The research approach and methodologies to be followed in this sub-project are outlined under each of the project objectives as under:

Objective 1:         Standardization of conditions for in vitro culture of bovine embryonic and spermatogonial stem cells.
Activity 1: In vitro production of buffalo blastocysts and isolation of ICM. (NDRI)

Buffalo blastocysts are being produced in vitro through a combination of the techniques of in vitro maturation, in vitro fertilization and in vitro culture of oocytes obtained from slaughterhouse ovaries using protocols standardized in our laboratory (Chauhan et al. 1998, Reprod Fertil Dev 10 (2): 173-77) and improved from time to time (Anand et al., 2008 Reprod Fertil Dev 20, 253-257). The blastocysts produced are used for the isolation of inner cell mass (ICM) cells. These cells are being subjected to long-term culture on appropriate feeder layers.

Isolation of spermatogonial cells (PDC)

Testis from buffalo slaughtered animal or biopsies from the cattle bull are beingobtained. Approximately 20 g of tissue from testis is enzymatic ally digested as described by O’Brien (1993) with modification as described by McLean et al (2002).Briefly, testis is removed, immersed in Hanks buffer, and the tunica is removed. Testes are transferred to tubes containing digestion medium, consisting of 0.5mg/mL collagenase type1V, 0.25mg/mL trypsin and 0.05 mg/mL DNase in Ca- and Mg- free Hanks buffer. Testes are digested for 15 min by shaking at 33 ˚C to dissociate tubules. The samples containing the tubule suspensions are transferred to ice and the tubules are allowed to sediment for 5 minutes. The supernant isremoved, fresh digestion medium added, and the tubules digested for 15 minutes by shaking at 32-37 ˚C . After the second digestion, the cell concentration is determined using a hematocytometer. The cell suspension is centrifuged again at 500×g for 4 minutes and then resuspended in DPBS or Enriched Krebs-Ringer Bicarbonate medium containing 0.03% trypan blue. Technique or methodology is being modified or changed as per the latest available information.

An enriched population of spermatogonial stem cells is critical because large number of contaminating testis somatic cells may have an adverse effect on SSC which will be maintained in vitro. The SSCs are being enriched by differential plating and purification are being carried out by discontinous percol density gradient centrifugation followed by cell viability testing by trypan blue exclusion method and flowcytometry.

Activity 2:       Selection of basic media for the culture of stem cells

Embryonic stem cells (NDRI)
The ES cells using media like DMEM, which had originally been developed for the general cell culture work using somatic cells. In the last few years, a number of new media have been developed which are tailor-made for the culture of ES cells. The efficacy of these media like knockout-DMEM needs to be evaluated for supporting the growth of buffalo ES cells. The buffalo ES cell like cells are being cultured for long term (i.e. 6-8 months) in the laboratory having dedicated facility  (laminar hood, Three gas CO2 incubator, zoom sterio microscope and inverted microscope, etc.). Various serum replacers in the long term culture have been reported to give good results, especially in mouse and human ES cells. To what extent can these serum replacers support the growth and proliferation of buffalo ES cells has already been examined.

Spermatogonial stem cell (PDC)

 Testicular germ cells, from buffalo and cattle, are being cultured in 24-well plates or in 6- well plates with basic stem cell culture medium consisting of Dulbecco’s minimal essential medium (DMEM or F12 or F10) supplemented with 10% fetal bovine serum (FBS), 12 ng/mL mLIF, 20 ng/mL bovine bFGF, 0.04 ng/mL h-IL-11, 10ng/mL hSCF, 1 mM sodium pyruvate, 2 mM L-glutamine,5.5 ×10-5 M 2-mercaptpethanol, 100 µg/mL streptomycin, 100 units/mL penicillin, 10 ng/mL OSM, 1 ng /mL platelet derived growth factor (PDGF) and 15 ng/mL human IGF-1.

