The Effects of Extracellular pH on Proliferation and Differentiation of human Bone Marrow Stem Cells

Yea Hyun Leem; Tae Suk Nam; Jung Hwa Kim; Kang Sik Lee; Dong Ho Lee; Juno Yun; Jae Suk Chang

doi:10.11005/kjbm.2012.19.1.35

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Leem, Nam, Kim, Lee, Lee, Yun, and Chang: The Effects of Extracellular pH on Proliferation and Differentiation of human Bone Marrow Stem Cells

Original Article

Korean Journal of Bone Metabolism

Korean Journal of Bone Metabolism 2012; 19(1): 35-46.

Published online: 18 May 2012

DOI: https://doi.org/10.11005/kjbm.2012.19.1.35

The Effects of Extracellular pH on Proliferation and Differentiation of human Bone Marrow Stem Cells

Yea Hyun Leem¹, Tae Suk Nam², Jung Hwa Kim¹, Kang Sik Lee¹, Dong Ho Lee¹, Juno Yun¹, Jae Suk Chang¹

¹Department of Orthopedic Surgery, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Korea.

²Nanoori Orthopedics Hospital, Seoul, Korea.

Address for Correspondence: Jae Suk Chang, Department of Orthopedic Surgery, Asan Medical Center, University of Ulsan, College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Korea. Tel: +82-2-3010-3530, Fax: +82-2-488-7877, jschang@amc.seoul.kr

Received 28 March 2012 Revised 19 April 2012 Accepted 30 April 2012

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/).

Abstract

Objectives

The purpose of this study is to identify whether the change of pH affects the proliferation and the differentiation of human bone marrow stem cells (hBMSCs) and what mechanism is underlied.

Methods

To achieve objective of this study, hBMSCs were cultivated in the conditioned media adjusted to potential of hydrogen (pH) ranging from 6.4 to 8.0 using addition of hydrochloric acid (HCl) and sodium hydroxide (NaOH). The ratio of proliferation of hBMSCs according to the change of pH was measured for 24 h, 48 h, and 72 h using water-soluble tetrazolium salt (WST)-8 method. To elucidate the mechanism involved, hBMSCs was subjected to blocking extracellular signal-regulated kinases (ERK) and calcium sensing receptor (CaSR) activation. The Osteogenic-related genes and alkaline phosphatase (ALP) activity were tested under the conditioned media.

Results

The proliferation of hBMSCs was promoted under extracellular alkali conditions (pH 7.6~8.0) via CaSR/ERK pathway. On the other hand, the differentiation was inhibited/delayed via decreased ALP activity besides gene expression at pH 8.0.

Conclusion

Extracellular alkali or acidic surrounding according to pH alteration can play a crucial role in hBMSC behavior including the proliferation and the differentiation.

Keywords: Bone marrow cells, Cell differentiation, Cell proliferation, pH, Stem cells

Figures and Tables

Fig. 1

The proliferation of human bone marrow stem cells (hBMSCs) according to medium potential of hydrogen in time-course dependent manner. (A) The proliferation of hBMSCs according to medium pH for 24 h incubation. (B) The proliferation of hBMSCs according to medium pH for 48 h incubation. (C) The proliferation of hBMSCs according to medium pH for 72 h incubation. The data are presented as means ± standard error of mean (SEM). ^* denotes a difference at P < 0.05.

Fig. 2

The expression of calcium-sensing receptor (CaSR) messenger ribonucleic acid (mRNA) and protein according to medium potential of hydrogen (pH) in time-course dependent manner. (A) The expression levels of CaSR mRNA according to medium pH in time-course dependent manner. (B) The expression levels of CaSR protein according to medium pH in time-course dependent manner. (GAPDH, glyceraldehyde-3-phosphate dehydrogenase)

Fig. 3

The suppression of proliferation in human bone marrow stem cells (hBMSCs) exposed by calcium-sensing receptor (CaSR) inbibitor. The data are presented as means ± standard error of mean (SEM). ^* denotes a difference at P < 0.05.

Fig. 4

The suppression of proliferation in human bone marrow stem cells (hBMSCs) exposed by extracellular signal-regulated kinases (ERK) inhibitor. The data are presented as means ± standard error of mean (SEM). ^* denotes a difference at P < 0.05.

Fig. 5

The expression of osteogenic-related molecules according to medium potential of hydrogen (pH). (A) The expression levels of runt-related transcription factor Runx2, alkaline phosphatase (ALP), and osteocalcin (OCN) messenger ribonucleic acid (mRNA) according to medium pH. (B) Quantitative analysis of Rnunx2, ALP, and OCN mRNA according to medium pH (Y-axis is a normalized level). The data are presented as means ± standard error of mean (SEM). ^* and ^** denotes a difference at P < 0.05 and P < 0.01, respectively. (GAPDH, glyceraldehyde-3-phosphate dehydrogenase)

Fig. 6

The activities of alkaline phosphatase (ALP) according to medium potential of hydrogen (pH). (A) Quantitative analysis of ALP activities according to medium pH for 7 days incubation. (B) Quantitative analysis of ALP activities according to medium pH for 14 days incubation (Y-axis is a normalized level). The data are presented as means ± standard error of mean (SEM). ^* denotes a difference at P < 0.05.

Table 1

Primers used for reverse transcription poly-merase chain reaction

GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase; OCN, osteocalcin; CaSR, calcium sensing receptor

Notes

This work was supported by Korean Ministry of Health, Welfare, and Family Affairs (MHWFA grant A084697).

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