Evaluating differentiation propensity of in-house derived human embryonic stem cell lines KIND-1 and KIND-2

Abstract

Human embryonic stem (hES) cells possess the ability to self-renew indefinitely and provide a potential source of differentiated progeny representing all three embryonic germ layers. Although hES cell lines share the expression of typical pluripotency markers, limited data is available regarding their differentiation capabilities. We have earlier reported the in-house derivation of two hES cell lines, KIND-1 and KIND-2 on human feeders. Here, we describe a comparative study carried out on both these cell lines to better understand the differentiation potential of KIND-1 and KIND-2 by gene expression analysis of representative gene transcripts reflecting pluripotency and the three germ layers viz. ectoderm, mesoderm, and endoderm. Gene expression analysis and immunolocalization studies were undertaken on (a) 7- and 14-d old embryoid bodies (EBs) (b) spontaneously differentiated cells from EBs, (c) cells derived from EBs under the influence of various growth factor treatments and (d) KIND-1 and KIND-2 cells co-cultured on mouse embryonic visceral endoderm-like feeder (END-2). Despite both the cell lines being XX, derived, passaged, and cultured similarly, KIND-1 exhibits preferential differentiation towards endodermal lineage whereas KIND-2 spontaneously forms beating cardiomyocytes. Perhaps the occurrence of discrete epigenetic profile in both the cell lines predisposes them to encompass different developmental potential in vitro. Our data provide evidence for existence of distinct differentiation propensity among hES cell lines and emphasizes the need to derive more hES cell lines for future regenerative medicine.

Polycomb group protein expression during differentiation of human embryonic stem cells into pancreatic lineage in vitro

“…For spontaneous differentiation, embryoid bodies (EBs) were generated from KIND1 cells as described earlier [38]. Three representative genes MAP2 (ectoderm lineage), HAND1 (mesoderm lineage) and MIXL (endoderm lineage) were studied by qRT-PCR from embryoid bodies harvested on day 7 and day 15.…”

“…For all the genes, the cycling parameters for PCR comprised of initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 sec, primer annealing at 62°C for 30 sec and extension at 72°C for 1 min using Dream Taq Polymerase as per manufacturer’s instructions (Thermo Scientific, IL, USA). The primers used for RT-PCR are as described earlier [38]. The PCR products were visualized by electrophoresis on 2% agarose gel, containing 0.5 μg/ml ethidium bromide (Bangalore Genei, Bangalore, India) and the product size was approximated using 100-bp DNA ladder (Bangalore Genei).…”

“…Embryoid bodies were prepared and cultured as described earlier [38] and EBs were harvested at Day 7 and Day 15. For plating, the EBs (7 days post suspension culture) were plated on gelatin coated dishes for 10 days in same media as used for EBs in suspension culture and later harvested for RNA extraction.…”

“…In the present study, in-house derived hES cell line KIND1 [30,38] was differentiated into pancreatic lineage under feeder-free culture system by two strategies viz spontaneous and directed differentiation. The expression of PcG protein transcript RING1A, RING1B, BMI1, CBX2, SUZ12, EZH2, EED and JARID2 was examined during differentiation of KIND1 and compared to the pattern in adult human pancreatic RNA.…”

Feeder & basic fibroblast growth factor-free culture of human embryonic stem cells

“…Upregulation of some endodermal genes, namely, FLT1, FOXA2 and GATA4 was observed in KIND-1 cells propagated in I-HFF-CM from group I when compared to positive control, but the difference was not significant between groups I and III in respect to positive control. This could be explained by the propensity of KIND-1 cells toward endodermal lineage33. Specific staining for ectodermal marker Tuj1was also confirmed by considering primary control (data not shown).…”