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<DIV id="id_1">
<P class="p0 ft0"><A href="http://www.nexusacademicpublishers.com/journal/4">Advances in Animal and Veterinary Sciences. </A>2 (3): 183 – 187</P>
<P class="p1 ft1"><A href="http://dx.doi.org/10.14737/journal.aavs/2014/2.3.183.187">http://dx.doi.org/10.14737/journal.aavs/2014/2.3.183.187</A></P>
<P class="p2 ft2">Research Article</P>
<P class="p3 ft3">Isolation and Enrichment of Spermatogonial Stem Cells with MACS in Cattle Calves by Autopsy in Vitro</P>
<P class="p4 ft4">Jitendra K Verma, Ankur Sharma, DK Mandal, SK Tyagi, AK Mathur, Mahesh Kumar*</P>
<P class="p5 ft5">NAIP on Stem Cell, Semen freezing Laboratory, Project Directorate on Cattle, Meerut (U.P.) *Corresponding author: mkpdcattle@gmail.com</P>
<TABLE cellpadding=0 cellspacing=0 class="t0">
<TR>
	<TD class="tr0 td0"><P class="p6 ft6">ARTICLE HISTORY</P></TD>
	<TD class="tr0 td1"><P class="p7 ft7">ABSTRACT</P></TD>
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<DIV id="id_2">
<DIV id="id_2_1">
<P class="p0 ft0">Received: <NOBR>2014–02–21</NOBR></P>
<P class="p0 ft8">Revised: <NOBR>2014–03–04</NOBR></P>
<P class="p1 ft5">Accepted: <NOBR>2013–03–04</NOBR></P>
<P class="p8 ft9">Key Words:</P>
<P class="p9 ft5">Spermatogonial stem cell, Magnatic activated cell sorting, CD9, FITC, DBA</P>
</DIV>
<DIV id="id_2_2">
<P class="p10 ft0">Spermatognial stem cells (SSCs) are unipotent adult stem cells which form the foundation of spermatogenesis for continuous production of sperms throughout the animal life. Due to their rare availability in the testis, we decided to target CD9 surface marker and magnetic– activated cells sorting MACS for enrichment of testicular cell suspension with the cells of stem cell properties. We have successfully characterized the isolated and enriched SSCs population with the <A href="http://en.wikipedia.org/wiki/Fluorescein_isothiocyanate">Fluorescein isothiocyanate </A>FITC tagged secondary antibody under fluorescent microscope at 500 nm wavelength. Isolated Cells could form the colonies resembling to SSCs colonies after one week of culturing in DMEM along with 10% ABS at 37<SPAN class="ft10">0</SPAN>C temperature, 5% CO<SPAN class="ft11">2 </SPAN>and 90%NO<SPAN class="ft11">2</SPAN>. <NOBR><SPAN class="ft12">α</SPAN>–<SPAN class="ft13">N</SPAN>–acetylgalactosamine</NOBR> (GalNAc) is reported to be present in SSCs. We have used FITC– DBA lectin to characterize the SSCs colonies after 14 days of culture. Our findings suggest the use of CD9 surface marker and MACS technology for the enrichment of SSCs and DBA for characterizing SSC colonies.</P>
<P class="p11 ft14">All copyrights reserved to Nexus® academic publishers</P>
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<DIV id="id_3">
<P class="p0 ft5"><SPAN class="ft15">ARTICLE CITATION</SPAN>: Verma JK, Sharma A, Mandal DK, Tyagi SK, Mathur AK, Kumar M (2014). Isolation and enrichment of spermatogonial stem cells with MACS in cattle calves by autopsy in vitro. <SPAN class="ft16">Adv. Anim. Vet. Sci. </SPAN>2(3): 183 – 187.</P>
</DIV>
<DIV id="id_4">
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<P class="p0 ft9">INTRODUCTION</P>
<P class="p12 ft0">Stem cells have the potential to revolutionize tissue regeneration and engineering. The hematopoietic stem cells were the first stem cells to be prospectively identified. Since then, an ever increasing number of new types of stem cells, including embryonic stem cells, spermatogonial stem cells (SSCs) and cancer stem cells, have been identified and characterized. They can be useful in understanding general cellular processes in, e.g., embryogenesis, organogenesis, cancer or ageing, but also as a vehicle for the generation of transgenic animals for functional gene analysis and disease models (Bosio et al., 2009).</P>
