Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications.

We have recently described manufacturing of human induced pluripotent stem cells (iPSC) master cell banks (MCB) generated by a clinically compliant process using cord blood as a starting material (Baghbaderani et al. in Stem Cell Reports, 5(4), 647-659, 2015). In this manuscript, we describe the detailed characterization of the two iPSC clones generated using this process, including whole genome sequencing (WGS), microarray, and comparative genomic hybridization (aCGH) single nucleotide polymorphism (SNP) analysis. We compare their profiles with a proposed calibration material and with a reporter subclone and lines made by a similar process from different donors. We believe that iPSCs are likely to be used to make multiple clinical products. We further believe that the lines used as input material will be used at different sites and, given their immortal status, will be used for many years or even decades. Therefore, it will be important to develop assays to monitor the state of the cells and their drift in culture. We suggest that a detailed characterization of the initial status of the cells, a comparison with some calibration material and the development of reporter sublcones will help determine which set of tests will be most useful in monitoring the cells and establishing criteria for discarding a line.

Developing Induced Pluripotent Stem Cell-Based Therapy for the Masses.

The discovery of induced pluripotent stem cells and the ability to manufacture them using clinically compliant protocols has the potential to revolutionize the field of regenerative medicine. However, realizing this potential requires the development of processes that are reliable, reproducible, and cost-effective and that at the same time do not compromise the safety of the individuals receiving this therapy. In the present report, we discuss how cost reductions can be obtained using our experience with obtaining approval of biologic agents, autologous therapy, and the recent approval of cord blood banks. Significance: For therapy to be widely available, the cost of manufacturing stem cells must be reduced. The steps proposed in the present report, when implemented, have the potential to reduce these costs significantly.

cGMP-Manufactured Human Induced Pluripotent Stem Cells Are Available for Pre-clinical and Clinical Applications.

The discovery of induced pluripotent stem cells (iPSCs) and the concurrent development of protocols for their cell-type-specific differentiation have revolutionized our approach to cell therapy. It has now become critical to address the challenges related to the generation of iPSCs under current good manufacturing practice (cGMP) compliant conditions, including tissue sourcing, manufacturing, testing, and storage. Furthermore, regarding the technical challenges, it is very important to keep the costs of manufacturing and testing reasonable and solve logistic hurdles that permit the global distribution of these products. Here we describe our efforts to develop a process for the manufacturing of iPSC master cell banks (MCBs) under cGMPs and announce the availability of such banks.

Research using Mesenchymal Stem/Stromal Cells: quality metric towards developing a reference material.

Mesenchymal stem/stromal cells (MSCs) have been extensively investigated for their regenerative, immune-modulatory, and wound healing properties. While the laboratory studies have suggested that MSC's have a unique potential for modulating the etiopathology of multiple diseases, the results from clinical trials have not been encouraging or reproducible. One of the explanations for such variability is explained by the "art" of isolating and propagating MSCs. Therefore, establishing more than minimal criteria to define MSC would help understand best protocols to isolate, propagate and deliver MSCs. Developing a calibration standard, a database and a set of functional tests would be a better quality metric for MSCs. In this review, we discuss the importance of selecting a standard, issues associated with coming up with such a standard and how these issues can be mitigated.

Can cord blood banks transform into induced pluripotent stem cell banks?

The discovery of induced pluripotent stem cells (iPSCs) and the rapid evolution of clinically compliant protocols to generate such lines from a variety of tissue sources has raised the possibility that personalized medicine may be achievable in the near future. Several strategies to deliver iPSCs for iPSC-derived cell-based therapy have been proposed: one such model has been the cell-banking model, using processes developed by the cord blood industry. The cord blood industry has evolved primarily as a banking model in which units of cord blood harvested from discarded placenta are stored either in a public or a private cord blood bank for future use. The consideration of a cord blood--like banking model has been further spurred by the realization that this population of cells is an ideal starting sample to generate pluripotent cells. Spurred by these technological advances, major efforts are underway to develop a current Good Manufacturing Practice--compliant protocol to generate iPSCs from cord blood and to develop a haplobanking strategy. In this article, we discuss the issues that may affect such an effort.

Concise review: making and using clinically compliant pluripotent stem cell lines.

The field of pluripotent stem cells (PSCs) is in a state of dynamic flux driven by significant advances in the derivation of specific phenotypes from embryonic stem cells, breakthroughs in somatic cell nuclear transfer, and dramatic improvements in generating induced PSCs using zero footprint methods. Spurred by these technological advances, companies have begun to plan clinical studies using human PSC derivatives manufactured in current Good Manufacturing Practice-compliant conditions. In the present review, we discuss the challenges in making these biological products, starting from tissue sourcing to the processes involved in manufacture, storage, and distribution. Additional challenges exist to meeting the regulatory requirements and keeping costs affordable. A model is described that has been proposed by the U.S. National Institutes of Health for reducing the costs and permitting flexibility and innovation by individual investigators. This model, combined with small adjustments in the regulatory processes tailored to address the unique properties of PSCs, has the potential of significantly accelerating the implementation of PSC-based cell therapy.

