GENERATION OF OOCYTE-LIKE CELLS FROM BUFFALO UMBILICAL CORD BLOOD- MESENCHYMAL STEM CELLS
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Date
2022
Authors
PRIYA SEHRWAT
Journal Title
Journal ISSN
Volume Title
Publisher
ICAR-NDRI, KARNAL
Abstract
The aim of present study was to perform in vitro differentiation of Umbilical Cord
Blood- Mesenchymal Stem Cells to Oocytes like cells. UCB-MSC were isolated
from the umbilical cord of calf just after a delivery with the help of 18-gauge syringe
and brought to lab for further process. Blood was equally diluted with DPBS and
loaded on HiSepTM LSM 1077 gradient and centrifuged at 400 g for 30 min and a
buffy coat was formed at interface was taken and seeded in flask containing
DMEM/F12, 20% FBS,1% P/S solution at 5% CO2 at 37°C in incubator. Media was
changed twice a week for 3 weeks. After 3-week RNA of cultured cells were
isolated and characterized for MSCs confirmation through RT-PCR, Alkaline
Phosphatase Assay and immunostaining for MSC specific gene. After
confirmation of MSCs which appear to be spindle in shape and adherent in nature
and having CD73, CD90 and CD105 surface markers present on them, were
seeded in well for differentiation into Primordial Germ Cell Like Cells (PGCLCs)
then to Oocyte Like Cells (OLCs) using differentiation media containing
DMEM/F12, FBS, BMP4 and P/S antibiotic solution. Bone Morphogenic Protein 4
(BMP4) is a member of the transforming growth factor-b (TGFb) group of proteins,
to initiate signal transduction in target cells. BMP4 due to its signal transduction
pathway, it activates different proteins used in germ cell development.
Confirmation of PGCLCs on 7th day was done by immunostaining using STELLA
and VASA as primary antibody, also by doing RT-PCR for PGCLCs specific genes
(Oct4, VASA, STELLA and SCY3). OCLs formed on 14th day were also confirmed
by immunostaining taking ZP2 as primary antibody, also confirming by RT-PCR
for OLCs specific genes (ZP2 and ZP3). The results were found positive for
PGCLCs and OLCs formation. It can be concluded that UCB-MSC can be directed
differentiation into Oocyte Like Cell.