<P class="p13 ft5">Spermatogonial stem cells (SSCs) are unipotent adult stem cells responsible for the maintenance of the spermatogenesis throughout the entire life of the male (Bosio et al., 2009). They are the only germ line stem cells in adult animals. The SSC may choose to <NOBR>self–renewal</NOBR> or generate a daughter cell committed to differentiation (Van der and Weiss, 2000). They form the foundation of spermatogenesis and are required for the continuous production of sperm through a balance between SSC self– renewal and differentiation in adult testis (Hofmann, 2008). Studies of SSCs are complicated because these cells are very few in number (Aponte et al., 2005). This provides an opportunity for methods like <NOBR>magnetic–activated</NOBR> cells sorting (MACS) and fluorescence activated cells sorting (FACS) which can enrich whole cell suspension with SSCs. SSCs provide the foundation for spermatogenesis throughout the life of male animals (De Rooij and Russell, 2000).</P>
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<P class="p14 ft0"><NOBR>Kanatsu–Shinohara</NOBR> et al. (2003) reported the isolation and <NOBR>long–term</NOBR> culture system for SSCs. Development of this culture systems provided possibilities to study SSCs <SPAN class="ft13">in vitro</SPAN>. However, the percentage of SSCs in GS cell culture was unexpectedly low, and only <NOBR>0.04–1.26%</NOBR> could colonize and reconstitute seminiferous tubules of infertile animals <NOBR>(Kanatsu–Shinohara</NOBR> et al., 2005).</P>
<P class="p15 ft0">For isolation of spermatogonial stem cells (SSCs) and undifferentiated spermatogonia, Dym et al. (2009) studied certain markers including CD9 and <NOBR>OCT–4</NOBR> in mammals. He et al. (2010) isolated human spermatogonial cells by MACS, using GPR125 as a specific marker for spermatogonial cells in mouse. Recently Piravar et al. (2013) have reported the efficient use of MACS using various surface markers such as CD 9, <NOBR>OCT–4</NOBR> etc. to enrich and culture the SSCs <SPAN class="ft13">in vitro </SPAN>without feeder cell lines. FACS and MACS are the techniques which had been used in past to enrich the SSCs population. <A href="http://www.ncbi.nlm.nih.gov/pubmed?term=Zhao%20XX%5BAuthor%5D&cauthor=true&cauthor_uid=12196708">Zhao </A>et al. (2002) and <A href="http://www.ncbi.nlm.nih.gov/pubmed?term=de%20Wynter%20EA%5BAuthor%5D&cauthor=true&cauthor_uid=8528102">De Wynter </A>et al. (1995) also have used MACS to purify and enrich of stem cells targeting various surface markers and found remarkable success in their experiment.</P>
<P class="p16 ft5">Lectins proteins such as peanut agglutinin (PNA), which recognizes <NOBR>D–(+)–</NOBR> galactose, has been used to fractionate bovine hematopoietic stem cells and also in the purification and identification of adult neural stem cells (Salner et al., 1982). In this experiment, we describe Dolichos biflorus agglutinin (DBA), which recognizes <NOBR><SPAN class="ft17">α</SPAN>–<SPAN class="ft16">N</SPAN>–</NOBR> acetylgalactosamine (GalNAc) as being highly reactive towards bovine SSCs. More recently Nash et al. (2007) reported the utility for DBA in the characterization of</P>
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<TR>
	<TD class="tr1 td2"><P class="p7 ft18">Verma et al (2014). Spermatogonial Stem Cells with MACS in Cattle</P></TD>
	<TD class="tr1 td3"><P class="p17 ft19">183</P></TD>
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<P class="p0 ft5">ISSN: <NOBR>2307–8316</NOBR> (Online); ISSN: <NOBR>2309–3331</NOBR> (Print)</P>
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<P class="p0 ft21"><A href="http://www.nexusacademicpublishers.com/journal/4">Advances in Animal and Veterinary Sciences. </A>2 (3): 183 – 187 <A href="http://dx.doi.org/10.14737/journal.aavs/2014/2.3.183.187"><SPAN class="ft20">http://dx.doi.org/10.14737/journal.aavs/2014/2.3.183.187</SPAN></A></P>
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<P class="p18 ft22">pluripotent cells because it can be used as a nondestructive marker and as a reliable readout for initial differentiation events, at a level of temporal resolution that was not previously possible.</P>
