Generation of Multipotent Foregut Stem Cells from Human Pluripotent Stem Cells

Abstract

SummaryHuman pluripotent stem cells (hPSCs) could provide an infinite source of clinically relevant cells with potential applications in regenerative medicine. However, hPSC lines vary in their capacity to generate specialized cells, and the development of universal protocols for the production of tissue-specific cells remains a major challenge. Here, we have addressed this limitation for the endodermal lineage by developing a defined culture system to expand and differentiate human foregut stem cells (hFSCs) derived from hPSCs. hFSCs can self-renew while maintaining their capacity to differentiate into pancreatic and hepatic cells. Furthermore, near-homogenous populations of hFSCs can be obtained from hPSC lines which are normally refractory to endodermal differentiation. Therefore, hFSCs provide a unique approach to bypass variability between pluripotent lines in order to obtain a sustainable source of multipotent endoderm stem cells for basic studies and to produce a diversity of endodermal derivatives with a clinical value.

Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation

“…1A) [6][7][8][9][10][11]. The DE differentiation basal medium in the modified D'Amour (Mod-D'Amour) protocol and three respective modified protocols (Protocol 7, Protocol 4, and Combined Protocol 4 and 7) was R medium, which comprised 1% nonessential amino acids, 2 mM l-glutamine, 50 units/mL of penicillin, and 50 mg/mL of streptomycin (P4333; Sigma-Aldrich) in RPMI-1640 medium (R8758; Sigma-Aldrich) ( Fig.…”

“…Primer sequences used in qRT-PCR were as follows: ACTB (forward, 5¢-CATGTACGTTGCTATCCAGGC-3¢, and reverse, 5¢-CTC CTTAATGTCACGCACGAT-3¢), OCT4 (forward, 5¢-GGG AGATTGATAACTGGTGTGTT-3¢, and reverse, 5¢-GTGTA TATCCCAGGGTGATCCTC-3¢), NA-NOG (forward, 5¢-TTT GTGGGCCTGAAGAAAACT-3¢, and reverse, 5¢-AGGGC TGTCCTGAATAAGCAG-3¢), ZIC1 (forward, 5¢-CGTTCG GAGCACTATGCTG-3¢, and reverse, 5¢-TGTTGCACGAC TTTTTGGGGT-3¢), Brachyury (BRA) (forward, 5¢-TATG AGCCTCGAATCCACAT AGT-3¢, and reverse, 5¢-CCTCG TTCTGATAAGCAGT CAC-3¢), SOX7 (forward, 5¢-ACGC CGAGCTCAGCAA GAT-3¢, and reverse, 5¢-TCCACGTA CGGCCTCTTCTG-3¢), GSC (forward, 5¢-AACGCGGAGA AGTGGAACAAG-3¢, and reverse, 5¢-CTGTCCGAGTCCA AATCGC-3¢), SOX17 (forward, 5¢-GGCGCAGCAGAATC CAGA-3¢, and reverse, 5¢-CCACGACTTGCCCAGCAT-3¢), FOXA2 (forward, 5¢-GG GAGCGGTGAAGATGGA-3¢, and reverse, 5¢-TCATGTTG CTCACGGAGGAGTA-3¢), insulin (INS) (forward, 5¢-AA GAGGCCATCAAGCAGATCA-3¢, and reverse, 5¢-CAGG AGGCGCATCCACA-3¢), PDX1 (forward, 5¢-AAGTCTAC CAAAGCTCACGCG-3¢, and reverse, 5¢-GTAGGCGCC GCCTGC-3¢), chromogranin A (CHGA) (forward, 5¢-TAAA GGGGATACCGAGGTGATG-3¢, and reverse, 5¢-TCGGA GTGTCTCAAAACATTCC-3¢), AFP (forward, 5¢-CTTTG [6][7][8][9][10][11]. In the modified D'Amour (Mod-D'Amour), Chetty, Teo, Tahamtani 1, and Tahamtani 2 protocols, growth factors, small molecules, and supplements were added to R medium (comprising 1% nonessential amino acids, 2 mM l-glutamine, 50 units/mL of penicillin, and 50 mg/mL of streptomycin in RPMI-1640 medium) in a two-step manner.…”

“…Various growth factors, small molecules, supplements, and basal media were applied in these protocols (Fig. 1A) [6][7][8][9][10][11]. Two parameters were used for evaluation: the total live cell number and eGFP + %.…”

“…The generation of DE, the first step in PSC differentiation, is extremely critical to obtain mature and fully functional endoderm lineages [5]. Various protocols have been developed to generate DE in vitro by recapitulating in vivo embryogenesis, but their efficiency varies widely [6][7][8][9][10][11]. It has been reported that the initiating DE density is influential for the final differentiation yield [12].…”

Culture of hESC‐derived pancreatic progenitors in alginate‐based scaffolds

“…All the primers used in the manuscript were validated by analyzing the dissociation curve and tested on multiple samples. Importantly, they were also used on time course where the expression of the corresponding gene gradually increased as shown by Cho et al [5] and Hannan et al [40]. RT-PCR was performed on Applied Biosystems® 7500…”