPSC culture system.

144 Pluripotent Stem Cells - From the Bench to the Clinic

The culture conditions that allow hPSCs to maintain their pluripotency is the complex environment and still under investigation. The researchers proved that the pluripotency of hPSCs is controlled by the TGF pathway which involves the cooperation of Activin and Nodal signaling pathway [38]. The pathways that related to the maintenance of the pluripotency of hPSCs might be provided by the secretion of cytokines, growth factors, extracellular matrix from the feeder cells, synthetic substrates, or culture medium. The improvement of culture system is not only necessary for maintenance of hPSC pluripotency but also for large-scale propagation of hPSCs prior to cell transplantation. The aims of our study were to determine (i) the effect of exogenous bFGF supplementation in the hPSC culture media on the morphol‐ ogy and gene expression of inactivated human cesarean scar fibroblasts (HSFs) and (ii) the feasibility of using the inactivated HSFs as the feeder cells for culturing the hPSCs. Our results showed that the cells shrunk and an increase in gap between the cells were observed in the inactivated HSFs that were cultured in 4 and 8 ng/ml but not in 0 ng/ml bFGF. Expression of Activin A, bFGF, TGF-β, and BMP4 was similar between inactivated HSFs cultured in 0, 4, and 8 ng/ml bFGF. After two hPSC lines including hESC line (Chula2.hES) and hiPSC line (PFX12) were cocultured with three conditions of inactivated HSFs for more than ten passages, the hPSCs lines were subjected to characterization. The results showed that the hESCs and hiPSCs cultured in 0, 4, and 8 ng/ml bFGF could maintain their undifferentiation state, differentiate in vitro into three embryonic germ layers, and maintain their normal karyotype. In conclusion, exogenous bFGF supplementation in the culture medium can be omitted when using HSFs as the feeder cells for culturing the hPSCs.

#### **5.1. Feeder-dependent culture system**

Feeder-dependent culture system referred to the culture system that the hPSCs grown by the support of feeder cells. The feeder-dependent culture system is the traditional system that was developed for generation and propagation of hPSCs [1–6]. Feeder cells secreted cytokines, extracellular matrices and provided the niches that support the growth and maintenance of pluripotency of hPSCs. Generally, MEFs are used as the feeder for culture of hPSCs. Due to, MEFs might stimulate the immune response of the patients after transplantation of hPSCs [22]. Therefore, the feeder cells that isolated from human tissues are the better choice for culture of the clinical-grade hPSCs. Several studies demonstrated that the supportive feeder cells of hPSCs could be derived from the human tissues. Hovatta et al. [3] firstly demonstrated that HFFs supported the derivation and culture of hESCs. HFFs supported the pluripotency of hPSCs by expression of the key genes such as TFG, FGF2, and Activin A which have been implicated in the maintenance of hPSC pluripotency [39]. Ma et al. [40], the HFFs produced interleukin-6 and can be used for coculture with mouse embryonic stem cells. However, not all the HFF lines exhibited the characteristics of supportive feeder cells. The data confirmed that the foreskin fibroblasts from the different donor secreted different amount of growth factors, cytokines, and specific genes related to pluripotency [39, 41]. Therefore, it is necessary to explore the new supportive feeder cell types in order to develop the xeno-free feeder dependent culture system of hPSCs. Up to date, feeder cells derived from other sources of human tissues, for example, human adult fallopian tubal fibroblasts, human fetal skin, human adult skin, human fetal muscle, human adult muscle, human adult marrow cells, human umbilical cord mesenchymal stem cells, human amniotic epithelial cells, and human iPS cell derived fibroblast-like cells had been reported as the supportive feeder cells for culture of the hPSCs [42–45]. In addition, genetic modification of human feeder cells such as immortal HFFs or bFGF-secreting HFFs has been developed and proved to be useful for culture of hPSCs [46, 47]. Although replacement of MEFs by human feeder cells can reduce the risk of contamination of mouse pathogens to the hPSCs, the use of FBS in the feeder cell culture medium does not meet the xeno-free condition of hPSCs. We recently demonstrated that HFFs cultured in the medium containing human umbilical cord blood serum (hUCBS) retain their supportive feeder characteristics for maintenance of hPSC lines [48]. In addition, we used hUCBS for isolation and culture of cesarean scar-derived fibroblasts (**Figure 1**). The xeno-free fibroblasts will be used for the feeder cells and the starting cells for generation of transgene-free hPSCs.

**Figure 1. Isolation of fibroblast cells from human cesarean scar tissue**. Fibroblast cells grew out from the tissue after culture for 7 days in the culture medium containing human umbilical cord blood serum (hUCBS). Scale bar = 100 μm.

To generate the clinical-grade human feeder cells, the cells should be derived under the culture system containing humanized or recombinant GMP materials and reagents. Interestingly, Prathalingam et al. [27] demonstrated that the clinical-grade feeder cells can be derived and cultured in the medium containing the FBS that was derived under the GMP condition. Prior to use of the feeder cells for derivation of the new hPSC lines or routinely coculture with the existing hPSC lines, the clinical-grade human feeder cells should be tested for the potential of the cells to support the proliferation of hPSCs. Although the standard protocol for character‐ ization of the feeder cells is lacking, several methods have been adopted. Inactivation of feeder cells by irradiation or Mitomycin C treatment is the method for inactivation of feeder cell proliferation prior to use for coculture with hPSCs. After being inactivated, the feeder cells should be able to secrete the enough amounts of cytokines and growth factors into the culture medium. Moreover, the extracellular matrices provided by the feeder play important roles for maintenance of the pluripotency of hPSCs [7]. The cells that easy to apoptosis after the inactivation process might probably not be a good supportive feeder cells.

One major concern of feeder-dependent culture system is coating the culture vessel with the xeno-free matrix in order to enhance the attachment of feeder cells to the surface of the culture vessel. Typically, porcine-skin-derived gelatin was used for coating the culture vessel prior of seeding the inactivated feeder cells. To meet the criteria of xeno-free condition, porcine-skinderived gelatin should be replaced by recombinant gelatin. Tannenbaum et al. [26] developed the platform of derivation of xeno-free and GMP-grade hESCs by using recombinant gelatin and they found no adverse effect of recombinant gelatin on the pluripotency of hESCs. In addition, Ding et al. [49] showed that human cord blood serum can be used for coating the culture vessels instead of the porcine-skin-derived gelatin for derivation and maintenance of hPSCs.

