Cobalt Chloride as a Hypoxia Mimicking Agent Induced HIF-1α and mTOR Expressions of Human Umbilical Cord Mesenchymal Stem Cells

ABSTRACT

Objective:

To assess the effects of cobalt chloride (CoCl2) as a hypoxia mimicking agent on human umbilical cord mesenchymal stem cells (hUCMSCs) expression of HIF-1α and mTOR for use in regenerative dentistry.

Material and Methods:

Human umbilical cord mesenchymal stem cells were isolated and then cultured. The characteristics of stemness were screened and confirmed by flow cytometry. The experiment was conducted on hypoxia (H) and normoxia (N) groups. Each group was divided and incubated into 24-, 48-, and 72-hours observations. Hypoxic treatment was performed using 100 µM CoCl2 on 5th passage cells in a conventional incubator (37°C; 5CO2). Then, immunofluorescence of HIF-1α and mTOR was done. Data was analyzed statistically using One-way ANOVA and Tukey’s HSD.

Results:

Significant differences were found between normoxic and hypoxic groups on HIF-1α (p=0.015) and mTOR (p=0.000) expressions. The highest HIF-1α expression was found at 48 hours in the hypoxia group, while for mTOR at 24 hours in the hypoxia group.

Conclusion:

Hypoxia using cobalt chloride was able to increase human umbilical cord mesenchymal stem cells expression of HIF-1α and mTOR.

Keywords:
Umbilical Cord; Mesenchymal Stem Cells; Stem Cell Research; Hypoxia; Regeneration

Introduction

Hypoxia is a condition where oxygen levels decrease in cells, tissues, or organs. The lowering of oxygen level could be caused by the decreased oxygen supply when there were decreased vasculature, healing process or inflammation [¹]. Other than that, hypoxia can be contributed to pathological factors involving microorganisms and immune responses [²,³]. Every tissue and cell have a different ability to adapt into a new environment ^[4]. In dentistry, hypoxic conditions can be found in gingivitis, periodontitis, periapical lesions, and peri-implant mucositis or peri-implantitis, as caused by periodontopathic biofilms ^[1]. This hypoxia condition in periodontal diseases has a correlation to systemic problems [³].

Tissue-engineering therapy using stem cells is well developed because of many advantages, such as cell regeneration, growth factor signaling, ability to differentiate, and biocompatibility [⁵]. The umbilical cord, one of the stem cell sources, has a significant number of mesenchymal stem cells (MSC) that could be easily acquired and cultured. In vitro, hUCMSCs has high proliferation and growth [⁶]. Previous in vitro researches have tried to explore and optimize the use of hUCMSCs for future application in regenerative dentistry [⁷,⁸,⁸,¹⁰]. Other study also showed that the proliferation potential of hUCMSCs when cultured in vitro was higher than other sources such as bone marrow MSCs [¹¹]. Many studies on in vitro culture and clinical application of MSC recommend to cultivate MSC under hypoxic conditions (1to 10O₂) [¹²]. Clinically, these will promote stem cell survival for application in hypoxic conditions, especially in regenerative dentistry, both for pediatric and adult patients.

Cobalt chloride (CoCl₂) as hypoxia-mimicking substance has been introduced and broadly used in many in vitro studies. Even though several researches have focused on the effect of hypoxia on dental derived stem cells, the effect of hypoxia on hUCMSCs need to be discussed. CoCl₂ solution is generally used as an alternative of modular incubator chamber [¹³]. Cobalt chloride is proven to improve the stemness of human dental pulp cells, this is an advantage as a method to increase the amount of cells without the loss of their capacity to differentiate in regenerative procedures [¹⁴]. CoCl₂ is water soluble which produced clear red solution when applied into cell culture to induce hypoxia [⁴]. CoCl₂ could mimic hypoxic condition through reduction or obstruction of HIF-1α degradation. The outcome of CoCl₂ was not significantly different from using hypoxic chamber [¹⁵,¹⁶] and in laboratory, it is hard to regulate and sustain steady oxygen tension [¹⁴]. Previous study with 100 μM of CoCl₂ concentration for hypoxia induction used other stem cells sources such as stem cells derived from human exfoliated deciduous teeth, adipose derived stem cells, and dental pulp stem cells [¹³,¹⁵,¹⁷], and one current study on hUCMSCs discuss a wide range of 10 μM and 250 μM but did not observe a longer time range [¹⁸].