<P class="p19 ft5">A number of different protocols have been published for the isolation and enrichment of stem cells including selective culturing, immunopanning, flow cytometric sorting, or magnetic sorting. Based on the available literature, we hypothesis that CD9 can serve as a good surface marker for enrichment by MACS. In this article, we discussed not only about the isolation procedures of SSCs but also about their enrichment with the help of magnetic assisted cell sorting because they are present only in small number in the testes. The colonies formed from SSCs were characterized by FITC tagged DBA lectin protein.</P>
<P class="p4 ft14">MATERIAL AND METHODS</P>
<P class="p0 ft23">Collection of Testes</P>
<P class="p18 ft22">The testes were collected from the recently dead cattle calves <NOBR>(6–12</NOBR> months old) by autopsy. The testes were brought to laboratory in the phosphate buffer saline supplemented with penicillin and streptomycin, under cold conditions within 2hrs.</P>
<P class="p20 ft24">Isolation of Spermatogonial Stem Cells (SSCs)</P>
<P class="p21 ft5">Visible connective tissue was first removed from the testis and then testes were washed three times in 0.9 % normal saline containing antibiotics and tunica albuginea was removed. About <NOBR>10–15</NOBR> g of testicular sample was processed for SSCs isolation. The chopped testicular samples were processed for enzymatic digestion as described by Van Pelt et al. (1996) with minor modifications. In brief, the testicular sample was chopped and minced into tiny pieces and later suspended in DPBS solution for washing. At last,</P>
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<P class="p22 ft0">the minced sample was suspended and washed with DMEM containing 14 mol NaHCO3/L, 4 mol Lglutamine/L, 1 ml/100 ml <NOBR>single–strength</NOBR> <NOBR>non–essential</NOBR> amino acids, 1000 IU/ml– 100 μg/ml <NOBR>penicillin–streptomycin</NOBR> and 15 mol/L Hepes. The Sample was then suspended in digestion media [DMEM containing 1 mg/ml collagenase and 5 μg/ml DNase I (all from Sigma– Aldrich, St. Louis, MO, USA)] and incubated at 37°C for 45 min in a shaking water bath operated at 140 cycles/min at 37<SPAN class="ft10">0</SPAN>C. After three washing in same medium the most of the interstitial cells were removed in supernatant. Seminiferous cord fragments were then given second digestion with 1 mg/ml collagenase, 1 mg/ ml hyaluronidase and 5 μg/ml DNase for 30 min. The sample was processed for third digestion with collagenase (0.5mg/ml), hyaluronidase (0.5mg/ml) and DNase (2.5 μg/ml) for thirty minutes. All the digestion steps were performed in water bath <NOBR>@120–140</NOBR> cycles/minute at 37<SPAN class="ft10">0</SPAN>C. The dispersed cells were transferred into a beaker kept in ice and then washed twice in DMEM medium supplemented with 10 % (v/v) adult bovine serum (ABS) to stop the enzymatic digestion. The cells were separated from the remaining tubule fragments by centrifugation at 500g for 5 min and after filtration through 100 and 60 μm nylon filters (Millipore</P>
<P class="p23 ft25">Corp., Bedford, MA, USA), the cells were pelleted. The isolated cells contained mixed population of cells of both the somatic cell population and stem cells.</P>
<P class="p24 ft26">Enrichment of Spermatogonial Stem Cells (SSCs) with</P>
<P class="p0 ft24">MACS</P>
<P class="p25 ft25">After enzymatic digestion, one part of the suspended cells at the concentration of <NOBR>1–2</NOBR> x 10<SPAN class="ft27">6 </SPAN>cells/ml was seeded in each well of culture plates. DMEM along with ABS (10%) was used as culture medium.</P>
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<TR>
	<TD class="tr2 td4"><P class="p26 ft5">Figure 1a</P></TD>
	<TD class="tr2 td5"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr2 td4"><P class="p27 ft5">Figure 1b</P></TD>
	<TD class="tr2 td6"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr2 td4"><P class="p28 ft5">Figure 1c</P></TD>
