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GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells.

ABSTRACT: BACKGROUND:Human mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments using mesenchymal stem cells, researchers have recently derived mesenchymal progenitors from human-induced pluripotent stem cells. Human-induced pluripotent stem cell-derived progenitors resemble adult mesenchymal stem cells in morphology, global gene expression, surface antigen profile, and multi-differentiation potential, but unlike adult mesenchymal stem cells, it can be produced in large numbers for every patient. For therapeutic applications, however, human-induced pluripotent stem cell-derived progenitors must be produced without animal-derived components (xeno-free) and in accordance with Good Manufacturing Practice guidelines. METHODS:In the present study we investigate the effects of expanding mesodermal progenitor cells derived from two human-induced pluripotent stem cell lines in xeno-free medium supplemented with human platelet lysates and in a commercial high-performance Good Manufacturing Practice-compatible medium (Unison Medium). RESULTS:The results show that long-term culture in xeno-free and Good Manufacturing Practice-compatible media somewhat affects the morphology, expansion potential, gene expression, and cytokine profile of human-induced pluripotent stem cell-derived progenitors but supports cell viability and maintenance of a mesenchymal phenotype equally well as medium supplemented with fetal bovine serum. CONCLUSIONS:The findings support the potential to manufacture large numbers of clinical-grade human-induced pluripotent stem cell-derived mesenchymal progenitors for applications in personalized regenerative medicine.

SUBMITTER: McGrath M

PROVIDER: S-EPMC6329105 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells.

McGrath Madison M Tam Edmund E Sladkova Martina M AlManaie Athbah A Zimmer Matthew M de Peppo Giuseppe Maria GM

Stem cell research & therapy 20190111 1

<h4>Background</h4>Human mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments ...[more]

PMID: 30635059

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Project description:A prerequisite for the realization of human pluripotent stem cell (hPSC) therapies is the development of bioprocesses for generating clinically relevant quantities of undifferentiated hPSCs and their derivatives under xeno-free conditions. Microcarrier stirred-suspension bioreactors are an appealing modality for the scalable expansion and directed differentiation of hPSCs. Comparative analyses of commercially available microcarriers clearly show the need for developing synthetic substrates supporting the adhesion and growth of hPSCs in three-dimensional cultures under agitation-induced shear. Moreover, the low seeding efficiencies during microcarrier loading with hPSC clusters poses a significant process bottleneck. To that end, a novel protocol was developed increasing hPSC seeding efficiency from 30% to over 80% and substantially shortening the duration of microcarrier loading. Importantly, this method was combined with the engineering of polystyrene microcarriers by surface conjugation of a vitronectin-derived peptide, which was previously shown to support the growth of human embryonic stem cells. Cells proliferated on peptide-conjugated beads in static culture but widespread detachment was observed after exposure to stirring. This prompted additional treatment of the microcarriers with a synthetic polymer commonly used to enhance cell adhesion. hPSCs were successfully cultivated on these microcarriers in stirred suspension vessels for multiple consecutive passages with attachment efficiencies close to 40%. Cultured cells exhibited on average a 24-fold increase in concentration per 6-day passage, over 85% viability, and maintained a normal karyotype and the expression of pluripotency markers such as Nanog, Oct4, and SSEA4. When subjected to spontaneous differentiation in embryoid body cultures or directed differentiation to the three embryonic germ layers, the cells adopted respective fates displaying relevant markers. Lastly, engineered microcarriers were successfully utilized for the expansion and differentiation of hPSCs to mesoderm progeny in stirred suspension vessels. Hence, we demonstrate a strategy for the facile engineering of xeno-free microcarriers for stirred-suspension cultivation of hPSCs. Our findings support the use of microcarrier bioreactors for the scalable, xeno-free propagation and differentiation of human stem cells intended for therapies.

Project description:Background Amnion-derived mesenchymal stem cells (AM-MSCs) are an attractive source of stem cell therapy for patients with irreversible liver disease. However, there are obstacles to their use due to low efficiency and xeno-contamination for hepatic differentiation. Methods We established an efficient protocol for differentiating AM-MSCs into hepatic progenitor cells (HPCs) by analyzing transcriptome-sequencing data. Furthermore, to generate the xeno-free conditioned differentiation protocol, we replaced fetal bovine serum (FBS) with polyvinyl alcohol (PVA). We investigated the hepatocyte functions with the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4. Finally, to test the transplantable potential of HPCs, we transferred AM-MSCs along with hepatic progenitors after differentiated days 11, 12, and 13 based on the expression of hepatocyte-related genes and mitochondrial function. Further, we established a mouse model of acute liver failure using a thioacetamide (TAA) and cyclophosphamide monohydrate (CTX) and transplanted AM-HPCs in the mouse model through splenic injection. Results We analyzed gene expression from RNA sequencing data in AM-MSCs and detected downregulation of hepatic development-associated genes including GATA6, KIT, AFP, c-MET, FGF2, EGF, and c-JUN, and upregulation of GSK3. Based on this result, we established an efficient hepatic differentiation protocol using the GSK3 inhibitor, CHIR99021. Replacing FBS with PVA resulted in improved differentiation ability, such as upregulation of hepatic maturation markers. The differentiated hepatocyte-like cells (HLCs) not only synthesized and secreted albumin, but also metabolized drugs by the CYP3A4 enzyme. The best time for translation of AM-HPCs was 12 days from the start of differentiation. When the AM-HPCs were transplanted into the liver failure mouse model, they settled in the damaged livers and differentiated into hepatocytes. Conclusion This study offers an efficient and xeno-free conditioned hepatic differentiation protocol and shows that AM-HPCs could be used as transplantable therapeutic materials. Thus, we suggest that AM-MSC-derived HPCs are promising cells for treating liver disease. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02470-y.

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GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells.

Publications

GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells.

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