In Vitro Identification And Bone Formation Of Human Embryonic Stem Cells Derived From Clonally Reconstituted Blastocyst

Objective: Nuclear cloning, which has also been accurately called somatic cell nuclear transfer (SCNT), involves the introduction of a nucleus from a donor cell into an enucleated oocyte to generate an embryo with a genetic makeup identical to that of the donor. In this study, an embryonic stem cell line----- (SCNT-rhESCs) was established from clonally reconstructed blastocyst by transferring human somatic nuclei derived from lymphocyte into healthy enucleated rabbit oocytes. The aim was to study the biological characteristics and osteogenic capacity of SCNT-rhESCs after subculture in vitro so as to explore the feasibility and application potential of SCNT-rhESCs-derived osteoblasts as seed cells for bone tissue engineering and clinical application. Methods: SCNT-rhESCs was established, cultured and expanded on MEF with reported protocols for hES cells before stem cell characteristic identification was carried out. After SCNT-rhESCs formed embryoid body in suspension culture for seven days, cells were transferred to cell culture dishes to allow attachment and cultured in mineral medium contained α-MEM with 15% refined fetal bovine serum and supplements as dexamethasone (1×10-8 mol/L),βglycerophosphate (10mmol/L) and ascorbic acid (50mg/L). Phenotype of cells after induction were identified and characterized with inverted microscope, Alizarin red staining, alkaline phosphatase staining by Gomori and immunofluorescence staining of typeⅠcollagen and osteocalcin (OCN) which show the main collagenous and noncollagenous components in bone matrix. Result: After continuous proliferation for more than 50 passages, SCNT-rhESCs displayed typical human ES cell morphology and maintained all human ES cell features, including positive staining for both ALP and cell surface markers including SSEA-3, SSES-4, TRA-1-60 and negative staining for mESCs surface maker SSEA-1 and were capable of differentiating into embryoid bodies in vitro. The cells maintained normal karyotypes and were proved genetically identical to the source cells. Mineralized cells from osteogenetic differentiation of SCNT-rhESCs can form bone nodules which could be clearly identified by Alizarin Red staining. Moreover, the major noncollagenous component of bone matrix osteocalcin, as well as the predominant collagenous component collagen type Ⅰ , were localized to the mineralized cells by immunohistochemistry and immunofluorescence detection. Conclusion: It has been demonstrated that SCNT-rhESCs have strong capacity for self-renew and tremendous potential for multilineage differentiation even after long term expansion and in vitro prolonged culture, and they could be induced to differentiate into mineralized osteoblasts under the influence of ascorbic acid,β-glycerophosphate and dexamethasone. The availability of human autologous embryonic stem cells ----SCNT-rhESCs and in vitro osteogenetic differentiation potential bring more light into cell-based regenerative medicine and thus minimized rejection and relative side effects of immunosuppressive medications and may provide a suitable seed cell source for bone tissue engineering as well as a suitable model for studying the molecular processes of osteoblastic development.