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Paper Information

Journal:   INTERNATIONAL JOURNAL OF REPRODUCTIVE BIOMEDICINE (IRANIAN JOURNAL OF REPRODUCTIVE MEDICINE)   SPRING 2011 , Volume 9 , Number SUPPL 2; Page(s) 27 To 27.
 
Paper: 

DIFFERENTIATION OF GERM LINE CELLS FROM BONE MARROW MESENCHYMAL STEM CELLS BY GENETIC MODIFICATION AND SORTING OF TRANSFECTED STRA-8 + CELLS

 
 
Author(s):  MAZAHERI Z.*, MOVAHEDIN M., RAHBARIZADEH F., AMANPOUR S., RASSOULZADEGAN M.
 
* DEPARTMENT OF ANATOMY, FACULTY OF MEDICAL SCIENCE, TARBIAT MODARES UNIVERSITY, TEHRAN, IRAN
 
Abstract: 
Introduction: Bone Morphogenetic Protein (BMP) -4 has a crucial role on Primordial Germ Cells (PGCs) development in vivo which can promote stem cell differentiation to PG-like cells. Establishing a practical method for purification of BMSCs-derived PG-like cells could leads to a possible cell based therapy for infertility treatment and avoiding tumorogenic effects of Bone marrow Mesenchymal Stem Cells (BMSCs). In this study, we investigated the BMSCs differentiation potential to Germ line Cells during in vitro and in vivo, and purified them by genetic modification and Magnetic Activated Cell Sorter (MACS).
Materials and Methods: Passage 4 murine BMSCs were characterized by CD44 and CD45 markers and osteo-adipogenic differentiation.
Different doses of BMP4 (0, 0.01, 0.1, 1, 5, 25, 50 and 100ng/ml) were added toBMSCs for PGCs differentiation. Viability, proliferation rates and expression of Mvh gene were analyzed by immunocytochemistery and RT-qPCR. For PG like cells enrichment, four groups were designed: Control, RA 3
mM (RA), Co-Culture (Co-C) and Co-Culture with RA 3mM (Co-C+RA). The inserts were placed in a 24 well plate containing mitomycin-treated MEF cell line (STO) and cultured for two days. Stra8-CD4HAglo was constructed and used for purification of premeiotic differentiated cells by MACS. Expression of pluripotency (Oct-4, Nanog, c-Myc) genes and specific germ cell (Mvh, Piwil 2, Stra-8) genes in BMSCs, PG like cells, PG like cells enrichment, purified Stra-8+cells were analyzed by RT-qPCR. Induction of purified Stra-8+cells into Spermatogonial Stem like Cells (SSCs) were designed in the following groups: Control, Inducer Cocktails (IC) [basic Fibroblast Growth Factor (bFGF) +RA+Lukemia Inhibitory Factor (LIF)], Co-C and Co-C+IC. For enriching of Stra-8+ cells derived SSCs, RA+Sertoli cell as a Co-Culture were used during 14 days culture. For assessment of functionality of SS like cells, 10 5 cells were transplanted to azospermia mice. Also, 2×106 cells were inoculated in athymic mice and tumorigenicity of BMSCs and Stra-8+ cells derived SSCs were compared, too.
Results: CD44+ and CD45 - BMSCs were able to differentiate to osteo-adipogenic lineages. There were significant differences (p
£0.05) in viability, proliferation rates and expression of Mvh between 25ng/ml BMP4 with other groups so 25 ng/ml was the selective dose of BMP-4 for 4 days. RT-qPCR results indicated that there were a significant decrease (p£0.05) in oct-4 and c-Myc after MACS (stra-8+ cells). RT-qPCR results indicated that there was a significant up regulation (p£0.05) in Stra-8, Mvh, Piwil2 expression in Co-C+RA group comparing to control during enrichment of PG like cells. Tratuma formation of BMSCs after 47 days in balb C athymic mice was observed with 50% take rate. The outcomes of RT-qPCR showed that expression of Oct-4 and c-Myc were significantly increased (p≤0.05) in BMSCs comparing to Stra-8+ cell. The following studies on differentiation and enrichment of SS like cells based on objective of this Project are ongoing.
Conclusion: Our results suggest that BMSCs can differentiate to PG-like cells by BMP-4. Selection and purification of PG-like cells can be effective on down regulation of pluripotency genes and decrease tumorogenisity in cell based therapies.
 
Keyword(s): BMSCS, BMP-4, PGCS
 
References: 
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