</TR>
<TR>
	<TD class="tr3 td7"><P class="p7 ft29">&nbsp;</P></TD>
	<TD class="tr4 td5"><P class="p7 ft30">&nbsp;</P></TD>
	<TD class="tr3 td7"><P class="p7 ft29">&nbsp;</P></TD>
	<TD class="tr4 td6"><P class="p7 ft30">&nbsp;</P></TD>
	<TD class="tr3 td7"><P class="p7 ft29">&nbsp;</P></TD>
</TR>
<TR>
	<TD class="tr5 td8"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr6 td9"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr5 td10"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr6 td11"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr5 td10"><P class="p7 ft28">&nbsp;</P></TD>
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	<TD class="tr0 td12"><P class="p7 ft5">Figure 1d</P></TD>
	<TD class="tr0 td13"><P class="p7 ft5">Figure 1e</P></TD>
	<TD class="tr0 td14"><P class="p7 ft31">Figure 1f</P></TD>
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<P class="p29 ft5">Figure 1a: 100 X live (color less) and dead (blue) in single cells suspension; Figure 1b: 100 X 0 day cultured cells where the isolated viable testicular cells are seeded; Figure 1c: 100 X Microbeads attached with CD9+ SSC; Figure 1d: 100 X Microbeads detached from CD9+SSCs; Figure 1e: 100 X SSCs bound with microbeads attached with FITC Tagged Secondary antibody under light microscopy; Figure 1f: 100 X SSCs bound with Microbeads attached with FITC Tagged Secondary antibody (green) under fluorescent Microscope</P>
<TABLE cellpadding=0 cellspacing=0 class="t4">
<TR>
	<TD class="tr1 td2"><P class="p7 ft18">Verma et al (2014). Spermatogonial Stem Cells with MACS in Cattle</P></TD>
	<TD class="tr1 td3"><P class="p17 ft19">184</P></TD>
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<P class="p0 ft5">ISSN: <NOBR>2307–8316</NOBR> (Online); ISSN: <NOBR>2309–3331</NOBR> (Print)</P>
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<P class="p0 ft0"><A href="http://www.nexusacademicpublishers.com/journal/4">Advances in Animal and Veterinary Sciences. </A>2 (3): 183 – 187</P>
<P class="p1 ft1"><A href="http://dx.doi.org/10.14737/journal.aavs/2014/2.3.183.187">http://dx.doi.org/10.14737/journal.aavs/2014/2.3.183.187</A></P>
<P class="p30 ft5">Figure 2a</P>
<TABLE cellpadding=0 cellspacing=0 class="t5">
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	<TD class="tr7 td15"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr7 td16"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr7 td17"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr8 td18"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr7 td19"><P class="p7 ft28">&nbsp;</P></TD>
</TR>
<TR>
	<TD class="tr9 td20"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr9 td21"><P class="p31 ft5">Figure 2b</P></TD>
	<TD class="tr9 td22"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr9 td23"><P class="p7 ft28">&nbsp;</P></TD>
	<TD class="tr9 td24"><P class="p32 ft5">Figure 2c</P></TD>
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	<TD class="tr10 td20"><P class="p7 ft32">&nbsp;</P></TD>
	<TD class="tr10 td21"><P class="p7 ft32">&nbsp;</P></TD>
	<TD class="tr10 td22"><P class="p7 ft32">&nbsp;</P></TD>
	<TD class="tr10 td23"><P class="p7 ft32">&nbsp;</P></TD>
	<TD class="tr10 td24"><P class="p7 ft32">&nbsp;</P></TD>
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<P class="p33 ft5">Figure 3a: Relative Difference in number of unsorted and sorted SSCs; Figure 2b and 2c: 100 X immunostaining of 14 days old SSCs colonies probed with FITC tagged DBA lectine protein under light (figure 2b) and florescent (figure 2c) microscope</P>