## **5.2. Feeder-free culture system**

factors, cytokines, and specific genes related to pluripotency [39, 41]. Therefore, it is necessary to explore the new supportive feeder cell types in order to develop the xeno-free feeder dependent culture system of hPSCs. Up to date, feeder cells derived from other sources of human tissues, for example, human adult fallopian tubal fibroblasts, human fetal skin, human adult skin, human fetal muscle, human adult muscle, human adult marrow cells, human umbilical cord mesenchymal stem cells, human amniotic epithelial cells, and human iPS cell derived fibroblast-like cells had been reported as the supportive feeder cells for culture of the hPSCs [42–45]. In addition, genetic modification of human feeder cells such as immortal HFFs or bFGF-secreting HFFs has been developed and proved to be useful for culture of hPSCs [46, 47]. Although replacement of MEFs by human feeder cells can reduce the risk of contamination of mouse pathogens to the hPSCs, the use of FBS in the feeder cell culture medium does not meet the xeno-free condition of hPSCs. We recently demonstrated that HFFs cultured in the medium containing human umbilical cord blood serum (hUCBS) retain their supportive feeder characteristics for maintenance of hPSC lines [48]. In addition, we used hUCBS for isolation and culture of cesarean scar-derived fibroblasts (**Figure 1**). The xeno-free fibroblasts will be

146 Pluripotent Stem Cells - From the Bench to the Clinic

used for the feeder cells and the starting cells for generation of transgene-free hPSCs.

**Figure 1. Isolation of fibroblast cells from human cesarean scar tissue**. Fibroblast cells grew out from the tissue after culture for 7 days in the culture medium containing human umbilical cord blood serum (hUCBS). Scale bar = 100 μm.

To generate the clinical-grade human feeder cells, the cells should be derived under the culture system containing humanized or recombinant GMP materials and reagents. Interestingly, It has been proposed that coculture of hPSCs with the feeder layer might contaminate the harmful substance and cause the risk of graft rejection, viral or bacterial infection, or zoonoses [50]. To culture hPSCs under the feeder-free condition, several media formulations have been developed that eliminate the use of fetal bovine serum and reduce or eliminate all animalderived components. Therefore, development of feeder-free and xeno-free culture system for hPSCs by the use of xeno-free reagents and matrices in every cell-handling process is important for obtaining clinical-grade hPSCs.

Feeder-free culture system is applied for culturing hPSCs without the feeder layers but the matrices such as Matrigel or recombinant human proteins, together with the commercially defined hPSC culture medium [51, 52]. Although the genetic and epigenetic instability could be detected in hPSCs grown under feeder-free condition [53–55], the feeder-free condition together with defined culture medium is still important because it allows the hPSCs to proliferate robustly.

In order to propagate and maintain the pluripotency of hPSCs under the feeder-free condition, it is necessary to provide the cell adhesion coating that support the proliferation of hPSCs. Matrigel, the solubilized basement matrix extracted from mouse Engelbreth-Holm-Swarm sarcoma is the widely used matrix. The major components of Matrigel include laminin, collagen IV, heparan sulfate proteoglycan, and entactin. In addition, Matrigel contains growth factors such as TGF-beta, fibroblast growth factor, TPA, and insulin-like growth factor [56, 57]. Matrigel provides the complex extracellular matrices that support the cell communication and the suitable microenvironment for the hPSCs to grow as well as maintain their pluripo‐ tency. Although hPSCs proliferated robustly on Matrigel, the origin of Matrigel makes this matrix unsuitable for maintaining the clinical-grade hPSCs. Recently, Ding et al. [49] devel‐ oped the humanized ECM by using umbilical cord blood serum (UCBS) as the matrix for derivation and culture of hPSCs. The best result was obtained when UCBS was used in combination with the basal medium supplemented with bFGF, fibronectin, and Y-27632. Other humanized matrices, for example, human placental-derived ECM and H9-hESC embryoid body–derived ECM have been proved to support the pluripotency of hPSCs [58, 59]. The hPSC lines cultured with the ECM derived from both types of human tissues or cells in the defined serum and xeno-free culture media such as TeSR2, sustained their pluripotency after pro‐ longed culture. However, lot-to-lot variations in the individual serum donor or tissue-derived ECM can affect the effectiveness of the serum or ECM in maintaining the pluripotent state of hPSCs. In order to develop the defined-feeder-free cell adhesion coating, individual extracel‐ lular matrix (ECM) proteins have been used instead of Matrigel. Several ECM including laminin, fibronectin, vitronectin, and collagen support the proliferation of hPSCs. Those ECM can be used as single or in various combinations. Interestingly, the studies have demonstrated that recombinant laminin-511 and recombinant vitronectin enable to support the growth and maintain the pluripotency of hPSCs [60, 61]. To improve feeder-free culture system, cell adhesion proteins can be added to ECM component. E-cadherin, the cell adhesion protein improved the attachment and expansion of hPSCs when the cells were cultured in defined media such as mTeSR1 and xeno-free media, TeSR2 [62]. Moreover, recombinant E-cadherin fusion protein supported the proliferation of the other pluripotent cells [63]. Additional developments in defined culture surfaces include synthetic peptide coatings such as Synthe‐ max, Peptide acrylate surfaces (PAS), or synthetic polymers have been proved to support the growth and pluripotency of hPSCs after prolonged culture [64]. The combination of human ECM proteins or cell adhesion molecules and synthetic biomaterials with well-designed surfaces and/or structures in the presence of a chemically defined medium containing recombinant growth factors would offer a xeno-free alternative to feeder cells for culturing hPSCs and maintaining their pluripotency. Because the xeno-free and feeder-free culture system is suitable for generation of hPSCs for clinical application, we reprogrammed CD34+ cells by the temperature-sensitive Sendai viral vectors carries OCT-4, SOX2, KLF4 and c-MYC, on Pronectin F plus (Sanyo Chemical Industries, Kyoto, Japan) coating matrix and cultured the cells in ReproFF (ReproCELL, Yokohama, Japan) culture medium (**Figure 2**). We will use this newly established hPSCs for further development of xeno-free culture system. In our study [48], the hPSC lines were derived and cultured on HFF feeder layer. The hPSC lines were continuously maintained on either mitomycin-C inactivated HFF-FBS or mitomycin-C inactivated HFF-hUCS in serum-free hPSC culture medium. The serum-free hPSC culture medium comprising 80% Knockout Dulbecco's modified Eagles' medium (KO-DMEM), 20% KO-SR, 1% nonessential amino acid, 1% Glutamax, 1% penicillin-streptomycin, 0.1 mM βmercaptoethanol (all from Invitrogen), and 8 ng/ml bFGF (R&D Systems, Minneapolis, MN, USA).

**Figure 2. Human-induced pluripotent stem cell culture on feeder-free condition**.The transgene-free human-induced pluripotent stem cells generated from CD34+ cells and culture in feeder-free culture system. Scale bar = 500 μm.

