Considerations for the development of iPSC-derived cell therapies: a review of key challenges by the JSRM-ISCT iPSC Committee.

Since their first production in 2007, human induced pluripotent stem cells (iPSCs) have provided a novel platform for the development of various cell therapies targeting a spectrum of diseases, ranging from rare genetic eye disorders to cancer treatment. However, several challenges must be tackled for iPSC-based cell therapy to enter the market and achieve broader global adoption. This white paper, authored by the Japanese Society for Regenerative Medicine (JSRM) - International Society for Cell Therapy (ISCT) iPSC Committee delves into the hurdles encountered in the pursuit of safe and economically viable iPSC-based therapies, particularly from the standpoint of the cell therapy industry. It discusses differences in global guidelines and regulatory frameworks, outlines a series of quality control tests required to ensure the safety of the cell therapy, and provides details and important considerations around cost of goods (COGs), including the impact of automated advanced manufacturing.

Characterizing Pluripotent Stem Cells Using the TaqMan® hPSC Scorecard(TM) Panel.

Rapid technological developments for the efficient generation of footprint-free induced pluripotent stem cells (iPSC) enabled the creation of patient-specific iPSC for downstream applications in drug discovery and regenerative medicine. However, the large number of iPSCs, generated from diverse genetic backgrounds using various methods and culture conditions, created a steep challenge for rapid characterization and a demand for standardized methods. Current methods rely on a combination of in vitro and in vivo cellular analyses based on the expression of markers of self-renewal and the ability of the cells to differentiate into cell types representative of the three germ layers as a confirmation of functional pluripotency. These methods, though informative and extensively used, are not ideal for parallel analyses of large numbers of samples and hence not amenable to high-throughput environments. Recently, genetic and epigenetic expression signatures were used to define and confirm cell states, thus providing a surrogate molecular assay that can potentially replace complex in vivo cellular assays such as teratoma formation. In this chapter, we describe a molecular assay for rapid characterization and standardization of pluripotent stem cells. The TaqMan(®) hPSC Scorecard™ Panel is a comprehensive gene expression real-time PCR assay that consists of 94 individual q-PCR assays comprised of a combination of control, housekeeping, self-renewal, and lineage-specific genes. The resulting expression data set is analyzed using cloud-based analysis software that compares the expression pattern against a reference standard composed of multiple functionally validated ESC and iPSC lines. This system was successfully used to test several ESC and iPSC lines in their undifferentiated states to confirm their signatures of self renewal, as well as their terminally differentiates states, via spontaneous differentiation and directed differentiation into specific lineages, to determine the lines' differentiation potential. This genetic analysis tool, together with the flexibility to utilize varying sample inputs and preparation methods, provides a rapid method to confirm functional pluripotency of ESCs and iPSCs.

Development and characterization of a clinically compliant xeno-free culture medium in good manufacturing practice for human multipotent mesenchymal stem cells.

Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, cartilage damage, and cardiac diseases. Despite remarkable progress in clinical trials, most applications still use traditional culture media containing fetal bovine serum or serum-free media that contain serum albumin, insulin, and transferrin. The ill-defined and variable nature of traditional culture media remains a challenge and has created a need for better defined xeno-free culture media to meet the regulatory and long-term safety requirements for cell-based therapies. We developed and tested a serum-free and xeno-free culture medium (SFM-XF) using human bone marrow- and adipose-derived MSCs by investigating primary cell isolation, multiple passage expansion, mesoderm differentiation, cellular phenotype, and gene expression analysis, which are critical for complying with translation to cell therapy. Human MSCs expanded in SFM-XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of MSCs expanded in traditional serum-containing culture medium (SCM). To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded in SFM-XF and SCM were compared, revealing relatively similar expression profiles. In addition, the SFM-XF supported the isolation and propagation of human MSCs from primary human marrow aspirates, ensuring that these methods and reagents are compatible for translation to therapy. The SFM-XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to serum-containing media for the production of large scale, functionally competent MSCs for future clinical applications.

