5 research outputs found
Cellular Properties and Expression of Pluripotent Markers in Human Dental Pulp Stem Cells Cultured in Serum-Free Medium
Introduction: The standard isolation and expansion of human
Dental Pulp Stem Cells (DPSCs) under invitro conditions
normally involve the usage of Fetal Bovine Serum (FBS).
However, its animal-origin poses possible concerns for clinically
relevant procedures. This critical issue compels the use of
Xenogeneic-Free (XF) or human-origin alternatives to FBS for
culture expansion and differentiation of DPSCs to determine the
usefulness for translating into therapeutic clinical applications.
Aim: To evaluate the cellular characteristics and expression of
pluripotent markers in DPSCs cultured using Serum-Containing
Medium (SCM-DPSCs) and Serum-Free Medium (SFMDPSCs).
Materials and Methods: This in-vitro descriptive study was
conducted at NITTE (Deemed to be University), Mangaluru,
Karnataka, India, from June 2019 to August 2020. DPSCs were
isolated from impacted third molars. The culture expanded DPSCs
in serum-containing and serum-free media were analysed on their
morphology, viability, proliferation rate, Population Doubling Time
(PDT), Alkaline Phosphatase (ALP) activity, cell surface markers
expression, osteogenic and adipogenic potential, and the relative
expression of selected pluripotent genes.
Results: The primary culture of DPSCs established in SCM and
SFM showed spindle shaped fibroblastic morphology with >80%
viability from passage 1 (P1) to P4. A significant (p-value<0.05)
difference in the proliferation rates in terms of cell numbers
between SCM-DPSCs and SFM-DPSCs was observed (day 6:
3×105
vs 0.8×105
; day 9: 5.8×105
vs 1.27×105
; day 12: 7.8×105
vs 1.56×105
, respectively). The average PDT values recorded
in SCM- and SFM-DPSCs were 44.33 hours and 58.41 hours,
respectively. A slightly higher expression of ALP activity was
observed in SCM-DPSCs than in SFM-DPSCs. Flow cytometry
analysis showed that both DPSCs were positive for CD29,
CD73, CD90, and negative for CD34 and CD45. The expression
of OCT4 and NANOG was relatively higher in SCM-DPSCs
compared to SFM-DPSCs. Further, SCM-DPSCs showed the
higher levels of SOX2 and SSEA4, but did not exhibit any
significant differences in their expression levels.
Conclusion: The results showed that DPSCs in FBS displayed
better growth kinetics and stemness markers expression along
with more propensities towards lineage differentiation. SFM can
be used to establish and expand DPSCs with characteristics
of multipotent stem cells, but needs further research for its
optimisation
Assessment of proliferation, clonogenic assay, and osteogenic differentiation of human periodontal ligament stem cells following application of orthodontic forces
Context: The proliferation and differentiation of human periodontal ligament stem cells (hPDLSC) into other cell types are also mediated by mechanical stresses; they might offer therapeutic benefits in tissue regeneration and angiogenesis. Objectives: The study was planned to assess the proliferation, clonogenic potential, and osteogenic differentiation of human periodontal ligament stem cells (PDLSC) following the application of light and heavy orthodontic forces. Materials and Methods: A couple forces of 50 gm (light force) were applied on the 1st premolar on the one side and 250 gm (heavy force) on the contralateral side in the upper arch of patients requiring orthodontic treatment with extraction of all 1st premolars. After 30 days, periodontal tissues were scrapped from extracted teeth for the establishment of PDLSC in vitro. PDLC from the lower premolar teeth where no orthodontic force was applied acted as the control group. Morphology, viability, proliferating rate and population doubling time, clonogenicity, and alkaline phosphatase activity were analysed. Result: The osteogenic potential was confirmed by Alizarin red staining and the expression of the osteogenic markers by qRT-PCR. The morphology, growth kinetics, potency, and osteogenic lineage characteristics inferred the application of high force reduced the proliferative ability and osteogenesis of PDLSC, though the difference was not significant. Conclusion: The established PDLSCs demonstrated their MSC-like properties based on morphology, growth kinetics, colony forming ability, and AP activity. The culture-expanded PDLSCs showed their differentiation potential into osteocytes. The application of high force reduced the proliferative ability and osteogenesis of PDLSCs, variations were not significant
Removal of cumulus cells before oocyte nuclear maturation enhances enucleation rates without affecting the developmental competence of porcine cloned embryos
The present study compared the efficiency of somatic cell nuclear transfer (SCNT) using porcine oocytes that were denuded of their cumulus cells at different maturation time. In pre-denuded group, the cumulus cells from cumulus-oocyte complexes (COCs) were removed at 29 hr post in vitro maturation (hpm) and followed by further culture for 12 hr. In control group, as a commonly followed procedure, cumulus cells were removed from COCs at 41 hpm. The majority of porcine oocytes at 29 hpm were observed in metaphase of the first meiotic division (MI). At 41 hpm, no significant (P > 0.05) differences were observed in nuclear maturation and mitochondrial distribution of oocytes between pre-denuded and control groups. However, in pre-denuded group oocytes, metaphase II (MII) plate and spindle were located closely as ‘adjacent’ to the first polar body (PB1), resulting in an increased enucleation rates than in control group oocytes by blind enucleation method. Following SCNT and parthenogenesis (PA) using pre-denuded group and control group oocytes, no significant (P > 0.05) differences were observed with respect to the development, total cell number, incidence of apoptosis and the expression profile of developmentally important genes (Pou5f1, Dnmt1, Dnmt3a, Igf2r, Bax, Bcl2 and Glut1) at the blastocyst stage. In conclusion, the removal of cumulus cells at 29 hpm in porcine oocytes increased the enucleation rates through proper positioning of PB1 without compromising the quality of SCNT embryos during preimplantation development. Hence, this could be a valuable strategy to improve the SCNT efficiency in a porcine model