## **6. Contamination concern**

factors such as TGF-beta, fibroblast growth factor, TPA, and insulin-like growth factor [56, 57]. Matrigel provides the complex extracellular matrices that support the cell communication and the suitable microenvironment for the hPSCs to grow as well as maintain their pluripo‐ tency. Although hPSCs proliferated robustly on Matrigel, the origin of Matrigel makes this matrix unsuitable for maintaining the clinical-grade hPSCs. Recently, Ding et al. [49] devel‐ oped the humanized ECM by using umbilical cord blood serum (UCBS) as the matrix for derivation and culture of hPSCs. The best result was obtained when UCBS was used in combination with the basal medium supplemented with bFGF, fibronectin, and Y-27632. Other humanized matrices, for example, human placental-derived ECM and H9-hESC embryoid body–derived ECM have been proved to support the pluripotency of hPSCs [58, 59]. The hPSC lines cultured with the ECM derived from both types of human tissues or cells in the defined serum and xeno-free culture media such as TeSR2, sustained their pluripotency after pro‐ longed culture. However, lot-to-lot variations in the individual serum donor or tissue-derived ECM can affect the effectiveness of the serum or ECM in maintaining the pluripotent state of hPSCs. In order to develop the defined-feeder-free cell adhesion coating, individual extracel‐ lular matrix (ECM) proteins have been used instead of Matrigel. Several ECM including laminin, fibronectin, vitronectin, and collagen support the proliferation of hPSCs. Those ECM can be used as single or in various combinations. Interestingly, the studies have demonstrated that recombinant laminin-511 and recombinant vitronectin enable to support the growth and maintain the pluripotency of hPSCs [60, 61]. To improve feeder-free culture system, cell adhesion proteins can be added to ECM component. E-cadherin, the cell adhesion protein improved the attachment and expansion of hPSCs when the cells were cultured in defined media such as mTeSR1 and xeno-free media, TeSR2 [62]. Moreover, recombinant E-cadherin fusion protein supported the proliferation of the other pluripotent cells [63]. Additional developments in defined culture surfaces include synthetic peptide coatings such as Synthe‐ max, Peptide acrylate surfaces (PAS), or synthetic polymers have been proved to support the growth and pluripotency of hPSCs after prolonged culture [64]. The combination of human ECM proteins or cell adhesion molecules and synthetic biomaterials with well-designed surfaces and/or structures in the presence of a chemically defined medium containing recombinant growth factors would offer a xeno-free alternative to feeder cells for culturing hPSCs and maintaining their pluripotency. Because the xeno-free and feeder-free culture system is suitable for generation of hPSCs for clinical application, we reprogrammed CD34+ cells by the temperature-sensitive Sendai viral vectors carries OCT-4, SOX2, KLF4 and c-MYC, on Pronectin F plus (Sanyo Chemical Industries, Kyoto, Japan) coating matrix and cultured the cells in ReproFF (ReproCELL, Yokohama, Japan) culture medium (**Figure 2**). We will use this newly established hPSCs for further development of xeno-free culture system. In our study [48], the hPSC lines were derived and cultured on HFF feeder layer. The hPSC lines were continuously maintained on either mitomycin-C inactivated HFF-FBS or mitomycin-C inactivated HFF-hUCS in serum-free hPSC culture medium. The serum-free hPSC culture medium comprising 80% Knockout Dulbecco's modified Eagles' medium (KO-DMEM), 20% KO-SR, 1% nonessential amino acid, 1% Glutamax, 1% penicillin-streptomycin, 0.1 mM βmercaptoethanol (all from Invitrogen), and 8 ng/ml bFGF (R&D Systems, Minneapolis, MN,

148 Pluripotent Stem Cells - From the Bench to the Clinic

USA).

Prior to clinical application, xeno-free hPSC lines should be tested and evaluated for their biological safety. The evaluation should follow the guidelines that developed for clinical trials or therapies. The hPSC lines should be free from serious pathogenic microorganisms such as human immunodeficiency virus (HIV) and human papilloma virus (HPV). On the other hand, hPSC lines should also be free from mycoplasma contamination. Although Romorini [65] demonstrated the successful elimination of the mycoplasma contamination in their newly established hPSC lines but the mycoplasma-free newly established cell line is the ideal cell line for therapeutic application. Moreover, the level of endotoxin in the culture medium of hPSCs should meet the requirement of the national or international standard. Other biological test, for example, injection of the hiPSCs into the chorioallantoic and yolk sac of the chicken for testing unknown pathogenic microorganisms can be performed [25].

## **7. Clinical trial and therapeutic application**

The clinical trial and therapeutic application requires hPSCs that were derived, cultured, and differentiated under the xeno-free and GMP conditions. Despite controversies and difficulties, the clinical trials using hESCs started in 2010 and continue ongoing. Geron Corporation, the biotechnology company based in California, USA began a clinical trial in patients with spinal cord injuries using hESCs in 2010. The patient was transplanted with oligodendrocyte precursor cells derived from hESCs. Unfortunately, the project was discontinued a year later because the company changed their business strategy. There is no official data that has been published from the study of Geron. However, the project has been started and continued again by Advance Cell Technology, the biotechnology company based in Massachusetts. The company just recently published their preclinical safety data [66]. In 2012, the clinical trials using hESC-derivative cells were initiated and conducted in the USA. Advance Cell Technol‐ ogy, performed the clinical trials in the patients with Stargardt's macular degeneration and age-related dry macular degeneration, which caused the loss of photoreceptor and results in blindness. Retinal-pigmented epithelial (RPE) cells derived from hESCs were transplanted to replace the degenerated cells. Interestingly, the visual improvement of the patients had been preliminarily reported [67, 68]. ViaCyte, the company based in California, USA reported the successful surgical transplantation of pancreatic precursor cells derived from hESCs for treatment of Type I diabetes [69]. Recently, the Japanese researchers from RIKEN institute performed the first clinical trial using hiPSC-derived cells for treatment of age-related macular degeneration. Autologous hPSCs were differentiated to retinal-pigmented epithelial (RPE) sheet and later transplanted to the patient. However, the second trial was cancelled due to the occurring of genetic instability of the cells prior to transplantation [70]. Besides the clinical trials mentioned above, the hPSC-based products for the treatment of Parkinson's disease and others diseases are currently in the pipeline [71].

## **8. Perspective of collaboration**

Although clinically compliant hPSC has been derived, limitations in scale of production and high costs for culture of hPSCs remain significant challenges. Besides efficiency and safety, the development of hPSC-based cell therapies are expected to be expensive, time consuming and might face the clinical failure. Therefore, the collaboration between academy, SME and large pharmaceutical company can take the advantage of opportunities to tap into various sources of support. Funding for basic research carried out in the academic setting is available from national research grant agencies and institutes. In the private sector, SMEs have the possibility to attract venture capital to fund efforts in regenerative medicine. In addition, the pharma‐ ceutical companies have the possibility to directly support the project using their internal resources. Besides individually applying for grants or securing other type of funding, the partners can also join together and obtain consortium grants or support based on collaborative networks from international initiatives such as the Framework Programs funded by the European Union. We had developed the similar model by carried out the project with the two European partners, The University of Copenhagen, Denmark and BioTalentum, Ltd, Hungary. The project was funded by the European Union under the Framework 7 through the Marie Curies Actions. The results of the project were not only the publications [72] but also trans‐ ferring the researchers and knowledge between the academy and industry. We believed that having research partners cover both non-profit and for-profit organizations is of great advantage when developing hPSC-based cell therapy.

