Hypoxic Preconditioning Promotes Survival of Human Adipose Derived Mesenchymal Stem Cell

Abstract

Background: Contributing factors for improved survival of human adipocytes mesenchymal stem cells (h-AMSCs) cultured through hypoxia preconditioning, in example apoptosis inhibition involving BCL2 and HSP27 expression, trigger signal expression (VEGF), SCF expression, OCT-4 expression, and CD44+ expression. The objective if this study was to explain the mechanism and role of hypoxic preconditioning and the optimal duration of hypoxic preconditioning exposure to improve survival of h-AMSCs. Methods: An experimental laboratory explorative study ( in vitro) with hypoxic preconditioning in h-AMSCs cultures. This research was conducted through four stages. First, isolation of h-AMSCs culture from adipose tissue of patients. Second, the characterization of h-AMSCs from adipose tissue by phenotype (flowcytometry) through CD44+, CD90+ and CD45-expression before being pre-conditioned for hypoxic treatment. Third, the hypoxic preconditioning in h-AMSCs culture ( in vitro) was performed with an oxygen concentration of 1% for 24, 48 and 72 hours. Fourth, observation of survival from h-AMSCs culture was tested on the role of CD44+, VEGF, SCF, OCT-4, BCL2, HSP27 with Flowcytometry and apoptotic inhibition by Tunnel Assay method. Results: The result of regression test showed that time difference had an effect on VEGF expression ( p<0.001; β=-0.482) and hypoxia condition also influenced VEGF expression ( p<0.001; β=0.774). The result of path analysis showed that SCF had effect on OCT-4 expression ( p<0.001; β=0.985). The regression test results showed that time effects on HSP27 expression ( p<0.001; β=0.398) and hypoxia precondition also affects HSP27 expression ( p<0.001; β=0.847). Pathway analysis showed that BCL2 expression inhibited apoptosis ( p=0.030; β=-0.442) and HSP27 expression also inhibited apoptosis ( p<0,001; β=-0.487). Conclusion: Hypoxic preconditioning of h-AMSC culture has proven to increase the expression of VEGF, SCF, OCT-4, and BCL2 and HSP27. This study demonstrated and explained the existence of a new mechanism of increased h-AMSC survival in cultures with hypoxic preconditioning (O2 1%) via VEGF, SCF, OCT-4, BCL2, and HSP 27.

Keywords: BCL-2; HSP27; SCF; VEGF expression; apoptosis; h-AMSCs.

Figure 1.

Isolation and culture of h-AMSCs from the patient's adipose tissue (human).

Figure 2.

Observation of survival of h-AMSCs in the form of CD44+, VEGF, SCF, OCT-4, BCL2, HSP27 expression, and apoptotic inhibition:

1. A.

   Phenotype expression of CD44+ was carried out by the flowcytometric method.

2. B.

   Immuno-cytochemical expression of VEGF.

3. C.

   Immunocytochemical expression of SCF from h-AMSCs culture.

4. D.

   Phenotype of OCT-4 expression (Immunocytochemistry and Immunofluorescence).

Figure 3.

Flowcytometry results from human AMSCs based on cell culture for CD44+ CD90+ CD45- expression.

Figure 4.

Immunohistochemical Characteristic of h-AMSCs based on VEGF expression at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours.

Figure 5.

Immunohistochemical Characteristic of h-AMSCs based on SCF expression at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours.

Figure 6.

Immunohistochemical Characteristic of h-AMSCs based on OCT-4 expression at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours. Immunofluorescence assay of h-AMSCs based on OCT-4 expression at: G) normoxic condition for 24 hours; H) normoxic condition for 48 hours; I) normoxic condition for 72 hours; J) hypoxic condition for 24 hours; K) hypoxic condition for 48 hours; L) hypoxic condition for 72 hours.

Figure 7.

Immunohistochemical Characteristic of h-AMSCs based on BCL2 expression at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours.

Figure 8.

Immunohistochemical Characteristic of h-AMSCs based on HSP27 expression at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours.

Figure 9.

Immunohistochemical Characteristic of h-AMSCs based on number of apoptotic cell amount at: A) normoxic condition for 24 hours; B) normoxic condition for 48 hours; C) normoxic condition for 72 hours; D) hypoxic condition for 24 hours; E) hypoxic condition for 48 hours; F) hypoxic condition for 72 hours.

Figure 10.

Path analysis with MANOVA and multiple linear regression analysis for hypoxic preconditioning in h-AMSCs survive.