Characterisation of induced mesenchymal stem cells and human foetal bone cells–implications for disease physiology and regenerative medicine

Abstract

The paucity of skeletal stem cell (SSC) progenitors, commonly referred to as mesenchymal stem cells (MSCs), makes the determination of a new source for SSCs necessary. MSCs derived from induced pluripotent stem cells (iPSC-MSCs) are regarded as such a potential new source. The presented work looked to generate iPSC-MSCs and to characterise iPSC-MSCs alongside human foetal bone cells (HFBCs) isolated from human foetal femurs 7 – 17 weeks post conception (wpc). A fully characterised control iPSC line was differentiated into iPSC-MSCs using a protocol published by Chen et al., (2012). After initial culture in small molecule SB431542 supplemented medium for 10 days, in vitro culture aimed to mature iPSC-MSCs for six passages did not succeed in the generation of mature iPSC-MSCs with elongated fibroblastic morphology and increased cell proliferation.In order to provide a base for future comparison of iPSC-MSC with HFBCs, human foetal femurs and HFBCs were characterised. Histological staining of human foetal femurs demonstrated a cartilage template for future bone with a thin bone collar surrounding the diaphysis at 8 wpc and, with ongoing development, a transition from a cartilage template toward a bone matrix with invading blood vessels (17 wpc). HFBCs isolated from 7 – 17 wpc human foetal femurs did not show any differences in morphology or proliferation capacity between developmental weeks. A temporal increase in chondrogenic gene expression was observed in HFBCs between 7 – 17 wpc with type IX collagen (COL9A1, p < 0.001) and aggrecan (ACAN, p < 0.001) demonstrating significantly enhanced expression. Osteogenic gene expression for OCN, OPN, COL1A1 and ALPL did not show a significant increase or decrease in HFBCs between 7 – 17 wpc. Two osteogenic media were tested on HFBCs isolated from 8 and 14 wpc human foetal femurs as well as iPSC-MSCs: i) standard osteogenic differentiation medium containing dexamethasone, ascorbate-2-phosphate, 1,25-(OH)2-vitamin D3 (referred to as osteogenic background medium, oBG) and, ii) oBG supplemented with small molecules smoothened agonist (SAG) and (4-(4-methoxyphenyl)pyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide) (TH). Comparison of the osteogenic potential of HFBCs isolated from 8 and 14 wpc human foetal femurs using both osteogenic differentiation media showed an advantage of HFBCs isolated from 8 wpc human foetal femurs cultured in oBG + TH + SAG for 19 days. Global DNA methylation showed no major significant decrease in HFBCs between 7 and 17 wpc. Bisulfite pyrosequencing demonstrated significant demethylation in proximal COL9A1 promoter at CpGs within SOX/Sry-binding sites correlating significantly with increase COL9A1 gene expression. CpG sites within the vitamin D3 response element in the OCN promoter were found to be strongly demethylated. Analysis of the regulatory function of miRNAs associated with chondrogenesis and osteogenesis was not successful. The current studies demonstrated a regulatory role of DNA methylation on COL9A1 expression in HFBCs isolated from a unique and developmentally relevant skeletal tissue, human foetal femurs from 7 - 17 wpc, thus providing an overview of foetal bone development during the first two trimesters. Although comparison between iPSC-MSCs and HFBCs was ultimately not possible, the acquired characterised sample set of HFBCs will prove advantageous in future comparison of generated iPSC-MSCs and HFBCs isolated from different developmental stages.

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Identifiers

ORCID for Richard Oreffo: orcid.org/0000-0001-5995-6726

ORCID for Rahul Tare: orcid.org/0000-0001-8274-8837

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