## **9. Conclusion**

cord injuries using hESCs in 2010. The patient was transplanted with oligodendrocyte precursor cells derived from hESCs. Unfortunately, the project was discontinued a year later because the company changed their business strategy. There is no official data that has been published from the study of Geron. However, the project has been started and continued again by Advance Cell Technology, the biotechnology company based in Massachusetts. The company just recently published their preclinical safety data [66]. In 2012, the clinical trials using hESC-derivative cells were initiated and conducted in the USA. Advance Cell Technol‐ ogy, performed the clinical trials in the patients with Stargardt's macular degeneration and age-related dry macular degeneration, which caused the loss of photoreceptor and results in blindness. Retinal-pigmented epithelial (RPE) cells derived from hESCs were transplanted to replace the degenerated cells. Interestingly, the visual improvement of the patients had been preliminarily reported [67, 68]. ViaCyte, the company based in California, USA reported the successful surgical transplantation of pancreatic precursor cells derived from hESCs for treatment of Type I diabetes [69]. Recently, the Japanese researchers from RIKEN institute performed the first clinical trial using hiPSC-derived cells for treatment of age-related macular degeneration. Autologous hPSCs were differentiated to retinal-pigmented epithelial (RPE) sheet and later transplanted to the patient. However, the second trial was cancelled due to the occurring of genetic instability of the cells prior to transplantation [70]. Besides the clinical trials mentioned above, the hPSC-based products for the treatment of Parkinson's disease and

Although clinically compliant hPSC has been derived, limitations in scale of production and high costs for culture of hPSCs remain significant challenges. Besides efficiency and safety, the development of hPSC-based cell therapies are expected to be expensive, time consuming and might face the clinical failure. Therefore, the collaboration between academy, SME and large pharmaceutical company can take the advantage of opportunities to tap into various sources of support. Funding for basic research carried out in the academic setting is available from national research grant agencies and institutes. In the private sector, SMEs have the possibility to attract venture capital to fund efforts in regenerative medicine. In addition, the pharma‐ ceutical companies have the possibility to directly support the project using their internal resources. Besides individually applying for grants or securing other type of funding, the partners can also join together and obtain consortium grants or support based on collaborative networks from international initiatives such as the Framework Programs funded by the European Union. We had developed the similar model by carried out the project with the two European partners, The University of Copenhagen, Denmark and BioTalentum, Ltd, Hungary. The project was funded by the European Union under the Framework 7 through the Marie Curies Actions. The results of the project were not only the publications [72] but also trans‐ ferring the researchers and knowledge between the academy and industry. We believed that having research partners cover both non-profit and for-profit organizations is of great

others diseases are currently in the pipeline [71].

advantage when developing hPSC-based cell therapy.

**8. Perspective of collaboration**

150 Pluripotent Stem Cells - From the Bench to the Clinic

To enable a xeno-free culture system capable of clinical use, it is important to examine the sources of components used in hPSC derivation, culture, including the reagents used for handling cells during passaging and the reagents used for cryopreservation. The sufficient protocols and documentations, development of hPSC derivation and culture conditions under the strict cleanroom and the use of GMP-grade reagents and materials should be prepared. In addition to the technical issues involved, establishment of the donor consent documentation appropriately and utilize forms that specifically states that the cells will be used for cell therapy purposes have to be prepared. The clinical grade hPSC lines that derived completely under the GMP conditions will be a valuable source for the future clinical therapies.

## **Acknowledgements**

This work was supported by Chulalongkorn University Research Grant (GRB\_APS\_03\_57\_30\_01).

## **Author details**

Kamthorn Pruksananonda\* and Ruttachuk Rungsiwiwut

\*Address all correspondence to<EMAIL_ADDRESS>
Human Embryonic Stem Cells Research Center, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

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## **Affordable Clean Air Facility for (Stem) Cell Therapy in an Academic Setting**

Una Chen

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62757

#### **Abstract**

Together with her colleagues, the author has worked extensively on the differentiation potential of mouse embryonic stem cells (ESCs) to blood islands containing embryoid bodies (EBs) in culture. Such EBs contain hematopoietic stem- precursor cells (HSCs) committed to lymphoid, myeloid lineages and cells possessing Natural Killer (NK) cell marker. Further differentiation of such EBs to mature B-lymphoid cells can be demonstrat‐ ed in a second stage of tissue culture, by co-culturing separated EBs with a mitogen, Lipopolysaccharide (LPS). The demonstration of differentiation of such cells to mature Tand B-lymphoid cells was performed in vivo, i.e., by implanting EBs subcutaneously in nude mice. The differentiation of ESCs to EBs needed to be scaled up to obtain enough HSCcontaining EBs for a second stage of differentiation, in vitro and in vivo. Selection of HSCcontaining EBs must be performed manually under a microscope outside the tissue culture incubator. For research purposes, the cell culture medium contains antibiotics to inhibit the growth of bacteria. However for clinical application, the medium should be free of antibiotics. A conventional research laboratory environment is not sufficient for such an application. A GMP (Good Manufacturing Production) facility with clean air became necessary. Commercial clean air facilities used in the operation room and/or by industry were over budget. The attention of the author has shifted to developing an affordable GMP facility for clinical research in academia. These efforts were successful and the price for such a GMP facility is 10% of a comparable industrial model. After that experience, "a GMP facility for cell therapy on wheels" was further developed. This chapter will describe and share with academic colleagues our experience of constructing such affordable GMP facilities from scratch, starting with the purchase of high-efficiency particulate arrestance (HEPA) filters for the clean air facility.

**Keywords:** cGMP, clinical application, mobile HEPA filter, mobile clean air facility, mini-clean air box

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

cGMP (current Good Manufacturing Production) for (stem) cell therapy is the making (growth, propagation, expansion, scaled up production) of (stem) cells for clinical purposes. The procedures and requirements include two parts: hardware and software.

Here, I shall discuss and share some experiences that we have conducted and performed by converting a regular research tissue culture room-facility, especially the hardware part, into

**Figure 1: GMP facility.** A: Biochemistry Institute Building in Uni. Clinic Giessen, cold needle graphic by U.C. Blue arrow points to the location of GMP facility. B: Planning of the GMP facility: Space 1-B-1: A front room; Space 1-B-2: Air lock; Space 1-B-3: main space of the GMP facility; Space 1-B-4: Soft wall where clean bench was located. Red long square: Mobile HEPA filter, Black long square: Hanging HEPA filter.**C. Washable wall and doors for the facility.** The existing wall of the space (1-B, purple color) was coated with transparent lacquer. The newly constructed wall and doors were these between entrance preparation room (1-B-1) and airlock (1-B-2); airlock and the main room (1-B-3) of the facility. The material was iron frame coated with white color paint and acryl glass (shown in 1-C). A row of brush‐ es (black color, indicated by a blue arrow) at the bottom of the door (blue triangles in 1-B-1 and 1-B-2) for directing the outward air flowing.

an open system clean air facility inside an old building (**Figure 1**), and then transferring the facility in a container (**Figure 5**) to become a "GMP facility on wheels".

