Application-Oriented Bulk Cryopreservation of Human iPSCs in Cryo Bags Followed by Direct Inoculation in Scalable Suspension Bioreactors for Expansion and Neural Differentiation.

Stem cell-based therapies are promising tools for regenerative medicine and require bulk numbers of high-quality cells. Currently, cells are produced on demand and have a limited shelf-life as conventional cryopreservation is primarily designed for stock keeping. We present a study on bulk cryopreservation of the human iPSC lines UKKi011-A and BIONi010-C-41. By increasing cell concentration and volume, compared to conventional cryopreservation routines in cryo vials, one billion cells were frozen in 50 mL cryo bags. Upon thawing, the cells were immediately seeded in scalable suspension-based bioreactors for expansion to assess the stemness maintenance and for neural differentiation to assess their differentiation potential on the gene and protein levels. Both the conventional and bulk cryo approach show comparative results regarding viability and aggregation upon thawing and bioreactor inoculation. Reduced performance compared to the non-frozen control was compensated within 3 days regarding biomass yield. Stemness was maintained upon thawing in expansion. In neural differentiation, a delay of the neural marker expression on day 4 was compensated at day 9. We conclude that cryopreservation in cryo bags, using high cell concentrations and volumes, does not alter the cells' fate and is a suitable technology to avoid pre-cultivation and enable time- and cost-efficient therapeutic approaches with bulk cell numbers.

Scalable expansion of iPSC and their derivatives across multiple lineages.

Induced pluripotent stem cell (iPSC) technology enabled the production of pluripotent stem cell lines from somatic cells from a range of known genetic backgrounds. Their ability to differentiate and generate a wide variety of cell types has resulted in their use for various biomedical applications, including toxicity testing. Many of these iPSC lines are now registered in databases and stored in biobanks such as the European Bank for induced pluripotent Stem Cells (EBiSC), which can streamline the quality control and distribution of these individual lines. To generate the quantities of cells for banking and applications like high-throughput toxicity screening, scalable and robust methods need to be developed to enable the large-scale production of iPSCs. 3D suspension culture platforms are increasingly being used by stem cell researchers, owing to a higher cell output in a smaller footprint, as well as simpler scaling by increasing culture volume. Here we describe our strategies for successful scalable production of iPSCs using a benchtop bioreactor and incubator for 3D suspension cultures, while maintaining quality attributes expected of high-quality iPSC lines. Additionally, to meet the increasing demand for "ready-to-use" cell types, we report recent work to establish robust, scalable differentiation protocols to cardiac, neural, and hepatic fate to enable EBiSC to increase available research tools.

Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells.

Organoids derived from human pluripotent stem cells are interesting models to study mechanisms of morphogenesis and promising platforms for disease modeling and drug screening. However, they mostly remain incomplete as they lack stroma, tissue resident immune cells and in particular vasculature, which create important niches during development and disease. We propose, that the directed incorporation of mesodermal progenitor cells (MPCs) into organoids will overcome the aforementioned limitations. In order to demonstrate the feasibility of the method, we generated complex human tumor as well as neural organoids. We show that the formed blood vessels display a hierarchic organization and mural cells are assembled into the vessel wall. Moreover, we demonstrate a typical blood vessel ultrastructure including endothelial cell-cell junctions, a basement membrane as well as luminal caveolae and microvesicles. We observe a high plasticity in the endothelial network, which expands, while the organoids grow and is responsive to anti-angiogenic compounds and pro-angiogenic conditions such as hypoxia. We show that vessels within tumor organoids connect to host vessels following transplantation. Remarkably, MPCs also deliver Iba1+ cells that infiltrate the neural tissue in a microglia-like manner.

Generation of two induced pluripotent stem cell lines from skin fibroblasts of sisters carrying a c.1094C>A variation in the SCN10A gene potentially associated with small fiber neuropathy.

Induced pluripotent stem cells (iPSC) were derived from human dermal fibroblasts (HDF) of two siblings with small fiber neuropathy (SFN) potentially based on the same variation in SCN10A but exhibiting diverse disease phenotypes. HDF were reprogrammed using a non-integrating mRNA approach and showed robust expression of pluripotency markers. iPSC displayed no chromosomal aberrations and were differentiated into all three germ-layers. These two cell lines with a familial genetic background may provide a useful in vitro tool to investigate the underlying mechanisms leading to different phenotypes caused by the same variation.

Generation of the human induced pluripotent stem cell line UKWNLi002-A from dermal fibroblasts of a woman with a heterozygous c.608 C>T (p.Thr203Met) mutation in exon 3 of the nerve growth factor gene potentially associated with hereditary sensory and autonomic neuropathy type 5.

In this study, we report the human induced pluripotent stem cell line (iPSC) HSAN5-T203 M-iPSC, generated from human dermal fibroblasts (HDF) of a woman carrying a heterozygous c.608C > T mutation in the nerve growth factor gene potentially associated with hereditary sensory and autonomic neuropathy type 5 (HSAN5). HDF were reprogrammed using transient expression of key transcription factors for pluripotency and immune evasion via transfection of synthetic mRNA and miRNA. HSAN5-T203 M-iPSC retained the disease-associated genotype c.608C > T, while maintaining a normal karyotype, showed robust and abundant expression of pluripotency-associated markers and could be differentiated into cells of all three germ layers.

Generation of Cardiomyocytes From Vascular Adventitia-Resident Stem Cells.

