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 o...
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doaj-d996237de46b4ee1ab3b0b7023b0c2822021-06-18T04:56:25ZengJCDR Research and Publications Private LimitedJournal of Clinical and Diagnostic Research2249-782X0973-709X2021-05-01155YC05YC0910.7860/JCDR/2021/48893.14864Cellular Properties and Expression of Pluripotent Markers in Human Dental Pulp Stem Cells Cultured in Serum-Free MediumChethan Kumar0Basan Gowda Sharanappa Kurkalli1Shishir Shetty2AKSHAY BAIRAPURA MANJA3Veena Shetty4 Mohana Kumar Basavarajappa5Research Scholar, Department of Nitte University Centre for Stem Cell Research and Regenerative Medicine, Nitte (Deemed to be University), Mangaluru, Karnataka, India.Professor, Department of Nitte University Centre for Stem Cell Research and Regenerative Medicine, Nitte (Deemed to be University), Mangaluru, Karnataka, India.Professor, Department of A. B. Shetty Memorial Institute of Dental Sciences, Nitte (Deemed to be University), Mangaluru, Karnataka, India.Research Scholar, Department of Nitte University Centre for Stem Cell Research and Regenerative Medicine, Nitte (Deemed to be University), Mangaluru, Karnataka, India.Professor, Department of Nitte University Centre for Stem Cell Research and Regenerative Medicine, Nitte (Deemed to be University), Mangaluru, Karnataka, India.Associate Professor, Department of Nitte University Centre for Stem Cell Research and Regenerative Medicine, Nitte (Deemed to be University), Mangaluru, Karnataka, India.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.https://www.jcdr.net/articles/PDF/14864/48893_CE[Ra1]_F[IK]_PF1(SC_RK)_PN(KM).pdfcellular therapyfetal bovine serumosteogenesisxenogeneic free |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Chethan Kumar Basan Gowda Sharanappa Kurkalli Shishir Shetty AKSHAY BAIRAPURA MANJA Veena Shetty Mohana Kumar Basavarajappa |
spellingShingle |
Chethan Kumar Basan Gowda Sharanappa Kurkalli Shishir Shetty AKSHAY BAIRAPURA MANJA Veena Shetty Mohana Kumar Basavarajappa Cellular Properties and Expression of Pluripotent Markers in Human Dental Pulp Stem Cells Cultured in Serum-Free Medium Journal of Clinical and Diagnostic Research cellular therapy fetal bovine serum osteogenesis xenogeneic free |
author_facet |
Chethan Kumar Basan Gowda Sharanappa Kurkalli Shishir Shetty AKSHAY BAIRAPURA MANJA Veena Shetty Mohana Kumar Basavarajappa |
author_sort |
Chethan Kumar |
title |
Cellular Properties and Expression of Pluripotent Markers in Human Dental Pulp Stem Cells Cultured in Serum-Free Medium |
title_short |
Cellular Properties and Expression of Pluripotent Markers in Human Dental Pulp Stem Cells Cultured in Serum-Free Medium |
title_full |
Cellular Properties and Expression of Pluripotent Markers in Human Dental Pulp Stem Cells Cultured in Serum-Free Medium |
title_fullStr |
Cellular Properties and Expression of Pluripotent Markers in Human Dental Pulp Stem Cells Cultured in Serum-Free Medium |
title_full_unstemmed |
Cellular Properties and Expression of Pluripotent Markers in Human Dental Pulp Stem Cells Cultured in Serum-Free Medium |
title_sort |
cellular properties and expression of pluripotent markers in human dental pulp stem cells cultured in serum-free medium |
publisher |
JCDR Research and Publications Private Limited |
series |
Journal of Clinical and Diagnostic Research |
issn |
2249-782X 0973-709X |
publishDate |
2021-05-01 |
description |
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. |
topic |
cellular therapy fetal bovine serum osteogenesis xenogeneic free |
url |
https://www.jcdr.net/articles/PDF/14864/48893_CE[Ra1]_F[IK]_PF1(SC_RK)_PN(KM).pdf |
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