Hypoxia Induce Stemness Gene Expression of h-UBCMSC
Arini Dewi Antari1,2, Agung Putra1,2*,, Azizah Retno Kustiyah1,2, Eko Setiawan1,2, Vito Mahendra1,2
*Corespondence [email protected]
1Medical faculty of Sultan Agung University, Semarang, Indonesia
2Stem Cell and Cancer Research Laboratory of Sultan Agung University
INTRODUCTION
Mesenchymal stem cell (MSC) provide a promising stem cell source for regenerative medicine due to their ability to self-renewal and differentiate toward various lineages, including adipocytes, osteoblast and chondrocytes (Samsonraj et al., 2017). MSC can be isolated from umbilical cord, adipose tissues, dental tissues and bone marrow (Ullah et al., 2015). Moreover, in clinical applications, systemic administration of MSC requires a large number of cells (1-10 × 106 cells / kg) to obtain the maximum therapeutic effect thus a large-scale expansion in vitro needed to produce large number of cells (Antebi et al., 2018). A previous study has shown that long-term in vitro culture may lead MSC to senescence, reduced potential differentiation, morphological abnormalities, low specific surface marker expression, and ends with the cessation of cell proliferation activity which results in spontaneous differentiation. Therefore, provide a specific niche is essential to extend the life span of stem cells. (Liu, 2017).
Previous study reported that MSCs are found in a niche under hypoxic conditions. Niche provides an environment wherein MSC can renew itself and maintain an undifferentiated state (Liu, 2017). Other studies also showed that MSCs that are cultured in hypoxic conditions (2-5%
oxygen) have a rapid cell growth and exhibit higher rates of proliferation and better retain their stem cell properties (Haque et al., 2015). Nanog, SOX2 and OCT4 are pluripotence genes that is essential for maintenance undifferentiated state and self-renewal of stem cell (Zomer et al., 2015). In this study, we investigate the effect of hypoxia (5% O2) 24h in enhancing MSC stemness through analyzing CDX Nanog, OCT4 and SOX2 relative expression.
METHODS
Mesenchymal Stem Cell Isolation and Cultivation
The Ethics Committe of the Medical Faculty Sultan Agung Islamic University of Semarang approved this study and all patients gave written inform consent. Umbilical cord were obtained from pregnancy women after caesarean deliveries. Umbilical cords were chopped into small pieces. Cord pieces were cultivated in Dulbecco`s Modified Eagle Media (DMEM) (Sigma-Aldrich, Louis St, MO) supplemented with 1% antibiotic/antimycotic (Gibco™
Invitrogen, NY, USA) and 10% Fetal Bovine Serum (FBS) (Gibco™ Invitrogen, NY, USA) at 37°C and 5% CO2. The medium changed every 3 days. When 80% confluence was reached, cells were passaged with trypsin EDTA. The fourth passage cells were used for experiments. Cell that had undergone 4 passage were use for this study.
MSCs characteristic
hUC-MSC differentiation potential was determined using osteogenic differentiation assay. hUC-MSCs-like at passage 4 were detached using enzymatic protocol and reculture in T25 flask containing standard medium. After 90% confluency, the standard medium was replaced 12 h incubation, hUC-MSCs were cultured in the medium of osteogenic differentiation containing DMEM (Sigma-Aldrich, Louis St, MO), supplemented with 10% FBS (Gibco™
Invitrogen, NY, USA), 10-7 mol/L/ 0.1 µM dexamethasone, 10 mmol/L ß glycerophosphate, and 50 µmol/L ascorbate-2phosphate (Sigma-Aldrich, Louis St, MO) in 5% CO2 and at 37°C for 21 day incubation. The fixed cells were stained with 0.2 % Alizarin Red solution (Sigma- Aldrich) to represent calcium deposition.