HIF-1α was initially thought to be the key oxygen sensing subunit, which could upregulate the secretion of fibroblast growth factor (FGF)-2, vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, transforming growth factor (TGF)-b, and platelet derived growth factor (PDGF)-b, under hypoxia condition [¹⁹]. HIF-1α activation would affect self-renewal, proliferation, and colony forming activity of MSCs [²⁰]. Mammalian target of rapamycin (mTOR) controls metabolism and cell growth in response to nutrient, including O₂. mTOR activation would lead to stem cells growth and quiescence [²¹]. mTOR pathway has a crucial role as an integrator and sensor of a great variation of environmental signs that are associated to metabolic quiescence, cell cycle arrest, and biosynthetic requirements [¹⁸].

Cultured hUCMSCs in hypoxic condition is expected to improve proliferation of the cells through HIF-1α and mTOR. MSC culture proved that some cells proliferation under low O₂ pressure compared to normal atmospheric pressure. hUCMSCs that is being cultured under 2,5or 5O₂ concentration showing HIF-1α in significant amount [²²]. The other marker which plays a chief role in cells growth, survival, metabolism and proliferation is mTOR [²³]. This study assessed the effects of cobalt chloride as hypoxia mimicking agent on human umbilical cord mesenchymal stem cells (hUCMSCs) towards HIF-1α and mTOR expressions at 24, 48, and 72 hours for future use in regenerative dentistry.

Material and Methods

Ethical Clearance

This study was approved by The Health Research Commission for Ethical Clearance, Universitas Airlangga, Faculty of Dental Medicine (630/HRECC.FODM/XII/2021).

Isolation of hUCMSCs

Isolation, expansion, and culture of umbilical cord was done according to previously published method [²⁴]. Umbilical cord was provided from healthy donor with Caesarean operation without complications conducted at the Integrated Surgical Center RSUD Dr. Soetomo, Surabaya. The donor has approved and signed the informed consent. Healthy donor was chosen because based on our unpublished study, health status of the donor would affect the quality of the umbilical cord and the stem cells.

Hypoxic Treatment on hUCMSCs using CoCl₂

Hypoxia on hUCMSCs was done at passage 5. Hypoxic condition was done using chemical inducer (Hypoxia mimicking agent) Cobalt chloride hexahydrate solution (CoCl₂) in concentration of 100 μM was applied in the culture media to induce hypoxia [²⁵]. Then, hUCMSCs was incubated for 24, 48, and 72 hours. For the control groups hUCMSCs were incubated in normoxia condition (37°C; 5CO₂).

Immunofluorescence Test of HIF-1α and mTOR Expression

Cultured hUCMSCs from treatment groups were examined with the following procedures: fixation buffer 200 μL was added, then incubation was done for 20 minutes at 4°C, and rinsed with flowing buffer solution. Cells were permeabilized by addition of perm buffer, then incubated for 20 minutes 4°C and washed with flowing buffer solution again. After cells were fixated and/or permeabilized, the first antibody was being put inside and incubated for 25 minutes at 4°C, then, the cells were rinsed with flowing buffer solution. After the first antibody was attached to the cells, the second antibody was introduced and incubated for another 25 minutes at 4°C. The cells were washed again with flowing buffer solution. The antibody used were FITC HIF-1α (Bioss Antibodies, USA) to observe the expression of HIF-1α and FITC mTOR (Bioss Antibodies, USA) to observe the expression of mTOR. Immunofluorescence imaging was seen as green colored luminescence and captured using fluorescence microscope at 200× magnification (Olympus CKX53, Japan).

Statistical Analysis

Statistical analysis was performed using SPSS software (SPSS, USA). Data were analyzed statistically using One-way ANOVA and Tukey’s HSD with level of significance α = 0.05. The data was tested for normality using Shapiro-Wilk.

Results

HIF-1α Expression

HIF-1α expression under normoxia and hypoxia conditions in 24-, 48-, and 72 hours can be seen in Figures 1 and 2. Data analysis of HIF-1α expression in the study can be seen in Table 1. The data were distributed normally (p>0.05). Homogeneity test showed the variants of those groups were homogeny (p>0.05). One-way ANOVA test showed p = 0.015 (p<0.05) which meant there were significant differences between HIF-1α count on control and treatment groups. From multiple comparison Tukey HSD test, there were significant differences of expression between groups. Significant difference was found among the control and treatment groups. The highest HIF-1α expression was found in hypoxia 48 hours group (H48). From the data analysis, HMA CoCl₂ significantly increase the expression of HIF-1α in 24-, 48- and 72 hours.

Figure 1
Comparison of HIF-1α expression on hUCMSCs between control and treatment groups. HIF-1α expression on hUCMSCs marked with the presence of green colored fluorescence.

Figure 2
Bar chart showing mean and standard deviation of HIF-1α and mTOR expressions.

Table 1
Tukey HSD test results between the control and treatment groups of HIF-1α and mTOR expressions.