Banking on iPSC--is it doable and is it worthwhile.

The discovery of induced pluripotent stem cells (iPSCs) and concurrent development of protocols for their cell-type specific differentiation have revolutionized studies of diseases and raised the possibility that personalized medicine may be achievable. Realizing the full potential of iPSC will require addressing the challenges inherent in obtaining appropriate cells for millions of individuals while meeting the regulatory requirements of delivering therapy and keeping costs affordable. Critical to making PSC based cell therapy widely accessible is determining which mode of cell collection, storage and distribution, will work. In this manuscript we suggest that moderate sized bank where a diverse set of lines carrying different combinations of commonly present HLA alleles are banked and differentiated cells are made available to matched recipients as need dictates may be a solution. We discuss the issues related to developing such a bank and how it could be constructed and propose a bank of selected HLA phenotypes from carefully screened healthy individuals as a solution to delivering personalized medicine.

Survival and engraftment of dopaminergic neurons manufactured by a Good Manufacturing Practice-compatible process.

BACKGROUND AIMS: We have previously reported a Good Manufacturing Practice (GMP)-compatible process for generating authentic dopaminergic neurons in defined media from human pluripotent stem cells and determined the time point at which dopaminergic precursors/neurons (day 14 after neuronal stem cell [NSC] stage) can be frozen, shipped and thawed without compromising their viability and ability to mature in vitro. One important issue we wished to address is whether dopaminergic precursors/neurons manufactured by our GMP-compatible process can be cryopreserved and engrafted in animal Parkinson disease (PD) models. METHODS: In this study, we evaluated the efficacy of freshly prepared and cryopreserved dopaminergic neurons in the 6-hydroxydopamine-lesioned rat PD model. RESULTS: We showed functional recovery up to 6 months post-transplantation in rats transplanted with our cells, whether freshly prepared or cryopreserved. In contrast, no motor improvement was observed in two control groups receiving either medium or cells at a slightly earlier stage (day 10 after NSC stage). Histologic analysis at the end point of the study (6 months post-transplantation) showed robust long-term survival of donor-derived tyrosine hydroxylase (TH)(+) dopaminergic neurons in rats transplanted with day 14 dopaminergic neurons. Moreover, TH(+) fibers emanated from the graft core into the surrounding host striatum. Consistent with the behavioral analysis, no or few TH(+) neurons were detected in animals receiving day 10 cells, although human cells were present in the graft. Importantly, no tumors were detected in any grafted rats, but long-term tumorigenic studies will need to determine the safety of our products. CONCLUSIONS: Dopaminergic neurons manufactured by a GMP-compatible process from human ESC survived and engrafted efficiently in the 6-OHDA PD rat model.

Stable enhanced green fluorescent protein expression after differentiation and transplantation of reporter human induced pluripotent stem cells generated by AAVS1 transcription activator-like effector nucleases.

Human induced pluripotent stem (hiPS) cell lines with tissue-specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno-associated virus integration site 1 (AAVS1) locus has been used as a "safe harbor" locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator-like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus-immediate early enhancer/chicken ß-actin/rabbit ß-globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage-specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1-CAG-EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high-efficiency targeting can be obtained with open-source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.

Soliciting strategies for developing cell-based reference materials to advance mesenchymal stromal cell research and clinical translation.

The mesenchymal stromal cell (MSC) field continues to rapidly progress with a number of clinical trials initiated and completed, with some reported successes in multiple clinical indications, and a growing number of companies established. The field, nevertheless, faces several challenges. Persistent issues include the definition of a MSC and comparability between MSC preparations. This is because of inherent cell heterogeneity, the absence of markers that are unique to MSCs, and the difficulty in precisely defining them by developmental origin. Differences in the properties of MSCs also depend on the site of tissue harvest, phenotypic and genotypic characteristics of the donor and the isolation, and storage and expansion methods used. These differences may be sufficient to ensure that attributes of the final MSC product could differ in potentially significant ways. Since there are currently no gold standards, we propose using a reference material to establish methods of comparability among MSC preparations. We suggest four possible "ruler scenarios" and a method for global distribution. We further suggest that critical to establishing a reference material is the need to define protocols for comparing cells. The main purpose of this article is to solicit input in establishing a consensus-based comparison. A comparative approach will be critical to all stages of translation to better clarify mechanisms of MSC actions, define an optimal cell manufacturing process, ensure best practice clinical investigations, extend the use of an MSC product for new indications, protect an MSC product from imitators, and develop uniform reimbursement policies. Importantly, a reference material may enable a consensus on a practical definition of MSCs.

Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies.

Human pluripotent stem cells (PSCs) are a leading candidate for cell-based therapies because of their capacity for unlimited self renewal and pluripotent differentiation. These advances have recently culminated in the first-in-human PSC clinical trials by Geron, Advanced Cell Technology and the Kobe Center for Developmental Biology for the treatment of spinal cord injury and macular degeneration. Despite their therapeutic promise, a crucial hurdle for the clinical implementation of human PSCs is their potential to form tumors in vivo. In this Perspective, we present an overview of the mechanisms underlying the tumorigenic risk of human PSC-based therapies and discuss current advances in addressing these challenges.

hESC-derived Olig2+ progenitors generate a subtype of astroglia with protective effects against ischaemic brain injury.

Human pluripotent stem cells (hPSCs) have been differentiated to astroglia, but the utilization of hPSC-derived astroglia as cell therapy for neurological diseases has not been well studied. Astroglia are heterogeneous, and not all astroglia are equivalent in promoting neural repair. A prerequisite for cell therapy is to derive defined cell populations with superior therapeutic effects. Here we use an Olig2-GFP human embryonic stem cell (hESC) reporter to demonstrate that hESC-derived Olig2(+) progenitors generate a subtype of previously uncharacterized astroglia (Olig2PC-Astros). These Olig2PC-Astros differ substantially from astroglia differentiated from Olig2-negative hESC-derived neural progenitor cells (NPC-Astros), particularly in their neuroprotective properties. When grafted into brains subjected to global ischaemia, Olig2PC-Astros exhibit superior neuroprotective effects and improved behavioural outcome compared to NPC-Astros. Thus, this new paradigm of human astroglial differentiation is useful for studying the heterogeneity of human astroglia, and the unique Olig2PC-Astros may constitute a new cell therapy for treating cerebral ischaemia and other neurological diseases.

Pluripotent stem cells in translation: a Food and Drug Administration-National Institutes of Health collaboration.

Recently, the U.S. Food and Drug Administration (FDA), the U.S. National Institutes of Health, and the stem cell research community have collaborated on a series of workshops that address moving pluripotent stem cell therapies into the clinic. The first two workshops in the series focused on preclinical science, and a third, future workshop will focus on clinical trials. This summary addresses major points from both of the recent preclinically focused meetings. When entering into a therapeutics developmental program based on pluripotent cells, investigators must make decisions at the very early stages that will have major ramifications during later phases of development. Presentations and discussions from both invited participants and FDA staff described the need to characterize and document the quality, variability, and suitability of the cells and commercial reagents used at every translational stage. This requires consideration of future regulatory requirements, ranging from donor eligibility of the original source material to the late-stage manufacturing protocols. Federal, industrial, and academic participants agreed that planning backward is the best way to anticipate what evidence will be needed to justify human testing of novel therapeutics and to eliminate wasted efforts.

Optimizing dopaminergic differentiation of pluripotent stem cells for the manufacture of dopaminergic neurons for transplantation.

BACKGROUND AIMS: We have previously described a xeno-free scalable system to generate transplantable dopaminergic neurons from human pluripotent stem cells. However, several important questions remain to be answered about our cell therapy efforts. These include determining the exact time at which cells should be transplanted and whether cells at this stage can be frozen, shipped, thawed and injected without compromising their ability to mature and survive the transplantation procedure. We also needed to determine whether further optimization of the culture process could shorten the development time and reduce variability and whether a current Good Manufacture Practice (CGMP) facility could manufacture cells with fidelity. METHODS: We developed an optimized protocol that included modulating the sonic hedgehog homolog gradient with bone morphogenetic proteins (BMP2) and addition of activin to the culture medium, which shortened the time to generate Lmx1A and FoxA2 immunoreactive cells by 4-6 days. RESULTS: We showed that cells at this stage could be safely frozen and thawed while retaining an excellent ability to continue to mature in vitro and survive transplant in vivo. Importantly, we successfully adapted this process to a CGMP facility and manufactured two lots of transplant-ready dopaminergic neurons (>250 vials) under CGMP-compatible conditions. In vitro characterization, including viability/recovery on thawing, whole genome expression as well as expression of midbrain/dopaminergic markers, showed that the cells manufactured under GMP-compatible conditions were similar to cells produced at lab scale. CONCLUSIONS: Our results suggest that this optimized protocol can be used to generate dopaminergic neurons for Investigational New Drug enabling studies.

A review of the methods for human iPSC derivation.

The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs) offers an opportunity to generate pluripotent patient-specific cell lines that can help model human diseases. These iPSC lines could also be powerful tools for drug discovery and the development of cellular transplantation therapies. Many methods exist for generating iPSC lines but those best suited for use in studying human diseases and developing therapies must be of adequate efficiency to produce iPSCs from samples that may be of limited abundance, capable of reprogramming cells from both skin fibroblasts and blood, and footprint-free. Several reprogramming techniques meet these criteria and can be utilized to derive iPSCs in projects with both basic scientific and therapeutic goals. Combining these reprogramming methods with small molecule modulators of signaling pathways can lead to successful generation of iPSCs from even the most recalcitrant patient-derived somatic cells.