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<P class="p14 ft0">The culture conditions were kept as 37<SPAN class="ft10">0</SPAN>C temperature, 5% CO<SPAN class="ft11">2 </SPAN>and 90%N<SPAN class="ft11">2</SPAN>. While another part of the suspended cells were processed for magnate assisted cell sorting (MACS) as it was described by Bosio et al. (2009) with slight modifications. <NOBR>CD–9</NOBR> cell surface marker was targeted to enrich the cell suspensions with stem cells. The CD9+ SSCs were obtained using the MACS kit according to the manufacturer's instructions (Invitogen). The cells were incubated with 100 μl microbeads directly conjugated to mouse monoclonal <NOBR>anti–human</NOBR> CD 9 antibody at 4˚C for 30 min. Subsequently, the suspended cells were added to a MACS column that was placed in the magnetic field of a MACS separator (Invitrogen). The labeled CD9 positive cells were retained on the column and the unlabeled cells were eluted; when the column was removed from the magnetic field, the magnetically retained CD9+ cells were collected as positively selected cells for further research (He et al., 2011). Microbeads were detached from purified cells by incubating in Dulbecco modified minimum essential medium (DMEM) + 5% fetal calf serum (FCS) for 6hrs at 37<SPAN class="ft10">0</SPAN>C. These cells were washed with DMEM + 10% ABS (adult bovine serum) three times.</P>
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<P class="p0 ft24">Phenotypic Characterization of SSCs Sorted by MACS</P>
<P class="p34 ft0"><NOBR>CD–9</NOBR> cell surface marker was targeted to enrich the cell suspensions with stem cells. Primary antibodies (0.5µg) against <NOBR>CD–9</NOBR> (Invitrogen, AHS0902) surface markers were added to the cell suspension (1 x 10<SPAN class="ft10">6</SPAN>/ml) and incubated for <NOBR>10–15</NOBR> minutes at <NOBR>2–8<SPAN class="ft10">0</SPAN>C.</NOBR> After the incubation, the cells were washed three times with <NOBR>buffer–1</NOBR> (PBS+1% BSA, <NOBR>pH–7.4)</NOBR> and 1 x 10<SPAN class="ft10">6 </SPAN>(25µl) microbeads bound with FITC tagged secondary antibody (Invitrogen, 110.31) were added. The cell suspension along with buffer and microbeads attached with FITC tagged secondary antibody was incubated for 30 min with continuous rotation @ 10 rotation per min at <NOBR>2–8<SPAN class="ft10">0</SPAN>C.</NOBR></P>
<P class="p4 ft26">Phenotypic Characterization of 14 Days Old Colonies</P>
<P class="p0 ft24">with FITC Tagged DBA Lectin</P>
<P class="p34 ft5"><NOBR>Fluorescent–labeled</NOBR> lectins were purchased from Invitrogen. Colonies formed after 14 days of culturing SSCs were stained with <A href="http://en.wikipedia.org/wiki/Fluorescein_isothiocyanate">Fluorescein isothiocyanate </A>(FITC) tagged lectin DBA with slight modifications from methods used by Nash et al. (2007). SSCs colonies were fixed with 4 % para formaldehyde for 30 minutes at room temperature after five times washing with Dulbeccos modified phosphate buffer saline (DPBS). Washing with DPBS (five</P>
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<TR>
	<TD class="tr1 td2"><P class="p7 ft18">Verma et al (2014). Spermatogonial Stem Cells with MACS in Cattle</P></TD>
	<TD class="tr1 td3"><P class="p17 ft19">185</P></TD>
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<P class="p0 ft5">ISSN: <NOBR>2307–8316</NOBR> (Online); ISSN: <NOBR>2309–3331</NOBR> (Print)</P>
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<P class="p0 ft21"><A href="http://www.nexusacademicpublishers.com/journal/4">Advances in Animal and Veterinary Sciences. </A>2 (3): 183 – 187 <A href="http://dx.doi.org/10.14737/journal.aavs/2014/2.3.183.187"><SPAN class="ft20">http://dx.doi.org/10.14737/journal.aavs/2014/2.3.183.187</SPAN></A></P>
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<P class="p12 ft5">minutes each) was repeated three times after fixing the colonies. The SSCs colonies were incubated in <NOBR>triton–x–100</NOBR> (0.1%) for 30 minutes at room temperature. Colonies were washed again three times with DPBS. Non specific sites were blocked with goat serum (4% in DPBS) at room temperature for 30 minutes. Blocking solution was decanted and <NOBR>FITC–DBA</NOBR> was added to the cultured colonies for <NOBR>1–1.5</NOBR> hr at room temperature in dark. Just before observation under fluorescent microscope at 500nm the colonies were washed three times with DPBS.</P>
<P class="p4 ft14">RESULTS</P>