Groups	N	HIF-1α Expression Mean (SD)	p-value	mTOR Expression Mean (SD)	p-value
N24	3	18.19 ± 2.62	0.015*	21.29 ± 0.65	0.000*
N48	3	21.24 ± 0.18		23.28 ± 1.05
N72	3	14.45 ± 1.57		17.98 ± 1.84
H24	3	21.27 ± 4.82		26.41 ± 2.30^
H48	3	23.06 ± 1.45^		21.27 ± 0.30
H72	3	17.99 ± 1.84		14.45 ± 1.57

• *Significant at α = 0.05 (One-way ANOVA); ^Highest average score (Tukey HSD).

mTOR Expression

mTOR expression under normoxia and hypoxia conditions in 24-, 48-, and 72 hours can be seen in Figures 2 and 3. Data analysis of mTOR expression in the study can be seen in Table 1. The data were distributed normally (p>0.05). Homogeneity test showed the variants of those groups were homogeny (p>0.05). One-way ANOVA test showed p=0.000, which meant there were significant differences between mTOR count on control and treatment groups. From multiple comparison Tukey HSD test, there were significant differences of expression between groups. The highest expression was found on H24 group. Based on the data analysis, HMA CoCl₂ significantly increase mTOR expression from 24 hours treatment group.

Figure 3
Comparison of mTOR expression on hUCMSCs between control and treatment groups. mTOR expression on hUCMSCs marked with the presence of green colored fluorescence.

Discussion

This study used human umbilical cord mesenchymal stem cells because MSCs from umbilical cord have multipotent properties as an alternative treatment option for many diseases and abnormalities, easy to culture, has broad application and beneficial for in vivo applications [²⁶]. In this study, one donor is sufficient to provide the cells for assessments, as hUCMSCs are easily cultured and highly expandable in a relatively short time [²⁷,²⁸]. In vitro researches were conducted to study the complex processes in application and preservation of stem cells [¹³,¹⁴,¹⁷]. In this study, CoCl₂ concentration of 100 μM was used and the observation is expanded from 24, 48 to 72 hours.

HIF-1α acted as the main regulator of genes that could be induced in hypoxic condition. Main target gene of HIF-1α is connected to angiogenesis, proliferation, survival of the cell, and metabolism [¹⁹,²⁰]. Higher HIF-1α would maintain hUCMSCs’ stemness and decrease cell aging, where quiescent adult stem cells sustain a slow cycling state to evade cellular damage and to safeguard tissue regeneration capacity [²⁹]. A study shown the potential when modulating nutrition sensing and pathway response to improve survival and therapeutic features of MSCs. Several metabolic regulators are pleiotropic and have more and unwanted effect against cell properties, so it is important to understand intracellular tissue and metabolism to do rational approach when applying stem cell in tissue engineering for optimal therapeutic effect [³⁰].

The amount of HIF-1α expression based on the result in group with hypoxic CoCl₂ treatment showed higher fluorescence than in control group without hypoxic treatment, at 24 hours, 48 hours, and 72 hours. But, the highest expression in hypoxic treatment was found at 48 hours. The lowest HIF-1α expression was found at 72 hours. This score was parallel with previous study that shown the stabilization of HIF-1α protein on low oxygen condition. HIF-1α activity depended on oxygen level and HIF-1α target is set under normoxic or hypoxic condition could be a potential therapy target [³¹]. Another study also shown that in hypoxic condition, HIF-1α increased survival and suppressed apoptosis of MSCs. This study elaborated protective effect of HIF-1α against MSCs survival under hypoxic condition [³²].

Overall, both control and treatment groups, the highest expression of HIF-1α was found at 48 hours. HIF-1α expression at 24- and 72-hours control group only have slight differences. Highest increase could be seen on 48 hours control group. HIF-1α expressions are higher on hypoxic treatment group than the normoxic control group. The highest expression is at 24 hours and the difference is significant between control and treatment groups. The lowest expression is at 48 hours. Hypoxic condition made most eukaryotic cells changed their main metabolism strategy from more dominant mitochondrial respiration into glycolysis escalation to be able to maintain ATP level. Deprogramming of metabolism that was induced by hypoxia was the key to fulfill cellular energy needs during acute hypoxic condition. On transcription level, this metabolic switch could be arranged by few ways including HIF-1α [³³]. HIF-1α played an important role in hypoxic signal transduction process physiologically and pathologically. Gene target from activation of HIF-1α has many functions, such as glycolysis, glucose transport, erythropoiesis, and angiogenesis. A lot of these genes involved in proliferation, survival, and differentiation of the cells, such as EPO, VEGF, and many others [³³].

