Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Differentiation of Human-Induced Pluripotent Stem Cell-Derived Endocrine Progenitors to Islet-like Cells Using a Dialysis Suspension Culture System.

ABSTRACT: The production of functional islet-like cells from human-induced pluripotent stem cells (hiPSCs) is a promising strategy for the therapeutic use and disease modeling for type 1 diabetes. However, the production cost of islet-like cells is extremely high due to the use of expensive growth factors for differentiation. In a conventional culture method, growth factors and beneficial autocrine factors remaining in the culture medium are removed along with toxic metabolites during the medium change, and it limits the efficient utilization of those factors. In this study, we demonstrated that the dialysis suspension culture system is possible to reduce the usage of growth factors to one-third in the differentiation of hiPSC-derived endocrine progenitor cells to islet-like cells by reducing the medium change frequency with the refinement of the culture medium. Furthermore, the expression levels of hormone-secretion-related genes and the efficiency of differentiation were improved with the dialysis suspension culture system, possibly due to the retaining of autocrine factors. In addition, we confirmed several improvements required for the further study of the dialysis culture system. These findings showed the promising possibility of the dialysis suspension culture system for the low-cost production of islet-like cells.

SUBMITTER: Choi H

PROVIDER: S-EPMC8392085 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Endodermal differentiation of human induced pluripotent stem cells using simple dialysis culture system in suspension culture.

Project description:A differentiation of human induced pluripotent stem cells (hiPSCs) into definitive endoderm linage is required for a preparation of metabolic organ derived cells. The differentiation consumed high-priced cytokines and small molecules, which have hampered the manufacturability of differentiated cells. Although the cytokines and small molecules are remained or cells produce the autocrine factors, daily culture medium change should be proceeded to remove toxic metabolites generated from cells. In this study, we developed a simple dialysis culture system to refine the medium during definitive endodermal differentiation. We demonstrated that dialysis culture prevented cell damage to remove lactate. The hiPSCs cultured with dialysis also differentiated similarly as usual differentiation without dialysis even if they were not supplied Activin A for latter culture days in the differentiation. With this dialysis culture system, hiPSCs were differentiated into endodermal lineage with medium refinement and recycling and autocrine factors as well as cytokines, which may lead to reduce differentiation cost.

| S-EPMC6933453 | biostudies-literature

Differentiation of stem cell-derived pancreatic progenitors into insulin-secreting islet clusters in a multiwell-based static 3D culture system.

Project description:Orbital shaker-based suspension culture systems have been in widespread use for differentiating human pluripotent stem cell (hPSC)-derived pancreatic progenitors toward islet-like clusters during endocrine induction stages. However, reproducibility between experiments is hampered by variable degrees of cell loss in shaking cultures, which contributes to variable differentiation efficiencies. Here, we describe a 96-well-based static suspension culture method for differentiation of pancreatic progenitors into hPSC-islets. Compared with shaking culture, this static 3D culture system induces similar islet gene expression profiles during differentiation processes but significantly reduces cell loss and improves cell viability of endocrine clusters. This static culture method results in more reproducible and efficient generation of glucose-responsive, insulin-secreting hPSC-islets. The successful differentiation and well-to-well consistency in 96-well plates also provides a proof of principle that the static 3D culture system can serve as a platform for small-scale compound screening experiments as well as facilitating further protocol development.

| S-EPMC10261893 | biostudies-literature

A massive suspension culture system with metabolic purification for human pluripotent stem cell-derived cardiomyocytes.

Project description:Cardiac regenerative therapy with human pluripotent stem cells (hPSCs), such as human embryonic stem cells and induced pluripotent stem cells, has been hampered by the lack of efficient strategies for expanding functional cardiomyocytes (CMs) to clinically relevant numbers. The development of the massive suspension culture system (MSCS) has shed light on this critical issue, although it remains unclear how hPSCs could differentiate into functional CMs using a MSCS. The proliferative rate of differentiating hPSCs in the MSCS was equivalent to that in suspension cultures using nonadherent culture dishes, although the MSCS provided more homogeneous embryoid bodies (EBs), eventually reducing apoptosis. However, pluripotent markers such as Oct3/4 and Tra-1-60 were still expressed in EBs 2 weeks after differentiation, even in the MSCS. The remaining undifferentiated stem cells in such cultures could retain a strong potential for teratoma formation, which is the worst scenario for clinical applications of hPSC-derived CMs. The metabolic purification of CMs in glucose-depleted and lactate-enriched medium successfully eliminated the residual undifferentiated stem cells, resulting in a refined hPSC-derived CM population. In colony formation assays, no Tra-1-60-positive colonies appeared after purification. The nonpurified CMs in the MSCS produced teratomas at a rate of 60%. However, purified CMs never induced teratomas, and enriched CMs showed proper electrophysiological properties and calcium transients. Overall, the combination of a MSCS and metabolic selection is a highly effective and practical approach to purify and enrich massive numbers of functional CMs and provides an essential technique for cardiac regenerative therapy with hPSC-derived CMs.

| S-EPMC4250209 | biostudies-literature

Protocol development to further differentiate and transition stem cell-derived pancreatic progenitors from a monolayer into endocrine cells in suspension culture.