Efficient generation of astrocytes from human pluripotent stem cells in defined conditions.

Astrocytes can be generated from various tissue sources including human pluripotent stem cells (PSC). In this manuscript, we describe a chemically defined xeno-free medium culture system for rapidly generating astrocytes from neural stem cells derived from PSC. We show that astrocyte development in vitro, mimics normal development in vivo, and also passes through a CD44(+) astrocyte precursor stage. Astrocytes generated by our method display similar gene expression patterns, morphological characteristics and functional properties to primary astrocytes, and they survive and integrate after xenotransplantation. Whole genome expression profiling of astrocyte differentiation was performed at several time points of differentiation, and the results indicate the importance of known regulators and identify potential novel regulators and stage-specific lineage markers.

Human neural crest stem cells derived from human ESCs and induced pluripotent stem cells: induction, maintenance, and differentiation into functional schwann cells.

The neural crest (NC) is a transient, multipotent, migratory cell population unique to vertebrates that gives rise to diverse cell lineages. Much of our knowledge of NC development comes from studies of organisms such as chicken and zebrafish because human NC is difficult to obtain because of its transient nature and the limited availability of human fetal cells. Here we examined the process of NC induction from human pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). We showed that NC cells could be efficiently induced from hESCs by a combination of growth factors in medium conditioned on stromal cells and that NC stem cells (NCSCs) could be purified by p75 using fluorescence-activated cell sorting (FACS). FACS-isolated NCSCs could be propagated in vitro in five passages and cryopreserved while maintaining NCSC identity characterized by the expression of a panel of NC markers such as p75, Sox9, Sox10, CD44, and HNK1. In vitro-expanded NCSCs were able to differentiate into neurons and glia (Schwann cells) of the peripheral nervous system, as well as mesenchymal derivatives. hESC-derived NCSCs appeared to behave similarly to endogenous embryonic NC cells when injected in chicken embryos. Using a defined medium, we were able to generate and propagate a nearly pure population of Schwann cells that uniformly expressed glial fibrillary acidic protein, S100, and p75. Schwann cells generated by our protocol myelinated rat dorsal root ganglia neurons in vitro. To our knowledge, this is the first report on myelination by hESC- or iPSC-derived Schwann cells.

Cell therapy in critical limb ischemia: current developments and future progress.

Critical limb ischemia (CLI) is a syndrome manifested by ischemic rest pain, non-healing ulcers and tissue loss. CLI patients are at very high risk of amputation and experience poor physical function, leading to severe morbidity and mortality. The fundamental goal for CLI treatment is to relieve ischemic rest pain, heal ulcers, prevent limb loss and improve the quality of life, thereby extending the survival of the patient. Surgical or endovascular revascularization aimed at increasing blood flow is currently available for limb salvage in CLI. However, up to 30% of CLI patients are not suitable for such interventions because of high operative risk or unfavorable vascular anatomy. Therefore exploring new and more effective strategies for revascularization of ischemic limbs is imperative for the establishment of a viable therapeutic alternative. With the emergence of new approaches, this review describes up-to-date progress and developments in cell-based therapy as a novel and promising alternative for CLI treatment. Preliminary clinical data have established the safety, feasibility and efficacy of stem cells, and numerous studies are underway to consolidate this evidence further. However, significant hurdles remain to be addressed before this research can be responsibly translated to the bedside. In particular, we need better understanding of the behavior of cells post-transplantation and to learn how to control their survival and migration proliferation/differentiation in the hostile pathologic environment. Future research should focus on methods of isolation, optimal dosage, appropriate cell type, route of administration, role of tissue-derived factors and supportive endogenous stimulation.

Assessing iPSC reprogramming methods for their suitability in translational medicine.

The discovery of the ability to induce somatic cells to a pluripotent state through the overexpression of specific transcription factors has the potential to transform the ways in which pharmaceutical agents and cellular transplantation therapies are developed. Proper utilization of the technology to generate induced pluripotent stem cells (iPSCs) requires that researchers select the appropriate reprogramming method for generating iPSCs so that the resulting iPSCs can be transitioned towards clinical applications effectively. This article reviews all of the currently available reprogramming techniques with a focus on critiquing them on the basis of their utility in translational medicine.

Stromal factors SDF1α, sFRP1, and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells.

Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson's disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by coculture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factors produced by stromal cells that result in SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 α (SDF1α), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified, and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1, and VEGFD to the culture medium, we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and ßIII-tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1, and VEGFD are major components of SDIA and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.