The isolated germ cells from one testes will be seeded into wells of the six well culture plates and incubated in a CO2 incubator at 32 degree C until the stem cells have colonized as a primary culture (approximately 7-12 days). Mitomycin C- treated feeder layer cells or fibroblast cells or sertoli cells are being used as a feeder cells as culture. The feeder cells are seeded on the culture surface at a concentration of 5× 106 cells/cm2 for 1-2 days before testes will be added. Medium is changed twice a week. For subculture, the colonies are agitated by pipetting after trypsin treatment for ten minutes at 37 degree C and they are harvested from the plate. The cells are centrifuged at 200×g for 5 minutes and placed into 2 fresh plates with mitoticaly inactivated feeder cells. The cells colonies will be pass aged at an interval of 7 to 14 days on average. All culture experiments are repeated 3 times; each experiment comprises triplicate cultures. The size of colonies is determined by their proliferation in cultures. Technique or methodology is being modified or changed as per the latest available information.

Activity 3:       Effect of media supplements like growth factors and different feeder layers on growth and proliferation  of stem cells

ES Cells (NDRI)

The identification of growth requirements and proliferation of ES cells for a long term culture are being standarized. Growth factors like basic fibroblast growth factor (bFGF) and glial-cell line derived neurotrophic factor (GDNF) have been reported to support the maintenance of pluripotency of ES cells during long term cultures in human and to a lesser extent in mouse ES cells. A number of other growth factors like EGF and PDGF have been reported to stimulate the proliferation of many different types of adult stem cells. The efficacy of these growth factors in stimulation of proliferation of buffalo ES cells needs to be tested. After examining various growth factors individually, they would be used in different combinations to develop a cocktail of growth factors which can stimulate the growth and proliferation of buffalo ES cells over long term cultures and multiple passages, without compromising their pluripotency. 

 Spermatogonial stem cells (SSCs) (PDC, NDRI)

The identification of growth requirements and development of a long term culture system for SSCs are being standarized. Appropriate growth factors are being selected based on the results reported in mouse, human and cattle and the efficacy of these growth factors are being compared in terms of supporting the growth of SSCs. The culture system is being developed for SSCs using serum free defined medium. The growth factors like bFGF, GDNF, glial-cell –line-derived neurotrophic factor-family receptor GFRalpha 1 are being added in the medium to see their effect on cell proliferation.

Objective 2:         Characterisation of stem using surface and intracellular markers (NDRI and PDC).

Activities 1 and 2:      Characterisation of stem cells using biochemical and surface markers

The ES cells produced are being characterized on the basis of a number of biochemical markers like alkaline phosphatase etc.. These cells are being characterized on the basis of surface markers like SSEA-1, SSEA-3, SSEA-4,TRA-1-60, TRA-1-81, TRA-2-49, TRA-2-54, CD9, CD30, Thy-1, Podoclyxin etc.

Alkaline Phosphatase Assay and Antibody Staining

Germ cell colonies are being fixed for detection of alkaline phosphates (AP) activity in 66%acetone/3%formaldehyde and then stained with napthol/AS-MX-alkaline AP substrate. For immunocyochemistry cells are being fixed with 3 %formaldehyde in PBS and the presence of GCNA1 and c-kit is being determined

β- Galactosidase Activity of spermatogonial cells

Cultured germ cell colonies are stained with 5- bromo4-chloro-3-indoylβ-galactoside(X-gal) to detect β- Galactosidase Activity to differentiate and identify germ cells colonies from feeder cells. To stain the cell colonies, medium is removed, the cell layer washed once with Phosphate Buffer Saline (PBS), fixed with .5% glutaraldhyde for 20 minutes in ice, washed twice with PBS, and then incubated with X-gal until blue color is developed.

Furthermore, characterization of spermatogonial stem cell like cells is being carried out by studying the expression of CD9, alpha6 and beta1 integrin etc.

