BMP9 induces osteogenesis and adipogenesis in the immortalized human cranial suture progenitors from the patent sutures of craniosynostosis patients

Abstract

The cranial suture complex is a heterogeneous tissue consisting of osteogenic progenitor cells and mesenchymal stem cells (MSCs) from bone marrow and suture mesenchyme. The fusion of cranial sutures is a highly coordinated and tightly regulated process during development. Craniosynostosis is a congenital malformation caused by premature fusion of cranial sutures. While the progenitor cells derived from the cranial suture complex should prove valuable for studying the molecular mechanisms underlying suture development and pathogenic premature suture fusion, primary human cranial suture progenitors (SuPs) have limited life span and gradually lose osteoblastic ability over passages. To overcome technical challenges in maintaining sufficient and long‐term culture of SuPs for suture biology studies, we establish and characterize the reversibly immortalized human cranial suture progenitors (iSuPs). Using a reversible immortalization system expressing SV40 T flanked with FRT sites, we demonstrate that primary human suture progenitor cells derived from the patent sutures of craniosynostosis patients can be efficiently immortalized. The iSuPs maintain long‐term proliferative activity, express most of the consensus MSC markers and can differentiate into osteogenic and adipogenic lineages upon BMP9 stimulation in vitro and in vivo. The removal of SV40 T antigen by FLP recombinase results in a decrease in cell proliferation and an increase in the endogenous osteogenic and adipogenic capability in the iSuPs. Therefore, the iSuPs should be a valuable resource to study suture development, intramembranous ossification and the pathogenesis of craniosynostosis, as well as to explore cranial bone tissue engineering.

Usefulness of Mesenchymal Cell Lines for Bone and Cartilage Regeneration Research

“…A reduction of osteogenic potential was also observed in some MSC cell lines immortalized with hTERT and SV40LT, such as hASCs-TS [85]. Song et al (2017) also pointed out that reversibly immortalized iSuPs (derived from coronal sutures) presented increased osteogenic potential when the immortalization gene SV40LT was removed [50]. Shu et al (2018) have proposed that MSCs with higher proliferative activity, such as SV40LT-transduced MSCs, may need a longer time to differentiate towards the osteogenic lineage [49].…”

“…Only 23 iMSC lines out of the 38 included in this review have been submitted for analysis of their chondrogenic potential. Two iMSC lines did not show any chondrogenic potential when assessed in two-dimensional culture: UE6E7T-2, derived from bone marrow and transduced with E6/E7 and hTERT [99]; and iSuPS, derived from coronal sutures and transduced with SV40LT [50]. Three other iMSC lines (SCP-1, BMA13H, and 3 Hits hMPC), all derived from bone marrow-MSCs, showed reduced chondrogenic potential in comparison to primary cells; all these iMSC lines were chondrogenically induced in three-dimensional culture and contained hTERT as immortalization gene, and two of them were incompletely immortalized [14,74,101].…”

“…Positive for ABS and ColII immunostaining in 3D alginate and PGA/PLLA scaffolds [17] Not tested/shown iUC-MSCs Positive for Runx2 and Osteocalcin upregulation [49] Positive for Sox9 upregulation [49] Positive for PPARγ upregulation [49] iSuPs Positive for ARS (increased if SV40LT is removed) and osteogenesis-related genes upregulation [50] No chondrogenic differentiation potential was found (also not shown) [50] Positive for OROS (increased if SV40LT is removed) [50] TAg cells…”

“…HPV E6/E7 proteins work in a similar manner, with p53 being the principal target of E6 and Rb being degraded via the ubiquitin proteasome pathway by the action of E7 [109]. SV40LT transduction has been employed for immortalizing MSCs derived from bone marrow from young and old donors [45,47], umbilical cord [49], cranial periosteum [51], coronal sutures [50], dental follicle [52], peripheral blood [48], and osteoarthritic cartilage [41]. SV40LT expression increases the lifespan of MSCs and usually raises its proliferation rate as well, but it has also been observed that this increased lifespan is not unlimited.…”

Niclosamide Exhibits Potent Anticancer Activity and Synergizes with Sorafenib in Human Renal Cell Cancer Cells

“…Crystal violet staining assay was conducted as described [43,44]. Briefly, subconfluent A498 and Caki-1 cells were treated with varied concentrations of Niclosamide or DMSO control.…”