## **2. Procedures and results**

#### **2.1. Software**

**1. Introduction**

160 Pluripotent Stem Cells - From the Bench to the Clinic

outward air flowing.

cGMP (current Good Manufacturing Production) for (stem) cell therapy is the making (growth, propagation, expansion, scaled up production) of (stem) cells for clinical purposes. The

Here, I shall discuss and share some experiences that we have conducted and performed by converting a regular research tissue culture room-facility, especially the hardware part, into

**Figure 1: GMP facility.** A: Biochemistry Institute Building in Uni. Clinic Giessen, cold needle graphic by U.C. Blue arrow points to the location of GMP facility. B: Planning of the GMP facility: Space 1-B-1: A front room; Space 1-B-2: Air lock; Space 1-B-3: main space of the GMP facility; Space 1-B-4: Soft wall where clean bench was located. Red long square: Mobile HEPA filter, Black long square: Hanging HEPA filter.**C. Washable wall and doors for the facility.** The existing wall of the space (1-B, purple color) was coated with transparent lacquer. The newly constructed wall and doors were these between entrance preparation room (1-B-1) and airlock (1-B-2); airlock and the main room (1-B-3) of the facility. The material was iron frame coated with white color paint and acryl glass (shown in 1-C). A row of brush‐ es (black color, indicated by a blue arrow) at the bottom of the door (blue triangles in 1-B-1 and 1-B-2) for directing the

procedures and requirements include two parts: hardware and software.

The software includes: Documentation, Standard Operational Procedures (S.O.P.), QA (Quality Assurance), QC (Quality Control) tests.

The criteria and requirements can vary from country to country and be updated periodically. One must be in contact with the relevant regulatory authority regularly to achieve the standard required. There are guidelines constantly updated from regulatory authorities such as FDA [1]. The information provided by the UK stem cell bank could also be of interest [2]. The appoint‐ ment of a QA person, possibly from industry working in the field, who can guide the produc‐ tion, give advice and suggestions, deal with update of the guidelines, and communicate with regulatory authorities, is highly recommended.

Standard Operation Procedure (S.O.P.)-1 (**Figure 7**) and S.O.P.-2 provide examples on how the clean air facility operation procedures were written, practiced, and controlled by the staff members. Our S.O.P. were updated whenever necessary and as often as possible to describe the real steps of practical exercise so that new/any staff members could hold a printout sheet in their hands, read, and follow the instructions, step by step, to meet the requirements and to operate properly.

"S.O.P. 1: Into the clean air room" (**Figure 7**) instructs the staff on what to do in the entrance preparation room, including how to change clothes, pin up hair, wear protective glasses; on how to take air shower when entering the airlock, on how to operate first in the clean air facility such as to measure particles counts, to use particle collectors, to place agar plates for collection of particles for microbiological sterile tests, before starting the cell culture experiments. Any leak and/or contamination of the air particles from outside will be detected by these proce‐ dures.

"S.O.P. 2: Out of the clean air room" (**Figure 8**) describes the reverse steps of "Into the clean air room", stating how to operate before leaving the facility, what to take out and where to place the samples collected, how to document the operation and to be double checked by a supervisor of the operation..

S.O.P. 3 (**Figure 9**) provides the regular and routine control of the clean air in the facility and describes, step by step, how and where (in clean bench, HEPA Filter 1, HEPA Filter 2, air lock) agar plates should be placed, when to be removed, where and how long they should be tested. The procedure describes how to clean the benches and what should be done before and after the cleaning, namely, agar plate tests. It describes when and where to measure particles using particle counter and particle collector. Any inappropriate operation in the entire procedure or contamination is documented and double-triple controlled by the operators and supervisor.

## **2.2. Hardware**

The hardware includes: standardization, inspection, and routinely periodic control of all equipment used for tissue culture, including temperature and cleanness control of refrigerator, accuracy control of pipette-aids, temperature and CO2 content of CO2 incubator, Laminar Flowclean bench, etc. One goes through such inspection routinely once a week, and documents the results on the so-called Monday S.O.P. (S.O.P. 3).

The equipment control is essential. The equipment do not have to be purchased new, though new equipments are under guarantee by the manufacturing companies and require less work, but the existing equipments must be inspected, (re)-standardized, and controlled routinely. Tissue culture working area should have no (bacterial–fungal) contamination (S.O.P. 3), i.e., clean air 100/ISO 6 (see below under criteria and in **Table 1**).

**Table 1.** ISO 14644-1 cleanroom standards vs US FED STD 209D standard.

#### **2.3. Clean air facility: closed vs open systems**

Two systems for cell culture work at the clean air bench: closed- system vs. open- system. Companies such as Phoenix in N.Y. sell such closed clean air benches. For our purpose of growing cells and selecting ES-cell derived embryoid bodies under a microscope, we could only use the open clean air bench located inside a clean air room.

Clean air rooms are classified according to the number and size of particles permitted per volume of air. There are basic codes of classification for cleanrooms:

1. US FED STD 209D and 209E cleanroom standards [3]:

It denotes the number of particles of size 0.5 μm or larger permitted per cubic foot (ft3 ) of air. For example "Class 100/A" is 100 particles/ft3 , "class 1000/B" is 1000 particles/ft3 , "class 10,000/ C" is the normal air environment. For a tissue culture bench, it is recommended to operate under condition A, i.e., 100 particles or less/ft3 . One m3 is 35 ft3 . This criteria of clean room classification was cancelled by the General Services Administration in 2001 [4, 5]. However, this system is still commonly used by many organizations, including us.

2. ISO 14644-1 standards [6]:

**2.2. Hardware**

results on the so-called Monday S.O.P. (S.O.P. 3).

162 Pluripotent Stem Cells - From the Bench to the Clinic

clean air 100/ISO 6 (see below under criteria and in **Table 1**).

**Table 1.** ISO 14644-1 cleanroom standards vs US FED STD 209D standard.

only use the open clean air bench located inside a clean air room.

volume of air. There are basic codes of classification for cleanrooms:

1. US FED STD 209D and 209E cleanroom standards [3]:

**2.3. Clean air facility: closed vs open systems**

For example "Class 100/A" is 100 particles/ft3

under condition A, i.e., 100 particles or less/ft3

**ISO 1** 10 2.37 1.02 0.35 0.083 0.0029 **ISO 2** 100 23.7 10.2 3.5 0.83 0.029

The hardware includes: standardization, inspection, and routinely periodic control of all equipment used for tissue culture, including temperature and cleanness control of refrigerator, accuracy control of pipette-aids, temperature and CO2 content of CO2 incubator, Laminar Flowclean bench, etc. One goes through such inspection routinely once a week, and documents the

The equipment control is essential. The equipment do not have to be purchased new, though new equipments are under guarantee by the manufacturing companies and require less work, but the existing equipments must be inspected, (re)-standardized, and controlled routinely. Tissue culture working area should have no (bacterial–fungal) contamination (S.O.P. 3), i.e.,

**Class Maximum particles/m3 FED STD 209D ≥0.1 μm ≥0.2 μm ≥0.3 μm ≥0.5 μm ≥1 μm ≥5 μm equivalent**

Two systems for cell culture work at the clean air bench: closed- system vs. open- system. Companies such as Phoenix in N.Y. sell such closed clean air benches. For our purpose of growing cells and selecting ES-cell derived embryoid bodies under a microscope, we could

Clean air rooms are classified according to the number and size of particles permitted per

C" is the normal air environment. For a tissue culture bench, it is recommended to operate

. One m3

, "class 1000/B" is 1000 particles/ft3

is 35 ft3

) of air.