Activity 3:       Characterisation of stem cells using intracellular markers (NDRI)

Characterization of ES cells is being also done based on intracellular transcription-based markers like Oct-4, NANOG, Sox-2, Rex-1, telomerase etc.

Activity 4:       Enrichment of stem cells by fluorescence-activated cell sorting (FACS)
                        (NDRI)

The surface markers are being to be identified using fluorescence-labeled antibodies. Once the profile of surface markers expressed by buffalo pluripotent ES cells is identified, the ES cells will be isolated and enriched by tagging them with appropriate fluorescent antibodies and sorting out the fluorescent cells by fluorescence-activated cell sorting (FACS) with the help of a flowcytometer.

For cell sorting experiments, cells will be suspended in PBS containing 4% FBS. Cell sorting and analysis will be analyzed by using a FACS Vantage flow cytometer/ cell sorter equipped (FACS machine) Sorted cells in DMEM medium containing 10% FBS will be washed once with the medium and prepared for further analysis or culture.This process would enable quick isolation and enrichment of buffalo ES cells expressing appropriate markers.

Similarly, characterization of SSCs is being carried out by immunosorting through phycoerythrin conjugated CD 9 monoclonal antibody.  The surface marker for bovine SSCs have not yet been identified. These marker will be identified in this program using several antigens (e.g. Thy-1, a6-integrin, Ep-CAM).

Activity 5:       Cloning and characterization of buffalo ortholog of POUfi (encoding transcription factor Oct-4 and NANOG) gene (NDRI)

Full length Oct-4 and NANOG cDNA and promoter sequence has been amplified using PCR techniques and cloned in standard cloning vector. Promoter sequence is sub cloned in reporter vector for functional analysis by Cell culture, transient transfections, and reporter assays are being performed.

Objective 3:         To elucidate the differentiation potential of stem cells

Activity 1:       Studies on spontaneous differentiation of stem cells (NDRI, PDC)

The ES cells produced are being subjected to culture in the absence of feeder layers and LIF for spontaneous differentiation for the formation of embryoid bodies, which contain all the three germ layers ectoderm, mesoderm and endoderm, will be studied. Similarly, spontaneous differentiation in the SSCs under the long term culture system. 

Activity 2:       Induced differentiation of stem cells (NDRI)

The ability of ES cells produced to form specific types of cells in the presence of specific stimulators would be examined. For example, their ability to form cardiomyocytes in the presence of nitric oxide or ascorbic acid had been examined. The SSCs would also be studied to induced differentiation to examine their differentiation potential in vitro.

Activity 3:       Transcriptional regulation network in self renewal and pluripotent (NDRI)

Expression pattern of various transcription-based pluripotency marker genes like Oct4, NANOG etc. are being studies in ES cells by real time PCR for understanding the basis of pluripotency in buffalo ES cells. Functional genomic approach involving RNAi mediated suppression of OCT-4/NANOG in buffalo stem cell and analysis of resulting transcriptional profile to identify OCT-4 and NANOG dependent genes in buffalo stem cell is being analyzed.  

Study of mutations in stem cells during multiple passasing using microsatellite markers

The suitable buffalo-specific microsatellite markers will be identified. DNA from stem cells at every 3rd passage of cultures will be isolated. Microsatellite genotyping will be done using automatic DNA sequencer.

Identified and characterized ES and SSCs are being cryopreserved in liquid nitrogen containers after different passaging for further study

Objective 4:         To test the developmental competence of stem cells using in vivo mouse and bovine models.

Activity 1:        Examination of developmental competence of ES cells in mouse model (NDRI)

The ES cells produced will be administered to immunosuppressed mice (nude mice) which will be kept in humidity and temperature controlled chamber in order to avoid any other infections. To examine their ability to form teratomas. Subsequently after 70-90 days the teratomas will be observed and subjected to serial histological analysis to find out the nature of germ layer cells (ectoderm, mesoderm and endoderm) they contain.