<P class="p0 ft24">Isolation of SSCs</P>
<P class="p35 ft5">Cell viability (Figure 1a) was found to be more that 80% in the single cell suspension after enzymatic digestion while the whole cell concentration was found to be <NOBR>1–2</NOBR> x 10<SPAN class="ft10">7</SPAN>/ml Single cell suspension was obtained without any undesired material from testis and seeded in each well at the concentration of <NOBR>1–2</NOBR> x 10<SPAN class="ft10">6</SPAN>/ml (Figure 1b).</P>
<P class="p0 ft24">Enrichment of SSCs with MACS</P>
<P class="p18 ft25">The micro beads could attach to CD9+ SSCs (Figure 1c). CD9+ SSCs were increased apparently in number after being sorted with MACS (Figure 2a).</P>
<P class="p0 ft24">Detachment of Microbeads from SSCs</P>
<P class="p12 ft5">The microbeads which were bound with CD9+ SSCs were removed completely for further culture (Figure 1d).</P>
<P class="p0 ft24">Phenotypic Characterization of SSCs with FITC Dye</P>
<P class="p18 ft22">The cells which were bound with microbeads were further processed for phenotypic characterization using FITC tagged secondary antibodies. They were photographed under light microscope and florescent microscope and green color florescent was obtained from the same cells where the microbeads were bound (Figure 1e and Figure 1f respectively).</P>
<P class="p36 ft24">SSCs Colonies and their Characterization with DBA</P>
<P class="p37 ft33">The MACS sorted CD9+ SSCs formed colonies which were characterized further by FITC tagged DBA lactin protein (Figure 2b Figure 2c).</P>
<P class="p38 ft9">DISCUSSION</P>
<P class="p39 ft0">The number of stem cells in testes is very low. According to Aponte et al. (2005), SSC comprise only 0.03% of all germ cells in testis and their isolation is often hindered by the presence of spermatogonial cells at different stages of differentiation. Our previous experiments conducted on the different age group of animals revealed that as the age of the animal increases the number of SSCs in testis decreases (unpublished data). Shinohara et al. (2000) have used CD9 cell surface marker to purify the SSCs form mixed cell population obtained from testis. Viability of the cells is another important criterion for culturing the SSCs. Viability of the cells decreases as the time lapses after animal’s death due to autolysis of the cells. Van Pelt et al. (1996) also mentioned about the importance of time gap between the death and isolation of SSCs. The testes were therefore, collected within 2 hr after the death of the animal. More than 80 % viability and <NOBR>1–2</NOBR> x 10<SPAN class="ft10">6</SPAN>Cells/ml concentration resulted in a very few <NOBR>(2–3)</NOBR> colonies resembling to SSCs colonies. The SSC isolated from seminiferous tubules is often contaminated by differentiating spermatogonial cells, sertoli cells and peritubular myoid cells. Most SSC culture systems are known to contain a mixture of testicular cells with about 1.33% SSC (Aponte et al., 2005). Luo et al.</P>
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<P class="p22 ft5">(2006) have used MACS to isolate the SSCs mixed cell population because identification and isolation of SSC had been difficult due to their rarity in testis and lack of SSC specific cell surface markers. <NOBR>Rodriguez–Sosa</NOBR> et al., (2006) also employed various methods such as differential plating, velocity sedimentation, elutriation, discontinuous gradient, Hoechst 33342 and rhodamine 123 side population, <NOBR>magnetic–activated</NOBR> cells sorting (MACS) and fluorescence activated cells sorting (FACS) to isolate SSC in different species. We could observe the direct effect of pH of the buffer used in attachment and detachment of microbeads with the cells. Buffer with pH 7.4 gave good binding results in comparison to the buffer with pH 7.2 and pH7.6. Li et al. (2013) reported that even after enriching the SSCs with percoll density gradient method, the cell suspension was still containing few contaminating testicular cells. Hence we used CD9 surface marker along with MACS to enrich the cell population with the SSCs. Similar to Kala et al. (2012), we also used FITC attached with secondary antibodies to characterize isolated SSCs under florescent microscope to confirm the purity of isolated CD9 positive SSCs.</P>