, "class 10,000/

. This criteria of clean room

It denotes the number of particles of size 0.5 μm or larger permitted per cubic foot (ft3

**ISO 3** 1000 237 102 35 8.3 0.29 Class 1 **ISO 4** 10,000 2370 1020 352 83 2.9 Class 10 **ISO 5** 100,000 23,700 10,200 3520 832 29 Class 100 **ISO 6** 1.0 × 106 237,000 102,000 35,200 8320 293 Class 1000 **ISO 7** 1.0 × 107 2.37 × 106 1,020,000 352,000 83,200 2930 Class 10,000 **ISO 8** 1.0 × 108 2.37 × 107 1.02 × 107 3,520,000 832,000 29,300 Class 100,000 **ISO 9** 1.0 × 109 2.37 × 108 1.02 × 108 35,200,000 8,320,000 293,000 Room air

International Organization for Standardization (ISO) codes ISO 14644-1 cleanroom standards are the most commonly used. ISO classifies cleanrooms on a scale from 1 to 9, with 1 being the "cleanest" air. ISO classification 1 cleanrooms have a maximum of 10 particles smaller than 0.1 micrometers in a cubic meter of airspace. Class 9 is used to describe normal room air.

3. BS 5295 cleanroom standards and standards of other countries:

There are many other standards, such as Britain BS 5295, Australia AS 1386, France AFNOR X44101, and Germany VD I.2083. Table 6 in [7] describes the details.

**Table 1** lists the comparison of US and ISO systems (1 and 2, above). Small numbers refer to ISO 14644-1 standards, which specify the decimal logarithm of the number of particles 0.1 μm or larger permitted per m<sup>3</sup> of air. The table provides an immediate comparison and under‐ standing of the criteria, such that an ISO class 5 clean room has at most 10<sup>5</sup> particles/m<sup>3</sup> . In our facility, we succeeded in achieving (marked in red color) class 1000/ISO 6 for clean air room, class 100/ISO 5 for clean bench open working area.

## **2.4. A mini-clean air box**

For the purpose of growing cells, flasks equipped with mini-filters are available. However, for picking ES-derived EBs, Petri dishes are preferable for the practical reason that an access to the open dish under the microscope is absolutely required. Therefore, we have developed and made "**mini-tissue culture boxes"**. They are self-made, from material such as ready-made plastic boxes or acryl glass derived from Mishell-Dutton cell incubation system, and equipped with an air in-let and out-let system for CO2 gassing before they are placed into the CO<sup>2</sup> incubator (**Figure 6**).

### **2.5. Open clean air facility**

The clean air facility was located on the second floor of an old building where the Biochemistry Institute of University clinic of Giessen is situated (**Figure 1-A**), having one window (blue arrow). The three windows to the right of this facility are those of my research laboratory.

### **2.6. Washable wall and doors for the facility**

Due to financial considerations, the option to have a commercial hardwall facility inside the existing building wall was not chosen. The existing wall of the space (**Figure 1-B**, purple color) was coated with a transparent vinyl lacquer/paint to reduce the release of particles from plaster.

The newly constructed wall and doors were those between the entrance preparation room (1- B-1) and air shower-airlock (1-B-2); air shower-air lock and the main clean air room (1-B-3) of the facility. The material of the newly constructed wall was white-painted iron frame and acryl glass (shown in **Figure 1-C**). The surface was smooth, washable, and inert, so that it will not attract and accumulate dust. A row of brushes (black color in **Figure 1-C**, indicated by a blue arrow) was fixed to the bottom of the door (blue triangles in 1-B-1 and 1-B-2) to direct the air flowing outward. Above the window was an air-conditioning apparatus (blue box).

The facility (**Figure 1-B**) was constructed and divided into the following spaces: an entrance preparation room for changing clothes and a storage shelf (Space 1-B-1), an air shower-airlock with a mobile high efficiency particulate air (HEPA) filter for taking air shower (Space 1-B-2), the main workspace (1-B-3) with a softwall cleanroom (Space B-4) with hanging HEPA filter from the ceiling, and a mobile HEPA filter moving inside and outside the softwall clean room. Inside the softwall clean room, a two-person-laminar-air-flow cabin/clean bench, and two chairs were placed for doing cell culture. The CO2 incubator could also be placed inside the softwall cleanroom. The red long box indicated the place where the mobile HEPA filter was located. The black long box indicated the place where the hanging HEPA filter was located.

## **2.7. HEPA filter on wheels**

The construction of the mobile HEPA filter is explained in this figure (**Figure 2**). It requires access to a metal workshop so that one can do the metal work by oneself, and/or contracting the design to a skilful mechanic who knows how to work with metal. A frame-in metal carrier with four wheels was designed with the precise size of the outer measurement of the HEPA filter. In this case, the HEPA filter from Zander (EU 3 and H 14, Zander, [8]) was purchased (**Figure 2-A** left image) and the designed construct was made by a mechanic (**Figure 2-A** right lower corner image). The HEPA filter could be lifted with the help of two persons and loaded to fit into/onto this carrier (**Figure 2-B**). The mounted HEPA filter could move around the space freely and securely, no extra screw was needed to fasten these two pieces together for this construct.

**Figure 2. HEPA Filter on the wheels.** The left image (A) contains two HEPA filters from Zander (No. EU 3 and H 14). To the right lower corner of these filters is the image of a self-made metal carrier with four wheels. The right image (B) shows a HEPA filter loaded on this wheeled metal carrier to become mobile.

#### **2.8. Soft-wall clean room self-construction**

#### *2.8.1. HEPA filter to hang from the ceiling*

glass (shown in **Figure 1-C**). The surface was smooth, washable, and inert, so that it will not attract and accumulate dust. A row of brushes (black color in **Figure 1-C**, indicated by a blue arrow) was fixed to the bottom of the door (blue triangles in 1-B-1 and 1-B-2) to direct the air

The facility (**Figure 1-B**) was constructed and divided into the following spaces: an entrance preparation room for changing clothes and a storage shelf (Space 1-B-1), an air shower-airlock with a mobile high efficiency particulate air (HEPA) filter for taking air shower (Space 1-B-2), the main workspace (1-B-3) with a softwall cleanroom (Space B-4) with hanging HEPA filter from the ceiling, and a mobile HEPA filter moving inside and outside the softwall clean room. Inside the softwall clean room, a two-person-laminar-air-flow cabin/clean bench, and two chairs were placed for doing cell culture. The CO2 incubator could also be placed inside the softwall cleanroom. The red long box indicated the place where the mobile HEPA filter was located. The black long box indicated the place where the hanging HEPA filter was located.

