Abstract
The well documented source for adult multipotent stem cells is spermatogonial stem cells (SSCs) of mammalian testis. It is foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. SSCs isolation from mammalian testis is difficult because of their scarcity and the lack of well characterized cell surface markers. Thus, the isolation of SSCs is of great interest for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. CD9 is a surface marker expressed in mouse and rat male germline stem cells. In this study, CD9 positive SSCs were successfully isolated from the goat testis using enzymatic digestion followed by three step purification: Differential plating, Percoll discontinuous density gradient followed by Magnetic activated cell sorting (MACS). Percoll discontinuous density gradient showed significant differences in the percentage of CD9+ SSCs across individual fraction. The fraction 36% and 40% gave the highest percentage of CD9+ SSCs i.e. 82% ± 1.2 and 9.2% ± 1.3 respectively. Magnetic activated cell sorting of CD9+ cells in the magnetic fraction of goat testes was in the range of 15% - 18% which is upto threefolds. CD9+ SSCs were further recovered with appreciable efficiency after immunomagnetic isolation by using various bead: cells ratio in which 4:1 ratio gave the highest yield of 69.06 × 105 with 18% of CD9+ SSCs. Magnetic activated cell sorting using anti-CD9 antibodies provides an efficient and fast approach as compared to conventional approaches such as differential plating and percoll discontinuous density gradient for enrichment strategy for spermatogonial stem cells from goat testes for undertaking research on basic and applied reproductive biology.
Highlights
A great excitement and expectation in today’s biomedical world is the study of stem cells, owning to their ability to exist in an undifferentiated state and transforming into differing tissue types, depending on what the cells ambient are
After filtration of the cell suspension final cell concentration was found in the range of 5 - 8 × 106 cells/g testicular tissue and a mean of 85.5 × 106 cells were isolated from a single testis
The cell suspension was checked for CD9+ spermatogonial stem cells and found to be 5.8 ± 1.09 percent
Summary
A great excitement and expectation in today’s biomedical world is the study of stem cells, owning to their ability to exist in an undifferentiated state and transforming into differing tissue types, depending on what the cells ambient are. Several surface protein markers are commonly found on stem cells such as ITGA6 ( known as α6-integrin), ITGB1 ( known as β1-integrin) [4] and CD9 [5]. These markers may facilitate interactions between stem cells and their cognate niches [6]. Flow cytometry and immunohistochemical technique revealed the expression of CD9 on mouse and rat testis cells. These cells were selected with the help of anti-CD9 antibody which resulted in an enrichment of spermatogonial stem cells from in-
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