mTOR expression on normoxic control group was higher than in hypoxic treatment group at 48 and 72 hours, whilst lower expression was found at 24 hours control group compared to 24 hours hypoxia treatment group. The highest expressions of mTOR in this study was at 24 hours hypoxic group. On the other hand, the lowest mTOR expression was at 72 hours hypoxic group. Previous study showed that mTOR function is regulated by hypoxic condition [³⁴]. The result of this study was in line with study about hypoxic effect on migration and proliferation of umbilical cord blood MSCs. Hypoxic treatment increased mTOR phosphorylation at 24- and 48-hours incubation. However, mTOR expression at 48 and 72 hours were decreased. This would explain that prolonged exposure to hypoxia would lead to decreased expression of mTOR, as cells need oxygen for their metabolism. Other possibility is the reduced number of cells or other factors which need further exploration. After 24 hours of hypoxic treatment, fluorescence amount of p-mTOR (Ser2481 and Ser2448) increased to 356and 153from the control group [³⁵].

mTOR expression at 48 hours control group was increased and then decreased at 72 hours control group. The highest score on the control group was at 48 hours group, whereas on control group the 72 hours showed the lowest mTOR expression. mTOR played a vital role in regulating growth, proliferation, and cell survival that depended on nutritional condition, stress signal, and growth factor [³⁶]. mTOR is one of the proteins which mechanism affected by hypoxic condition. Hypoxic condition affected almost all aspects of cellular life, from cytokine secretion, mitogen, and extracellular matrix modulator. Hypoxic condition on mTOR could stimulate autophagy [³⁷]. Research on amnion-derived MSCs (AD-MSCs) showed in vitro hypoxic condition increased trophoblast proliferation and autophagy which could be observed from escalation of mTOR expression [³⁶].

mTOR expression at 24 hours hypoxia group was higher than 24 hours control group. This range was the highest between the normoxic and hypoxic group. On the other hand, the lowest expression was found at 48 hours. The 72 hours normoxic and hypoxic have the lowest expressions of mTOR. The result of previous study indicated that hypoxic condition increased proliferation and stem cell migration through FN-IN β1 expression that passed through PI3K/Akt, HIF-1α, and mTOR pathway. This research shown mTOR expression reached maximum after being given hypoxic treatment for more than 120 minutes. Besides that, HIF-1α expression also started to increase after hypoxic treatment for 6 hours [³⁸]. A study on placental chorionic plate-derived MSCs (CP-MSCs) also shown that hypoxic treatment induced stem cell factors (SCF) expression on CP-MSCs. Especially on SCF/c-kit pathway that increased self-renewal activity of CP-MSCs through paracrine / autocrine mechanism by autophagy which kept the balance of death and survival activity. Autophagy was an effective way to preserve healthy stem cell population by balancing elimination of abnormal cell with normal cell proliferation. Furthermore, this activity happened on the higher level on CP-MSCs if being compared with BM-MSCs by regulating mTOR phosphorylation. This research also shown that HIF-1α affected mTOR activity in hypoxic condition [³⁹].

In general, this study elucidated that in hypoxic condition, the expression of HIF-1α increased higher than in normoxic condition and mTOR expression on hypoxic condition also increased on early treatment if compared to in normoxic condition. Latest research shown that HIF-1α and mTOR were the main pathway to connect metabolism affected by hypoxia and proliferation and migration of hUCMSCs [³⁵]. This study proven that there is a connection between HIF-1α and mTOR. The possibility to use hUCMSCs at 48 hours of hypoxia pretreatment where both HIF-1α and mTOR were at the highest expression, as this is important factors in stem cell application, mainly in hypoxia microenvironment. This study used one donor for homogeneity, but this can also be the limitation of this study. The evaluation of hypoxia on heterogenous hUCMSCs from multiple donors can be suggested for future studies. Current study using hUCMSCs from one donor is sufficient to positively affect implant osseointegration [⁴⁰]. More donor might be required if there is a need for massive hUCMSCs productions or trials and for heterogeneity purposes. This research only focuses on the expression of HIF-1α and mTOR, therefore needed further researches to find other factors or expression as to optimize hUCMSCs use in regenerative dentistry.

Conclusion

CoCl₂ that is applied to human umbilical cord mesenchymal stem cells (hUCMSCs) could increase HIF-1α and mTOR expressions. The highest expression of HIF-1a was at 48 hours hypoxia and the lowest expression was at 72 hours hypoxia. Hypoxia increased mTOR expression at 24 hours and reached the lowest expression at 72 hours.

Data Availability

The data used to support the findings of this study can be made available upon request to the corresponding author.

• Financial Support
  None.

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Edited by

Academic Editor: Wilton Wilney Nascimento Padilha

Publication Dates

• Publication in this collection
  02 Feb 2024
• Date of issue
  2024

History

• Received
  07 July 2022
• Reviewed
  08 Dec 2022
• Accepted
  28 June 2023