<P class="p40 ft0">Lengner et al. (2007) observed very low levels of OCT– 4 expression in SSCs as compared with ESCs. Hence, it cannot serve as a good marker for enrichment of SSCs. <NOBR>Kanatsu–Shinohara</NOBR> et al. (2004) concluded that <NOBR>CD–9</NOBR> is a better surface marker in comparison to <NOBR>OCT–4</NOBR> in testicular samples. We found that MACS can be used to enrich the culture with stem cell properties if the surface markers are known. Recently, Sheng et al. (2013) have also used MACS to enrich prostate cancerous cells which have the stem cell like properties. Patrawala et al. (2006) and Guo et al. (2012) have also used MACS for enrichment of stem cells in combination with antibodies against CD 9 surface marker. CD 9 is a stem cell surface marker which may act as a good choice of surface marker to enrich stem cells along with MACS (Cui et al., 2004). We were able to get relatively good number of CD9 positive cells when the cell suspension was sorted with MACS.</P>
<P class="p41 ft5">DBA also has been used as a marker for prespermatogonia, the precursors of bovine spermatogonia present until the onset of spermatogenesis at week 30 of age (Ertl et al., 1992). Here, we have hypothesized that DBA will be expressed in the SSCs of young bulls aged around 6 months. Hence, the colony formed from the SSCs isolated from young calves should be positive for presence of DBA marker. Our results were in support of our hypothesis where colonies were found to be positive for <NOBR>FITC–DBA.</NOBR> Nash et al. (2007) also reported the stem cells exhibiting affinity towards lectin DBA protein.</P>
<P class="p42 ft9">CONCLUSION</P>
<P class="p43 ft0">MACS in combination with stem cell marker CD9 provide an efficient tool for enrichment of cattle calves’ SSCs.</P>
<P class="p44 ft33">Further research may be carried out to compare the efficacy of FACS and MACS.</P>
<P class="p4 ft9">ACKNOWLEDGEMENT</P>
<P class="p45 ft33">Authors are very thankful to <NOBR>NAIP–ICAR</NOBR> for providing funding and also to <NOBR>PDC–ICAR</NOBR> for providing facility for conducting research.</P>
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	<TD class="tr1 td2"><P class="p7 ft18">Verma et al (2014). Spermatogonial Stem Cells with MACS in Cattle</P></TD>
	<TD class="tr1 td3"><P class="p17 ft19">186</P></TD>
</TR>
</TABLE>
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<P class="p0 ft21"><A href="http://www.nexusacademicpublishers.com/journal/4">Advances in Animal and Veterinary Sciences. </A>2 (3): 183 – 187 <A href="http://dx.doi.org/10.14737/journal.aavs/2014/2.3.183.187"><SPAN class="ft20">http://dx.doi.org/10.14737/journal.aavs/2014/2.3.183.187</SPAN></A></P>
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<P class="p0 ft34">REFERENCES</P>
<P class="p46 ft35">Aponte PM, Van Bragt MP, De Rooij DG and Van Pelt AM (2005). Spermatogonial stem cells: characteristics and experimental possibilities. APMIS . <NOBR>113:727–742.</NOBR></P>
<P class="p46 ft35">Bosio A, Huppert V, Donath S, Hennemann P, Malchow M, and Heinlein UAO (2009). Isolation and Enrichment of Stem Cells. Adv. Biochem. Engin/Biotechnol. <NOBR>114:23–72.</NOBR></P>
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<TABLE cellpadding=0 cellspacing=0 class="t1">
<TR>
	<TD class="tr1 td2"><P class="p7 ft18">Verma et al (2014). Spermatogonial Stem Cells with MACS in Cattle</P></TD>
	<TD class="tr1 td3"><P class="p17 ft19">187</P></TD>
</TR>
</TABLE>
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<DIV id="id_4">
<P class="p0 ft5">ISSN: <NOBR>2307–8316</NOBR> (Online); ISSN: <NOBR>2309–3331</NOBR> (Print)</P>
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