The construction of the mobile HEPA filter is explained in this figure (**Figure 2**). It requires access to a metal workshop so that one can do the metal work by oneself, and/or contracting the design to a skilful mechanic who knows how to work with metal. A frame-in metal carrier with four wheels was designed with the precise size of the outer measurement of the HEPA filter. In this case, the HEPA filter from Zander (EU 3 and H 14, Zander, [8]) was purchased (**Figure 2-A** left image) and the designed construct was made by a mechanic (**Figure 2-A** right lower corner image). The HEPA filter could be lifted with the help of two persons and loaded to fit into/onto this carrier (**Figure 2-B**). The mounted HEPA filter could move around the space freely and securely, no extra screw was needed to fasten these two pieces together for this

**Figure 2. HEPA Filter on the wheels.** The left image (A) contains two HEPA filters from Zander (No. EU 3 and H 14). To the right lower corner of these filters is the image of a self-made metal carrier with four wheels. The right image (B)

shows a HEPA filter loaded on this wheeled metal carrier to become mobile.

flowing outward. Above the window was an air-conditioning apparatus (blue box).

**2.7. HEPA filter on wheels**

164 Pluripotent Stem Cells - From the Bench to the Clinic

construct.

The same type of HEPA filter from Zander (EU 3 and H 14) was used for this construction (**Figure 3-A**). The HEPA filter was lifted onto self-constructed metal in-frames and suspended with additional metal support from the ceiling of the room (**Figure 3-B**).

#### *2.8.2. Self-constructed metal frame and heavy plastic hanging strips*

If there is sufficient budget, one could purchase and install the commercially available softwall clean room, such as the one from the company, Cleanairproducts [9]. However, we have chosen a lower budget option: to purchase the HEPA filter and to have a skilful worker perform the rest of the construction.

For the construction of the softwall clean room, the additional material such as metal frame, ceiling metal hangers for suspension, heavy plastic strips, were purchased and constructed. The design of metal frame for the softwall clean room depends on the size of the room and the type of metal pieces available locally. **Figure 3-B** shows one option. Around the metal frames were heavy plastic sheets with free flip strips a few centimeters away from the floor to allow the air to escape.

#### *2.8.3. Outfit for working in the clean air facility*

Among all the items to enter the clean room, human beings carry the most dust. Thus, it is essential to remove as many dust particles as possible from the workers in the air lock before entering the clean room.

As shown in **Figure 4**, the complete impermeable synthetic suit, either of tightly woven polyester or a nonwoven material, such as Tyvek, is preferable: Examples are polyester suit (Countdown clean systems, Derby, England), Jumpsuit (Tyvek Pro-Tech Classic, DuPontTM, US) was the recommended gown for working in the clean air room [10, 11]. In addition, hair cap, eye glasses (for non-glasses wearing workers), mouth mask, hand gloves, and washable plastic shoes were worn by the worker in the entrance preparation room before taking an air shower in the airlock. Most items were used once and disposed. Exceptions to this general rule were the protecting glasses and plastic shoes. The gown might be recycled a few times, if an X-ray machine is accessible to allow irradiation for sterilization.

**Figure 4. Outfits for working in the clean air facility.** A: A staff member wearing glasses, mouth mask, hair hood to cover the hair and skin in the entrance preparationroom before entering the airlock. **Tyvek® Coveralls** gown was the clothes of choice [11]. B: A backview of the clean air gown with shoes (derived from [10]).

#### **2.9. Mobile clean air facility/A clean air facility on wheels**

Two containers, Variante 6 from ALHO [12] (**Figure 5-A**), were rented to place in a parking lot of Europaviertal, an industrial area outside the city of Giessen. The enlarged image (**Figure 5-B**) shows the location of HEPA filters (in red) and other items in the facility. The container was built from smooth surface metal material so that no extra coating process to repel dust was performed. The space was divided into the following working areas: The container to the left was the preparation space for storage and for changing clothes (Space B-1); an airlock with a mobile HEPA filter for taking air shower (Space B-2); the container to the right was the main clean air working space (B-3). The ceiling of the container was not high enough to accommodate any hanging HEPA filter. Instead, two mobile HEPA filters were placed inside this container. The two-person-clean bench (in black) and two chairs were placed there for cell culture. A CO2 incubator was placed next to the clean bench for growing cells.

As shown in **Figure 4**, the complete impermeable synthetic suit, either of tightly woven polyester or a nonwoven material, such as Tyvek, is preferable: Examples are polyester suit (Countdown clean systems, Derby, England), Jumpsuit (Tyvek Pro-Tech Classic, DuPontTM, US) was the recommended gown for working in the clean air room [10, 11]. In addition, hair cap, eye glasses (for non-glasses wearing workers), mouth mask, hand gloves, and washable plastic shoes were worn by the worker in the entrance preparation room before taking an air shower in the airlock. Most items were used once and disposed. Exceptions to this general rule were the protecting glasses and plastic shoes. The gown might be recycled a few times, if an

**Figure 4. Outfits for working in the clean air facility.** A: A staff member wearing glasses, mouth mask, hair hood to cover the hair and skin in the entrance preparationroom before entering the airlock. **Tyvek® Coveralls** gown was the

Two containers, Variante 6 from ALHO [12] (**Figure 5-A**), were rented to place in a parking lot of Europaviertal, an industrial area outside the city of Giessen. The enlarged image (**Figure 5-B**) shows the location of HEPA filters (in red) and other items in the facility. The container was built from smooth surface metal material so that no extra coating process to repel dust

clothes of choice [11]. B: A backview of the clean air gown with shoes (derived from [10]).

**2.9. Mobile clean air facility/A clean air facility on wheels**

X-ray machine is accessible to allow irradiation for sterilization.

166 Pluripotent Stem Cells - From the Bench to the Clinic

**Figure 5. A hard wall clean air facility on wheels.** Panel A shows the detailed plan of two containers with windows and doors from ALHO, Type Variante 6 [12]. The enlarged image (panel B) shows how the space was divided. The space was divided into: Space B-1: storage and clothes changing; Space B-2: air lock with a mobile HEPA filter (in red) for air shower; Space B-3: Main clean air facility equipped with two mobile HEPA filters (in red). A two-person-clean air bench (in black), two chairs, and a CO2 incubator. Panel C shows one container from outside.

**Figure 6. A clean air minibox**.

#### **3. Conclusion**

It took the entire group almost two years to convert a regular cell culture laboratory in an old building located in a clinic campus into a clean air facility. It is well worth the effort to have undertaken such a project. A professional from Heraeus came to visit our clean air facility and did the particle count and particle collection himself. When he was about to leave the airlock, he shook his head and said that it is incredible that our clean air facility is so clean that even the industrial clean air facility could hardly achieve such a clean level. We consider our experiences valuable and worthwhile to share with members of the research community who wish to work in the direction of clinically-oriented projects such as ES cells and iPS. The total cost is estimated to be 10% of the commercial products, an affordable facility for many. In addition, we have moved the clean air facility into two containers and continued the work there. The advantage of "clean air facility on wheels" is that it can be shared by several laboratories in a nearby region for performing similar projects such as establishment of clinical grade stem cell lines. Our research work originated in 1987 while I was a scientific member of the Basel Institute for Immunology, and continued through decades in Germany from 1996, exploring different research directions and clinical applications including this clean air facility. For an overview of our research aims and outcome, please see review articles, Discussion Forum, and a World Patent on "Methods for growing stem cells" [13–16].

