The Future State of Newborn Stem Cell Banking.

Newborn stem cell banking began with the establishment of cord blood banks more than 25 years ago. Over the course of nearly three decades, there has been considerable evolution in the clinical application of stem cells isolated from newborn tissues. The industry now finds itself at an inflection point as personalized medicine and regenerative medicine continue to advance.

INDEX-db: The Indian Exome Reference Database (Phase I).

Deep sequencing-based genetic mapping has greatly enhanced the ability to catalog variants with plausible disease association. Confirming how these identified variants contribute to specific disease conditions, across human populations, poses the next challenge. Differential selection pressure may impact the frequency of genetic variations, and thus detection of association with disease conditions, across populations.

Repurposing the Cord Blood Bank for Haplobanking of HLA-Homozygous iPSCs and Their Usefulness to Multiple Populations.

Although autologous induced pluripotent stem cells (iPSCs) can potentially be useful for treating patients without immune rejection, in reality it will be extremely expensive and labor-intensive to make iPSCs to realize personalized medicine. An alternative approach is to make use of human leukocyte antigen (HLA) haplotype homozygous donors to provide HLA matched iPSC products to significant numbers of patients. To establish a haplobank of iPSCs, we repurposed the cord blood bank by screening ∼4,200 high resolution HLA typed cord blood samples, and selected those homozygous for the 10 most frequent HLA-A,-B,-DRB1 haplotypes in the Korean population.

Illustrating the potency of current Good Manufacturing Practice-compliant induced pluripotent stem cell lines as a source of multiple cell lineages using standardized protocols.

We have previously reported the generation of a current Good Manufacture Practice (cGMP)-compliant induced pluripotent stem cell (iPSC) line for clinical applications. Here we show that multiple cellular products currently being considered for therapy can be generated from a single master cell bank of this or any other clinically compliant iPSC line METHODS: Using a stock at passage 20 prepared from the cGMP-compliant working cell bank (WCB), we tested differentiation into therapeutically relevant cell types of the three germ layers using standardized but generic protocols. Cells that we generated include (i) neural stem cells, dopaminergic neurons and astrocytes; (ii) retinal cells (retinal pigment epithelium and photoreceptors); and (iii) hepatocyte, endothelial and mesenchymal cells.

Human-Induced Pluripotent Stem Cells Manufactured Using a Current Good Manufacturing Practice-Compliant Process Differentiate Into Clinically Relevant Cells From Three Germ Layers.

The discovery of reprogramming and generation of human-induced pluripotent stem cells (iPSCs) has revolutionized the field of regenerative medicine and opened new opportunities in cell replacement therapies. While generation of iPSCs represents a significant breakthrough, the clinical relevance of iPSCs for cell-based therapies requires generation of high-quality specialized cells through robust and reproducible directed differentiation protocols. We have recently reported manufacturing of human iPSC master cell banks (MCB) under current good manufacturing practices (cGMPs).

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.

Derivation, Characterization, and Neural Differentiation of Integration-Free Induced Pluripotent Stem Cell Lines from Parkinson's Disease Patients Carrying SNCA, LRRK2, PARK2, and GBA Mutations.

We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson's disease (PD) patients carrying SNCA, PARK2, LRRK2, and GBA mutations, and one age-matched control. After validation of pluripotency, long-term genome stability, and integration-free reprogramming, eight of these lines (one of each SNCA, LRRK2 and GBA, four PARK2 lines, and the control) were differentiated into neural stem cells (NSC) and subsequently to dopaminergic cultures. We did not observe significant differences in the timeline of neural induction and NSC derivation between the patient and control line, nor amongst the patient lines, although we report considerable variability in the efficiency of dopaminergic differentiation among patient lines.

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.

Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System.

Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor.

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.

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.

Comparative neurotoxicity screening in human iPSC-derived neural stem cells, neurons and astrocytes.

Induced pluripotent stem cells (iPSC) and their differentiated derivatives offer a unique source of human primary cells for toxicity screens. Here, we report on the comparative cytotoxicity of 80 compounds (neurotoxicants, developmental neurotoxicants, and environmental compounds) in iPSC as well as isogenic iPSC-derived neural stem cells (NSC), neurons, and astrocytes. All compounds were tested over a 24-h period at 10 and 100 μM, in duplicate, with cytotoxicity measured using the MTT assay.

Mitochondrial alterations by PARKIN in dopaminergic neurons using PARK2 patient-specific and PARK2 knockout isogenic iPSC lines.

In this study, we used patient-specific and isogenic PARK2-induced pluripotent stem cells (iPSCs) to show that mutations in PARK2 alter neuronal proliferation. The percentage of TH(+) neurons was decreased in Parkinson's disease (PD) patient-derived neurons carrying various mutations in PARK2 compared with an age-matched control subject. This reduction was accompanied by alterations in mitochondrial:cell volume fraction (mitochondrial volume fraction).

A platform for rapid generation of single and multiplexed reporters in human iPSC lines.

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript, we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics.

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.

Transfection, selection, and colony-picking of human induced pluripotent stem cells TALEN-targeted with a GFP gene into the AAVS1 safe harbor.

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors.

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.

Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells.

The potential applications of human embryonic stem cells (hESCs) in regenerative medicine and developmental research have made stem cell biology one of the most fascinating and rapidly expanding fields of biomedicine. The first clinical trial of hESCs in humans has begun, and the field of stem cell therapy has just entered a new era. Here, we report seven hESC lines (SEES-1, -2, -3, -4, -5, -6, and -7).

Enabling consistency in pluripotent stem cell-derived products for research and development and clinical applications through material standards.

There is a need for physical standards (reference materials) to ensure both reproducibility and consistency in the production of somatic cell types from human pluripotent stem cell (hPSC) sources. We have outlined the need for reference materials (RMs) in relation to the unique properties and concerns surrounding hPSC-derived products and suggest in-house approaches to RM generation relevant to basic research, drug screening, and therapeutic applications. hPSCs have an unparalleled potential as a source of somatic cells for drug screening, disease modeling, and therapeutic application.

Rescue of an in vitro neuron phenotype identified in Niemann-Pick disease, type C1 induced pluripotent stem cell-derived neurons by modulating the WNT pathway and calcium signaling.

Niemann-Pick disease, type C1 (NPC1) is a familial disorder that has devastating consequences on postnatal development with multisystem effects, including neurodegeneration. There is no Food and Drug Administration-approved treatment option for NPC1; however, several potentially therapeutic compounds have been identified in assays using yeast, rodent models, and NPC1 human fibroblasts. Although these discoveries were made in fibroblasts from NPC1 subjects and were in some instances validated in animal models of the disease, testing these drugs on a cell type more relevant for NPC1 neurological disease would greatly facilitate both study of the disease and identification of more relevant therapeutic compounds.

Need for DISCUSS(ion).

Dr. Rao comments on the article “The DISCUSS Project: Revised Points to Consider for the Derivation of Induced Pluripotent Stem Cell Lines From Previously Collected Research Specimens” that appears in this issue.

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.

Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons.

Human primary neural tissue is a vital component for the quick and simple determination of chemical compound neurotoxicity in vitro. In particular, such tissue would be ideal for high-throughput screens that can be used to identify novel neurotoxic or neurotherapeutic compounds. We have previously established a high-throughput screening platform using human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) and neurons.

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.