Co‐culture of mesenchymal‐like stromal cells derived from human foreskin permits long term propagation and differentiation of human embryonic stem cells

Abstract

Among the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal-derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF-MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin-C treatment. Growth pattern, gene profiling (Oct-4, Nanog, Sox-2, Rex-1), cytoskeletal protein expression (vimentin, nestin) and tri-lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF-MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA-4, and negative for CD34, CD45, HLA-DR cell-surface markers and were found to exhibit BM-MSC-like characteristics. hESC lines co-cultured with HF-MSC feeders showed expression of expected pluripotent transcription factors Oct-4, Nanog, Sox-2, GDF-3, Rex-1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA-4, TRA-1-81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical-grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications.

Developmental and Functional Nature of Human iPSC Derived Motoneurons

“…Alternatively, hESCs can be cultured permanently on feeder cells from human tissues like human foreskin fibroblasts [127,147], human bone marrow stromal cells or human amniotic mesenchymal cells (hAMCs) [227]. Feeder cells support the survival and the preservation of an undifferentiated state via supply of some critical factors either to promote selfrenewal or to suppress differentiation [214,217].…”

Comparative global gene expression profile of human limbal stromal cells, bone marrow mesenchymal stromal cells, adipose-derived mesenchymal stromal cells and foreskin fibroblasts

“…Morphologically, these cells resembled the fibroblasts with a slight difference in their size and shape. The MSCs derived from various sources are known for their multipotential differentiation towards adipocytes, osteocytes and chondrocytes [42][43][44]. However, they differ in terms of growth factor, cytokine secretion and immunomodulatory properties [45].…”

The advantages of co-culture over mono cell culture in simulating in vivo environment

“…The techniques of co-culture involved varied but this technique has been performed for the purpose of analyzing "cultivation of embryonic stem cells on feeder cells", "examination of immune cell interactions", and "evaluation of mesenchymal-stromal interactions" [5,[35][36][37][38][39]. Direct co-culture can be performed in nearly all cell culture dishes, for instance by layering two cell types one on top of the other.…”