## **Acknowledgements**

The author wishes to thank the entire team members for engagement. The author is grateful for the support and understanding of Professor Ewald Beck of Biochemistry Institute and Professor K. Knorr, Dean of the Uniclinic Giessen. We thank Dr. Donald Gerson of Canada for his advice and in serving as a QA person for this project. This project was financed by WP, ISPI e.V. internal grants. Dr. Richard Pink of Switzerland helps with the reading, suggestion, and editing of this article.

**Appendix 1**: Examples of S.O.P.s on how to write, to operate, and to control the practice of a clean air facility. This appendix will have the following three figures:

#### **Figure 7. S.O.P. -1. Into the clean air room**

About one hour before the operation, one must turn on the HEPA filters to let them running (register in the log book).

#### *In the entrance preparation room:*

**Figure 6. A clean air minibox**.

168 Pluripotent Stem Cells - From the Bench to the Clinic

It took the entire group almost two years to convert a regular cell culture laboratory in an old building located in a clinic campus into a clean air facility. It is well worth the effort to have undertaken such a project. A professional from Heraeus came to visit our clean air facility and did the particle count and particle collection himself. When he was about to leave the airlock, he shook his head and said that it is incredible that our clean air facility is so clean that even the industrial clean air facility could hardly achieve such a clean level. We consider our experiences valuable and worthwhile to share with members of the research community who wish to work in the direction of clinically-oriented projects such as ES cells and iPS. The total cost is estimated to be 10% of the commercial products, an affordable facility for many. In addition, we have moved the clean air facility into two containers and continued the work

**3. Conclusion**

1. Lab coat, jacket, sweater, etc. remain in the entrance room (The clothes hangers are on the right side).

2. Step onto the sticky mattress and take off the regular shoes and change to clean air room shoes right there.

3. Disinfect hands with 70% alcohol.

4. Tight hair and put on hair net.

5. Disinfect hands, put on hand gloves, and sterilize again.

6. Wear the hair cap over the hair net. Hair must be completely covered!

7. Put on clean room gown.

8. Put on mouth mask and eye glasses (when not wearing glasses).

9. Disinfect hands.

10. The material, which should be brought into the clean room, should be disinfected and put into the basket to carry in.

## *In air shower-airlock***:**

11. Enter the airlock and close the door to the preparation room.

12. Stay at least 5 min in the air shower-airlock and turn around regularly.

13. Meanwhile carry out one measurement using the particle-counter and one measurement using the air-collector.

14. Put on the clean room gown hanging on the door to the clean air room.

15. Stand on the sticky mattress, then enter the clean air room.

#### *In clean air room (HEPA filter 1, mobile):*

16. Run through once the particle counter and once the air collector.

17. Before entering the softwall clean air space (HEPA-Filter 2, hanging), step on the sticky mattress.

#### *In softwall cleanroom (HEPA-Filter 2, hanging):*

18. Before starting to work, carry out once the particle count and once the air collection.

#### **Figure 8. S.O.P. -2. Out of clean air room**

1. The total surface area inside the soft-wall clean room should be sprayed with 70% alcohol and wait a few minutes to let it absorb.

2. Turn on UV light with the key provided.

3. Turn off clean bench.

4. Record the data in the log book (Hera safe) and put the log book back to the book shelf.

5. Bring the garbage and agar plates to the entrance-preparation room. (Let agar plates incubate and check for growth).

6. Hang the clean air coat back on the door hook.

7. Take off all other clothes in the entrance preparation room and hang on the hooks or dispose. (Dirty clothes should be autoclaved).

8. Register in the log-book.

9. Turn off HEPA filters.

10. Turn off light.

5. Disinfect hands, put on hand gloves, and sterilize again.

7. Put on clean room gown.

170 Pluripotent Stem Cells - From the Bench to the Clinic

into the basket to carry in.

*In air shower-airlock***:**

using the air-collector.

mattress.

9. Disinfect hands.

6. Wear the hair cap over the hair net. Hair must be completely covered!

10. The material, which should be brought into the clean room, should be disinfected and put

13. Meanwhile carry out one measurement using the particle-counter and one measurement

17. Before entering the softwall clean air space (HEPA-Filter 2, hanging), step on the sticky

1. The total surface area inside the soft-wall clean room should be sprayed with 70% alcohol

valid until: 16.08.03

At:\_\_\_\_\_\_\_\_\_ Until:\_\_\_\_\_\_\_\_\_hr

18. Before starting to work, carry out once the particle count and once the air collection.

G.M.P./S.O.P.2 J.B. J.B. / AG Chcn written on: 16.08.01

8. Put on mouth mask and eye glasses (when not wearing glasses).

11. Enter the airlock and close the door to the preparation room.

15. Stand on the sticky mattress, then enter the clean air room.

16. Run through once the particle counter and once the air collector.

*In clean air room (HEPA filter 1, mobile):*

**Figure 8. S.O.P. -2. Out of clean air room**

and wait a few minutes to let it absorb.

2. Turn on UV light with the key provided.

**Titel: Out of the cleanair room**

3. Turn off clean bench.

*In softwall cleanroom (HEPA-Filter 2, hanging):*

12. Stay at least 5 min in the air shower-airlock and turn around regularly.

14. Put on the clean room gown hanging on the door to the clean air room.

Name 1 \_\_\_\_\_\_\_\_\_

Name 2 \_\_\_\_\_\_\_\_\_

#### **Figure 9. S.O.P.-3. Routine clean air room control**

**Important:** Turn on both HEPA filters in Room 130 and Room 131 at least one hour before the Monday S.O.P. routine control.

Change to clean room gown.

**Room 130 (HEPA-Filter 2**, i.e. inside the soft wall)

**Room 130: (HEPA-Filter 1**, i.e. mobile one outside the soft wall)

### **Air lock:**

#### **Entrance preparation room**

## **Author details**

Una Chen

Address all correspondence to<EMAIL_ADDRESS>
International Senior Professional Institute e.V. Grünbergerstrsse, Giessen, Germany

## **References**

International Senior Professional Institute e.V. Grünbergerstrsse, Giessen, Germany

[1] http://www.fda.gov/drugs/developmentapprovalprocess/manufacturing/

[3] Cleanroom Classification/Particle Count/FS209E/ISO TC209/

[5] http://www.wbdg.org/ccb/FEDMIL/notices.pdf, p. 148

[6] https://en.wikipedia.org/wiki/Cleanroom

[2] http://www.nibsc.org/UK\_Stem\_Cell\_Bank/Cell\_lines/EUTCD\_Cell\_Lines.aspx.

[4] Cancellation of FED-STD-209E - Institute of Environmental Sciences and Technology

and colony counts similar to the above procedure

172 Pluripotent Stem Cells - From the Bench to the Clinic

**Room 130: (HEPA-Filter 1**, i.e. mobile one outside the soft wall)

G.M.P./S.O.P.13)

**Air lock:**

(see G.M.P./S.O.P.13).

**Entrance preparation room**

Address all correspondence to<EMAIL_ADDRESS>
**Author details**

Una Chen

**References**

ucm169105.htm.

## **Induced Pluripotent Stem Cells for Clinical Use**

Tomohisa Seki and Keiichi Fukuda

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62505

#### **Abstract**