RATIONALE: Regeneration of lost cardiomyocytes is a fundamental unresolved problem leading to heart failure. Despite several strategies developed from intensive studies performed in the past decades, endogenous regeneration of heart tissue is still limited and presents a big challenge that needs to be overcome to serve as a successful therapeutic option for myocardial infarction. OBJECTIVE: One of the essential prerequisites for cardiac regeneration is the identification of endogenous cardiomyocyte progenitors and their niche that can be targeted by new therapeutic approaches. In this context, we hypothesized that the vascular wall, which was shown to harbor different types of stem and progenitor cells, might serve as a source for cardiac progenitors. METHODS AND RESULTS: We describe generation of spontaneously beating mouse aortic wall-derived cardiomyocytes without any genetic manipulation. Using aortic wall-derived cells (AoCs) of WT (wild type), αMHC (α-myosin heavy chain), and Flk1 (fetal liver kinase 1)-reporter mice and magnetic bead-associated cell sorting sorting of Flk1+ AoCs from GFP (green fluorescent protein) mice, we identified Flk1+CD (cluster of differentiation) 34+Sca-1 (stem cell antigen-1)-CD44- AoCs as the population that gives rise to aortic wall-derived cardiomyocytes. This AoC subpopulation delivered also endothelial cells and macrophages with a particular accumulation within the aortic wall-derived cardiomyocyte containing colonies. In vivo, cardiomyocyte differentiation capacity was studied by implantation of fluorescently labeled AoCs into chick embryonic heart. These cells acquired cardiomyocyte-like phenotype as shown by αSRA (α-sarcomeric actinin) expression. Furthermore, coronary adventitial Flk1+ and CD34+ cells proliferated, migrated into the myocardium after mouse myocardial infarction, and expressed Isl-1+ (insulin gene enhancer protein-1) indicative of cardiovascular progenitor potential. CONCLUSIONS: Our data suggest Flk1+CD34+ vascular adventitia-resident stem cells, including those of coronary adventitia, as a novel endogenous source for generating cardiomyocytes. This process is essentially supported by endothelial cells and macrophages. In summary, the therapeutic manipulation of coronary adventitia-resident cardiac stem and their supportive cells may open new avenues for promoting cardiac regeneration and repair after myocardial infarction and for preventing heart failure.

Generation of the human induced pluripotent stem cell line (UKWNLi001-A) from skin fibroblasts of a woman with Fabry disease carrying the X-chromosomal heterozygous c.708 G > C (W236C) missense mutation in exon 5 of the alpha-galactosidase-A gene.

Human dermal fibroblasts (HDF) were isolated from the skin punch biopsy of a 25-year-old woman with Fabry disease (FD), carrying a heterozygous c.708 G > C missense mutation in the alpha-galactosidase A gene. HDF were reprogrammed to induced pluripotent stem cells (iPSC) using synthetic mRNA, preventing the alteration of the genome and retaining the original genotype. FD-W236C-iPSC (UKWNLi001-A) showed typical human embryonic stem cell (hESC)-like morphology, expressed all analyzed pluripotency-associated markers, could be differentiated into cells from all three germ layers, and demonstrated a normal female karyotype. We provide a novel patient-specific cell line, allowing further insights into the pathophysiology of FD. Resource table.

Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation.

Neural stem cell (NSC) transplantation can influence immune responses and suppress inflammation in the CNS. Metabolites, such as succinate, modulate the phenotype and function of immune cells, but whether and how NSCs are also activated by such immunometabolites to control immunoreactivity and inflammatory responses is unclear. Here, we show that transplanted somatic and directly induced NSCs ameliorate chronic CNS inflammation by reducing succinate levels in the cerebrospinal fluid, thereby decreasing mononuclear phagocyte (MP) infiltration and secondary CNS damage. Inflammatory MPs release succinate, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, leading them to secrete prostaglandin E2 and scavenge extracellular succinate with consequential anti-inflammatory effects. Thus, our work reveals an unexpected role for the succinate-SUCNR1 axis in somatic and directly induced NSCs, which controls the response of stem cells to inflammatory metabolic signals released by type 1 MPs in the chronically inflamed brain.

Generation of a human induced pluripotent stem cell (iPSC) line from a 51-year-old female with attention-deficit/hyperactivity disorder (ADHD) carrying a duplication of SLC2A3.

Fibroblasts were isolated from a skin biopsy of a clinically diagnosed 51-year-old female attention-deficit/hyperactivity disorder (ADHD) patient carrying a duplication of SLC2A3, a gene encoding neuronal glucose transporter-3 (GLUT3). Patient fibroblasts were infected with Sendai virus, a single-stranded RNA virus, to generate transgene-free human induced pluripotent stem cells (iPSCs). SLC2A3-D2-iPSCs showed expression of pluripotency-associated markers, were able to differentiate into cells of the three germ layers in vitro and had a normal female karyotype. This in vitro cellular model can be used to study the role of risk genes in the pathogenesis of ADHD, in a patient-specific manner.

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single-use bioreactors.

The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell-based therapies and cell-loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400 ml. The simple and robust two-step process reported here first generates hiPSC aggregates of 324 ± 71 µm diameter in 7 days in 125 ml spinner flasks (100 ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000 ml bioreactors (1000 ml volume), finally yielding hiPSC aggregates of 198 ± 58 µm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40 days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16-fold increase in hiPSC quantity at the 100 ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000 ml scale, an additional 10-fold increase was achieved. Taken together, 16 × 106 hiPSCs were expanded into 2 × 109 hiPSCs in 14 days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell-based therapies and brings their clinical potential closer to fruition.