Int J Stem Cells.  2020 Nov;13(3):432-438. 10.15283/ijsc20074.

Human Placenta-Derived ECM Supports Tri-Lineage Differentiation of Human Induced Pluripotent Stem Cells

• Murchison AC
• Odanga JJ
• Treadwell ML
• Breathwaite EK
• Weaver JR
• Lee JB

• KMID: 2508917
• DOI: http://doi.org/10.15283/ijsc20074

Abstract

Human pluripotent stem cells (hPSCs) hold great promise for future applications in drug discovery and cell therapies. hPSC culture protocols require specific substrates and medium supplements to support cell expansion and lineage specific differentiation. The animal origin of these substrates is a severe limitation when considering the translation of hPSC derivatives to the clinic and in vitro disease modeling. The present study evaluates the use of a human placenta-derived extracellular matrix (ECM) hydrogel, HuGentra^Ⓡ , to support tri-lineage differentiation of human induced pluripotent stem cells (hiPSCs). Lineage-specific embryoid bodies (EBs) were plated onto three separate matrices, and differentiation efficiency was evaluated based on morphology, protein, and gene expression. HuGentra was found to support the differentiation of hiPSCs to all three germ layers: ectodermal, mesodermal, and endodermal lineages. hiPSCs differentiated into neurons, cardiomyocytes, and hepatocytes on HuGentra had similar morphology, protein, and gene expression compared to differentiation on Matrigel or other cell preferred matrices. HuGentra can be considered as a suitable human substrate for hiPSC differentiation.

Keyword

hiPSC; Tri-lineage; Differentiation; Human ECM

Figure

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  Fig. 1 Characterization of hiPSC lines and differentiation schematic. (a) Three hiPSC lines derived from adult dermal fibroblasts, neonatal foreskin fibroblasts, and adult human osteoblasts display the typical hiPSC morphology and stain for Tra-1-81. Representative images were taken 10×. (b) Each stage of differentiation is displayed with the respective media supplements used to induce that stage of the differentiation as indicated by the numbering. Representative images are shown of the three lineages during each stage of the differentiations. Representative images were taken 4×, 10×, and 20×.

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  Fig. 2 Characterization of hiPSC-derived neurons on matrices. (a) The ectodermal lineage marker A2B5 was expressed at a frequency of 12%±2.3% by dissociated EBs. (b) A2B5 expression following differentiation on gelatin, Matrigel, or HuGentra. *p<0.05 to hiPSCs differentiated on HuGentra versus Matrigel. Flow analysis for neuronal markers. (c) NES and (d) Tuj-1. (e) ICC of Tuj-1 and representative morphological images. Representative images were taken 20×. (f) The gene expression of OLIG2 in Matrigel and HuGentra normalized to expression on gelatin. Error bars represent standard deviation. *p<0.05 to hiPSCs differentiated on gelatin. n≥3.

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  Fig. 3 Characterization of hiPSC-derived cardiomyocytes on matrices. (a) Following 14 days of differentiation, contracting colonies were found on all matrices as indicated by arrows. Representative images were taken 4×. (b) No significant differences in the frequency of the contracting colonies relative to the total attached colonies when quantitated on day 30. (c) After dissociation, the cells were assessed for expression cTnT via flow cytometry. The level of cTnT was similar between all three matrices. (d) cTnT expression was confirmed with ICC on the three matrices. Representative images were taken 20×. (e) Relative gene expression of the mature cardiac marker, TNNT2, was also shown and normalized to expression of gelatin. Error bars represent standard deviation. n≥3.

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  Fig. 4 Characterization of hiPSC-derived hepatocytes on matrices. (a) The frequency of the attached EBs and the resulting colonies were observed and recorded as containing the morphology of hepatic cells. Representative images were taken 10× (top row) and 20× (bottom row). (b) A representative plate with labeled hepatic colonies in white and non-hepatic colonies in red. (c) The percentage of EBs containing the characteristic morphology were calculated as a percentage of the total attached EBs. (d) Quantification of ALB, HNF4, and AFP on the dissociated cells via flow cytometry showed no significant differences in expression efficiencies across the three matrices. (e) ICC analysis for ALB and AFP was shown on differentiated hepatic like cells. Representative images were taken 10×. (f) Gene expression of HNF4 and ALB on Collagen I and HuGentra normalized to expression on Matrigel. Error bars represent standard deviation. *p<0.05 to hiPSCs differentiated on Matrigel. n≥3.

Reference

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