Project description:The generation of functional β-cells from human pluripotent stem cells (hPSCs) for cell replacement therapy and disease modeling of diabetes is being investigated by many groups. We have developed a protocol to harvest and aggregate hPSC-derived pancreatic progenitors generated using a commercially available kit into near uniform spheroids and to further differentiate the cells toward an endocrine cell fate in suspension culture. Using a static suspension culture platform, we could generate a high percentage of insulin-expressing, glucose-responsive cells. We identified FGF7 as a soluble factor promoting aggregate survival with no inhibitory effect on endocrine gene expression. Notch inhibition of pancreatic progenitor cells during aggregation improved endocrine cell induction in vitro and improved graft function following implantation and further differentiation in mice. Thus we provide an approach to promote endocrine formation from kit-derived pancreatic progenitors, either through extended culture or post implant.

| S-EPMC10235054 | biostudies-literature

Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells in scalable suspension culture.

Project description:To harness the potential of human pluripotent stem cells (hPSCs), an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion). Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity) was enabled that were directly applicable to bioartificial cardiac tissue formation.

| S-EPMC4264033 | biostudies-literature

Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells.

Project description:To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2×10(9) CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

| S-EPMC4600677 | biostudies-literature

Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture.

Project description:Endothelial cells (ECs) are involved in a variety of cellular responses. As multifunctional components of vascular structures, endothelial (progenitor) cells have been utilized in cellular therapies and are required as an important cellular component of engineered tissue constructs and in vitro disease models. Although primary ECs from different sources are readily isolated and expanded, cell quantity and quality in terms of functionality and karyotype stability is limited. ECs derived from human induced pluripotent stem cells (hiPSCs) represent an alternative and potentially superior cell source, but traditional culture approaches and 2D differentiation protocols hardly allow for production of large cell numbers. Aiming at the production of ECs, we have developed a robust approach for efficient endothelial differentiation of hiPSCs in scalable suspension culture. The established protocol results in relevant numbers of ECs for regenerative approaches and industrial applications that show in vitro proliferation capacity and a high degree of chromosomal stability.

| S-EPMC5995343 | biostudies-literature

Generation of Human iMelanocytes from Induced Pluripotent Stem Cells through a Suspension Culture System.

Project description:Melanocytes, derived from neural crest cells, are involved in melanin production. This protocol describes a method to generate induced melanocytes (iMelanocytes) from human induced pluripotent stem cells (iPSCs) using a suspension culture system, which considerably improves the differentiation efficiency. The most critical parts of this protocol are the selection of a reliable iPSC line with strong potential to differentiate into melanocytes and their stemness maintenance. For complete information on the use and generation of this protocol, please refer to our Cell Reports article, Liu el al. (2019).

| S-EPMC7580196 | biostudies-literature

Bioreactor Suspension Culture: Differentiation and Production of Cardiomyocyte Spheroids From Human Induced Pluripotent Stem Cells.

Project description:Human induced-pluripotent stem cells (hiPSCs) can be efficiently differentiated into cardiomyocytes (hiPSC-CMs) via the GiWi method, which uses small-molecule inhibitors of glycogen synthase kinase (GSK) and tankyrase to first activate and then suppress Wnt signaling. However, this method is typically conducted in 6-well culture plates with two-dimensional (2D) cell sheets, and consequently, cannot be easily scaled to produce the large numbers of hiPSC-CMs needed for clinical applications. Cell suspensions are more suitable than 2D systems for commercial biomanufacturing, and suspended hiPSCs form free-floating aggregates (i.e., spheroids) that can also be differentiated into hiPSC-CMs. Here, we introduce a protocol for differentiating suspensions of hiPSC spheroids into cardiomyocytes that is based on the GiWi method. After optimization based on cardiac troponin T staining, the purity of hiPSC-CMs differentiated via our novel protocol exceeded 98% with yields of about 1.5 million hiPSC-CMs/mL and less between-batch purity variability than hiPSC-CMs produced in 2D cultures; furthermore, the culture volume could be increased ∼10-fold to 30 mL with no need for re-optimization, which suggests that this method can serve as a framework for large-scale hiPSC-CM production.

| S-EPMC8226172 | biostudies-literature

Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors.

Project description:Human induced pluripotent stem cells (hiPSCs) are a potential source for cell therapy of Duchenne muscular dystrophy. To reliably obtain skeletal muscle progenitors from hiPSCs, we treated hiPS cells with a Wnt activator, CHIR-99021 and a BMP receptor inhibitor, LDN-193189, and then induced skeletal muscle cells using a previously reported sphere-based culture. This protocol greatly improved sphere formation efficiency and stably induced the differentiation of myogenic cells from hiPS cells generated from both healthy donors and a patient with congenital myasthenic syndrome. hiPSC-derived myogenic progenitors were enriched in the CD57(-) CD108(-) CD271(+) ERBB3(+) cell fraction, and their differentiation was greatly promoted by TGF-β inhibitors. TGF-β inhibitors down-regulated the NFIX transcription factor, and NFIX short hairpin RNA (shRNA) improved the differentiation of iPS cell-derived myogenic progenitors. These results suggest that NFIX inhibited differentiation of myogenic progenitors. hiPSC-derived myogenic cells differentiated into myofibers in muscles of NSG-mdx 4Cv mice after direct transplantation. Our results indicate that our new muscle induction protocol is useful for cell therapy of muscular dystrophies.

| S-EPMC5920060 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data