BacMam-mediated gene delivery into multipotent mesenchymal stromal cells.

Baculoviruses have been used over the last several decades for high-level protein production in insect cells. Recently, modified baculovirus containing a mammalian promoter, known as BacMam virus, has been shown to give high transduction efficiencies across several cell types with minimal cytopathic effects. Cell types amenable to BacMam transduction include primary and adult stem cells. The shuttle vectors used in the construction of BacMam viruses can hold gene fragments up to 38 kb in size, and multiple BacMam viruses can be used in a single transduction for the delivery of more than one gene. BacMam technology has been used in the delivery and expression of targeted fluorescent protein cellular markers, small interfering RNAi, and extensively in the development of cell-based assays. BacMam offers an ideal method for the delivery and expression of large genes in hard-to-transfect cells such as primary and adult stem cells. In this chapter, we describe methods of generating high titer stocks of BacMam for transducing MSC and their derivatives.

A novel serum-free medium for the expansion of human mesenchymal stem cells.

INTRODUCTION: Human multipotent mesenchymal stem cell (MSC) therapies are being tested clinically for a variety of disorders, including Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, and cartilage defects. However, despite the remarkable clinical advancements in this field, most applications still use traditional culture media containing fetal bovine serum. The ill-defined and highly variable nature of traditional culture media remains a challenge, hampering both the basic and clinical human MSC research fields. To date, no reliable serum-free medium for human MSCs has been available. METHODS: In this study, we developed and tested a serum-free growth medium on human bone marrow-derived MSCs through the investigation of multiple parameters including primary cell isolation, multipassage expansion, mesoderm differentiation, cellular phenotype, and gene-expression analysis. RESULTS: Similar to that achieved with traditional culture medium, human MSCs expanded in serum-free medium supplemented with recombinant human platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF), and transforming growth factor (TGF)-beta1 showed extensive propagation with retained phenotypic, differentiation, and colony-forming unit potential. To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded under serum-free and serum-containing conditions were compared, revealing similar expression profiles. In addition, the described serum-free culture medium supported the isolation of human MSCs from primary human marrow aspirate with continual propagation. CONCLUSIONS: Although the described serum-free MSC culture medium is not free of xenogeneic components, this medium provides a substitute for serum-containing medium for research applications, setting the stage for future clinical applications.

A simplified culture and polymerase chain reaction identification assay for quality control performance testing of stem cell media products.

BACKGROUND AIMS: With the growing use of stem cell media technologies in research and clinical settings, there has been an increased demand for validated cell-based quality control tools that can first, routinely test performance of stem cell media products, second, verify stem cell line identity, and third, demonstrate differentiation potential. As a significant amount of time and effort is required to verify these aspects separately, especially with classic functional stains that take as along as 28 days to perform, there is a need for a quick, sensitive and validated assay with short turn around time. METHODS: Culture, gene microarray and polymerase chain reaction (PCR) methodologies were utilized in the design, development and testing of a standardized performance assay for the expansion, identity and differentiation potential of human multipotent mesenchymal stromal cells (MSC). RESULTS: A simplified culture- and PCR-based assay was validated and transferred into a quality control setting for performance testing of human MSC under uninduced and adipogenesis-induced conditions. CONCLUSIONS: An effective strategy has been demonstrated for identifying candidate genes, validating a gene of interest and creating an inexpensive low-technology PCR assay for distinguishing uninduced and early stage differentiating stem cells. This approach extends published criteria guidelines for routinely detecting uninduced human MSC and their differentiated progeny.

A single EBV-based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells.