Examination of developmental competence of SSCs in bovine model (PDC)
The SSCs produced would be administered to mice and histological analysis performed for the formation of cattle spermatogonia. Less valuable but healthy bulls donating poor quality semen will be used as recipients. All procedures with live animals will be done as per the gudelines of Animal Care Committee of the Institute.
Briefly, frozen thawed or fresh SSCs will be used for rete testis injection. The SSCs will be centrifuged and incubated in and labelled (CFDA-SE solution). After labeling, these will be centrifuged, re-suspended in ice-cold medium (EMEM) on ice until transplantation. Endogenous spermatogonia of the recipients will be abolished by chemotherapy using Busulfan treartment. Immunosuppression of recipients will be done by cyclosporin/corticoids. Flourescence-labelled donor stem cells will be injected into the rete testis of recipient animals through ultrasonography-guided approachThese bulls will be injected with xylazine IM as pre-anesthetic, will be positioned on their backs and fully anaesthetized with ketamine and diazepam IV as per the standard protocol. A 20 ga needle will be guided by ultrasound through the scrotum and into the extra-testicular rete, and a second 26 ga (or suitable) needle will be inserted through it. A total of 20- 30 × 106 cells in 5 mL will be injected through the 26 ga needle into the extra-testicular rete at 1 mL/min by gentle pressure on a 12 cm3 syringe. Both testes will be injected. Around 2–30 weeks after transplantation, colonization of the transplanted cells will be studied by observing the lacalization of PKH 26- labeled cells in the testicular tissues and further differentiation of SSCs into spermatogonia, spermatocytes or sperm. Technique or methodology will be modified or changed as per the latest available information.

Activity 2.  Identifying strategies for suitability of stem cells for cloning and transgenesis        (NDRI)

The ES and spermatogonial cells are being transfected with GFP tagged DNA (Factor IX/ or other) and subsequently cultured in vitro. After the multiple passging the GFP tagged DNA expressed cells are being separated. These cells are subsequently used for nuclear transfer (NT) or hand made cloning to see their viability and efficiency for producing cloned and transgenic animal.

Efficiency of ES cells through nuclear transfer

Briefly, the buffalo oocytes are being matured in vitro using standard protocol of our laboratory (Chauhan et al., 1998). The matured oocyte are enucleated by pushing out the first polar body and the metaphase II plate, plus a small amount of the cytoplasm that surroun them, with glass pipette into SOF or BO or TALP medium supplemented with cytochalasin B. After insertion of donor ES cells into the perivitelline space of oocytes, cell and cytoplasts are fused in fusion medium using electrofusion machine. The method is being modified as per the requirement.

Efficiency of ES cells through hand made cloning

The efficiency of the ES cell in hand made cloning has been evaluated in vitro. The matured oocytes are bisected and subsequently stained with Hoechst stain. Two cytoplast  without chromatin material are used. ES cells are placed between the two cytoplast obtained from two different oocytes and fused in fusion medium using electro fusion machine. Technique or methodology is modified or changed as per the latest available information.

Embryos generated through NT or hand made cloning are subsequently cultured in medium (SOF, mCR1aa or mCR2aa) for 9 days in three gas CO2 incubator. GFP expressing embryos are evaluated under the fluroscence inverted microscope for their integration (transgenic embryos).

All the aspects like, ES fusion with oocyte/cytoplast and their activation, integration of cytoplast with ES cells, merging of cells, culture of these ES fused oocytes and cytoplast and their subsequent development to embryos and other related to NT, GFP expression pattern of ES cells and their efficiency/compatibility for cloning and transgenesis are being determined.

The approach to be followed for filling the gaps with emphasis on novelties is presented below. The research gaps have been presented as question marks.

? Basics about methodologies, culture conditions media, FCS/FBS, feeder layer, cell numbers, pluripotency, cell - cell interactions, charecterization, telomerase activity,  transcription  factors, LIF, in vitro and in vivo differentiation etc. will be determined.