AIM: Stable expression of transgenes in stem cells has been a challenge due to the nonavailability of efficient transfection methods and the inability of transgenes to support sustained gene expression. Several methods have been reported to stably modify both embryonic and adult stem cells. These methods rely on integration of the transgene into the genome of the host cell, which could result in an expression pattern dependent on the number of integrations and the genomic locus of integration. To overcome this issue, site-specific integration methods mediated by integrase, adeno-associated virus or via homologous recombination have been used to generate stable human embryonic stem cell (hESC) lines. In this study, we describe a vector that is maintained episomally in hESCs. METHODS: The vector used in this study is based on components derived from the Epstein-Barr virus, containing the Epstein-Barr virus nuclear antigen 1 expression cassette and the OriP origin of replication. The vector also expresses the drug-resistance marker gene hygromycin, which allows for selection and long-term maintenance of cells harboring the plasmid. RESULTS: Using this vector system, we show sustained expression of green fluorescent protein in undifferentiated hESCs and their differentiating embryoid bodies. In addition, the stable hESC clones show comparable expression with and without drug selection. Consistent with this observation, bulk-transfected adipose tissue-derived mesenchymal stem cells showed persistent marker gene expression as they differentiate into adipocytes, osteoblasts and chondroblasts. CONCLUSIONS: Episomal vectors offer a fast and efficient method to create hESC reporter lines, which in turn allows one to test the effect of overexpression of various genes on stem cell growth, proliferation and differentiation.

Global Characterization and Genomic Stability of Human MultiStem, A Multipotent Adult Progenitor Cell.

The therapeutic benefits of adult adherent stem cells are currently being investigated in clinical trials for a variety of diseases. Data from initial clinical studies are promising and as a consequence of moving to later stage clinical studies, have resulted in larger scale clinical-grade cell production strategies. Therefore it becomes imperative to examine the epigenetic flux and genomic stability of stem cells in long-term culture to determine that minimal risk is associated with these therapies. Multipotent adult progenitor cells (MAPC) are an adherent adult stem cell population that can be derived from bone marrow and was the first of a class of adult stem cells that have broad developmental potential both in vitro and in vivo. Here, we report a panel of tests to characterize MultiStem, a multipotent adult stem cell type based on MAPC, and establish its genomic stability during culture expansion. A variety of techniques were employed that consisted of miRNA expression to characterize and define the cell population; chromosomal SNP analysis and G-banding to determine karyotypic stability; and methylation pattern and telomerase expression to examine potential changes in epigenetic and chromosomal stability with prolonged in vitro culture of cells. This panel of test was applied to cultures at early isolation stages and compared to cultures harvested at population doublings greater than those reached in current MultiStem clinical trials. These tests also provide a baseline for quality control of cells prepared from various biological donor sources for subsequent large scale propagation and preparation of cell banks for downstream applications.

Differentiating human multipotent mesenchymal stromal cells regulate microRNAs: prediction of microRNA regulation by PDGF during osteogenesis.

OBJECTIVE: Human multipotent mesenchymal stromal cells (MSC) have the potential to differentiate into multiple cell types, although little is known about factors that control their fate. Differentiation-specific microRNAs may play a key role in stem cell self-renewal and differentiation. We propose that specific intracellular signaling pathways modulate gene expression during differentiation by regulating microRNA expression. MATERIALS AND METHODS: Illumina mRNA and NCode microRNA expression analyses were performed on MSC and their differentiated progeny. A combination of bioinformatic prediction and pathway inhibition was used to identify microRNAs associated with platelet-derived growth factor (PDGF) signaling. RESULTS: The pattern of microRNA expression in MSC is distinct from that in pluripotent stem cells, such as human embryonic stem cells. Specific populations of microRNAs are regulated in MSC during differentiation targeted toward specific cell types. Complementary mRNA expression analysis increases the pool of markers characteristic of MSC or differentiated progeny. To identify microRNA expression patterns affected by signaling pathways, we examined the PDGF pathway found to be regulated during osteogenesis by microarray studies. A set of microRNAs bioinformatically predicted to respond to PDGF signaling was experimentally confirmed by direct PDGF inhibition. CONCLUSION: Our results demonstrate that a subset of microRNAs regulated during osteogenic differentiation of MSCs is responsive to perturbation of the PDGF pathway. This approach not only identifies characteristic classes of differentiation-specific mRNAs and microRNAs, but begins to link regulated molecules with specific cellular pathways.

PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages.

We compared the transcriptomes of marrow-derived mesenchymal stem cells (MSCs) with differentiated adipocytes, osteocytes, and chondrocytes derived from these MSCs. Using global gene-expression profiling arrays to detect RNA transcripts, we have identified markers that are specific for MSCs and their differentiated progeny. Further, we have also identified pathways that MSCs use to differentiate into adipogenic, chondrogenic, and osteogenic lineages. We identified activin-mediated transforming growth factor (TGF)-beta signaling, platelet-derived growth factor (PDGF) signaling and fibroblast growth factor (FGF) signaling as the key pathways involved in MSC differentiation. The differentiation of MSCs into these lineages is affected when these pathways are perturbed by inhibitors of cell surface receptor function. Since growth and differentiation are tightly linked processes, we also examined the importance of these 3 pathways in MSC growth. These 3 pathways were necessary and sufficient for MSC growth. Inhibiting any of these pathways slowed MSC growth, whereas a combination of TGF-beta, PDGF, and beta-FGF was sufficient to grow MSCs in a serum-free medium up to 5 passages. Thus, this study illustrates it is possible to predict signaling pathways active in cellular differentiation and growth using microarray data and experimentally verify these predictions.

Concise review: MicroRNA expression in multipotent mesenchymal stromal cells.

Mesenchymal stem cells, or multipotent mesenchymal stromal cells (MSC), isolated from various adult tissue sources have the capacities to self-renew and to differentiate into multiple lineages. Both of these processes are tightly regulated by genetic and epigenetic mechanisms. Emerging evidence indicates that the class of single-stranded noncoding RNAs known as microRNAs also plays a critical role in this process. First described in nematodes and plants, microRNAs have been shown to modulate major regulatory mechanisms in eukaryotic cells involved in a broad array of cellular functions. Studies with various types of embryonic as well as adult stem cells indicate an intricate network of microRNAs regulating key transcription factors and other genes, which in turn determine cell fate. In addition, expression of unique microRNAs in specific cell types serves as a useful diagnostic marker to define a particular cell type. MicroRNAs are also found to be regulated by extracellular signaling pathways that are important for differentiation into specific tissues, suggesting that they play a role in specifying tissue identity. In this review, we describe the importance of microRNAs in stem cells, focusing on our current understanding of microRNAs in MSC and their derivatives.

Qualification of embryonal carcinoma 2102Ep as a reference for human embryonic stem cell research.

As the number of human embryonic stem cell (hESC) lines increases, so does the need for systematic evaluation of each line's characteristics and potential. Comparisons between lines are complicated by variations in culture conditions, feeders, spontaneous differentiation, and the absence of standardized assays. These difficulties, combined with the inability of most labs to maintain more than a few lines simultaneously, compel the development of reference standards to which hESC lines can be compared. The use of a stable cell line as a reference standard offers many advantages. A line with a relatively unchanging hESC-like gene and protein expression pattern could be a positive control for developing assays. It can be used as a reference for genomics or proteomics studies, especially for normalizing results obtained in separate laboratories. Such a cell line should be widely available without intellectual property restraints, easily cultured without feeders, and resistant to spontaneous changes in phenotype. We propose that the embryonal carcinoma (EC) line 2102Ep meets these requirements. We compared the protein, gene, and microRNA expression of this cell line with those of hESC lines and alternative reference lines such as the EC line NTERA-2 and the karyotypically abnormal hESC line BG01V. The overall expression profiles of all these lines were similar, with exceptions reflecting the germ cell origins of EC. On the basis of global gene and microRNA expression, 2102Ep is somewhat less similar to hESC than the alternatives; however, 2102Ep expresses more hESC-associated microRNAs than NTERA-2 does, and fewer markers of differentiated fates.

Micro RNA profiling: an easy and rapid method to screen and characterize stem cell populations.

MicroRNAs (miRNAs) are small regulatory RNAs varying in length between 20 and 24 nucleotides. They are thought to play a key role during development by negative gene regulation at the post-transcriptional level. Recent studies using quantitative polymerase chain reaction (QPCR) and northern blot analysis have reported the presence of several miRNA unique to specific cell types. The NCode multispecies miRNA array provides a means for simultaneously profiling the expression patterns of hundreds of known miRNAs in a given cell type or biological sample. Using this method, miRNA expression patterns in embryonic and adult stem cell lines can be characterized and compared with each other. The accuracy of NCode miRNA array data can be further confirmed by QPCR analysis of putative array hits. This array-based screening platform is a fast and easy to use analytical tool that allows one to asses the state of stem cell lines following multiple passages in culture as well as a discovery tool that eliminates the need to screen large numbers of candidate regulatory miRNAs by northern blot or PCR. In this chapter, we describe in detail the method to carry out miRNA array analysis in human embryonal carcinoma cells and confirm the array results using QPCR.

Gene transfer via nucleofection into adult and embryonic stem cells.

The use of embryonic and adult stem cells as therapeutic agents is gaining momentum. A major impediment in the use of stem cells for genetic disorders is their ability to undergo genetic modification. The recognition of various site-specific integration methods open up a new avenue for gene therapy in stem cells. However, this necessitates efficient delivery of DNA molecule into cells. Most commercially used liposome-mediated transfection reagents are toxic or work poorly with stem cells. Electroporation, while effective in transfecting stem cells, is rather harsh and leads to excessive cell death. Nucleofection, a technology by Amaxa, uses a combination of electric pulse in an appropriate media, which decreases the toxicity and promotes efficient transfection of stem cells. Various types of adult and embryonic stem cells can be successfully transfected using this method, as described in this chapter.

Stem cell plasticity.

The central dogma in stem cell biology has been that cells isolated from a particular tissue can renew and differentiate into lineages of the tissue it resides in. Several studies have challenged this idea by demonstrating that tissue specific cell have considerable plasticity and can cross-lineage restriction boundary and give rise to cell types of other lineages. However, the lack of a clear definition for plasticity has led to confusion with several reports failing to demonstrate that a single cell can indeed differentiate into multiple lineages at significant levels. Further, differences between results obtained in different labs has cast doubt on some results and several studies still await independent confirmation. In this review, we critically evaluate studies that report stem cell plasticity using three rigid criteria to define stem cell plasticity; differentiation of a single cell into multiple cell lineages, functionality of differentiated cells in vitro and in vivo, robust and persistent engraft of transplanted cells.

Efficient transfection of embryonic and adult stem cells.

The ability of embryonic stem cells and adult stem cells to differentiate into specific cell types holds immense potential for therapeutic use in cell and gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells. In the study reported here, we demonstrate the use of nucleofection as a method to introduce plasmid DNA into embryonic and adult stem cells with significantly greater efficiency than electroporation or lipid-based transfection methods have. Using enhanced green fluorescent protein (eGFP) as a reporter gene, mouse embryonic stem cells were transfected both transiently and stably at a rate nearly 10-fold higher than conventional methods. The transfected cells retained their stem cell properties, including continued expression of the stem cell markers SSEA1, Oct4, and Rex1; formation of embryoid bodies; differentiation into cardiomyocytes in the presence of appropriate inducers; and, when injected into developing blastocysts, contribution to chimeras. Higher levels of transfection were also obtained with human embryonic carcinoma and human embryonic stem cells. Particularly hard-to-transfect adult stem cells, including bone marrow and multipotent adult progenitor cells, were also transfected efficiently by the method of nucleofection. Based on our results, we conclude that nucleofection is superior to currently available methods for introducing plasmid DNA into a variety of embryonic and adult stem cells. The high levels of transfection achieved by nucleofection will enable its use as a rapid screening tool to evaluate the effect of ectopically expressed transcription factors on tissue-specific differentiation of stem cells.