The immunophenotypes of hUC-MSCs were analyzed at fourth passage. UC-MSCs were stained with antibodies conjugated: fluorescein isothiocyanate (FITC)-conjugated CD90, Allophycocyanin (APC) conjugated CD73, Peridinin Chlorophyll Protein Complex (PerCP)- conjugated CD105 and phycoerythrin (PE)-conjugated Lin monoclonal antibodies for 30 min at 4°C in the dark. The fluorescence intensity of the cells was evaluated through flow cytometry (BD Bioscience, Franklin Lakes, NJ, USA).
RT-qPCR
RNA was isolated from cultured cells using TRI Reagent® (Sigma-Aldrich). The extracted RNA was measured by QuantusTMFluorometer. 1 µg of total RNA sample was used for cDNA synthesis using Enhanced Avian RT First Strand Synthesis Kit (Sigma - Aldrich). cDNA sample was amplified and quantified using KAPA SYBR® FAST qPCR Master Mix (2X) Kit.
Each cycle consisted of predenaturation of 95°C for 10 min, denaturation at 95°C for 15 s and annealing at 57°C for 1 min. For each sample, a non-RT control was used in parallel to detect any potential nonspecific amplification of contaminated genomic DNA. The genes of interest that were analyzed by RT-qPCR are listed in Table1. The relative expression of gene was determined using Eco Real-Time PCR Illumina®.
Table 1
Gene Primer Sequence
β actin Forward 5’-CACCATTGGCAATGAGCGGTTC-3’
Reverse 5’-AGGTCTTTGCGGATGTCCACGT-3’
NANOG Forward 5’-TTTGTGGGCCTGAAGAAAACT-3’
Reverse 5’-AGGGCTGTCCTGAATAAGCAG-3’
OCT 4 Forward 5’-TGCAGAAAGAACTCGAGCAA-3’
Reverse 5’-ACACTCGGACCACATCCTTC-3’
SOX 2 Forward 5’-AGAACCCCAAGATGCACAAC-3’
Reverse 5’-ATGTAGGTCTGCGAGCTGGT-3’
RESULT
Characterization of the MSCs
The hUC-MSC cells presented the capacity for a plastic attachment and show spindle-like morphology. hMSCs isolation was carried out based on the plastic adherent capability under standard conditions (37°C, 5% CO2). The cells showed fibroblast-like (spindle shape) and peculiar morphology (Figure 1A). Osteocyte differentiations were determined using a standard protocol. The calcium deposits were confirmed by Alizarin Red. The positive osteogenic cells
under the microscope were stained bright red (Fig. 1). The mean of the mesenchymal surface markers was99,9% for CD90, 99,2% for CD73, and 95,9% for CD105(Fig.2).
A
B C D
Figure 1: a) HUC-MSCs-like showed fibroblast-like or spindle-shaped characteristic; b) Red- bright colour after Osteogenic differentiation hMSCs-like was evidenced by mineralised matrix formation using Alizarin red staining
Pluripotence Genes Expression
There was a significant downregulation both the angiogenic and pluripotence gene, VEGF, OCT- 4, Nanog, CDX2 and SOX2 in MSCs cultured under both 5% Oxygen (p < 0.0001) at 24 h.
A B C
C D DISCUSSION
Aerobic organisms require oxygen (O2) to produce energy. For this reason, O2deprivation creates significant stress in living cells. O2deprivation is also paradoxically linked to the inappropriate accumulation of free radicals, which cause additional stress on proteins and DNA in the cell. During low O2(hypoxic) conditions, therefore, cells activate a number of adaptive responses to match O2supply with metabolic, bioenergetic, and redox demands. Cells temporarily arrest in the cell cycle, secrete survival and proangiogenic factors, and reduce energy consumption. These events are coordinated by various cellular pathways, including the gene regulation by hypoxia inducible factors (HIFs). HIF is recognized as a key modulator of the transcriptional response to hypoxic stress. Besides its adaptive function in cellular stress responses, recent work has also revealed important roles for HIF in both physiological and pathological processes.
Cells adapt to low oxygen levels by altering the expression of genes involved in cell life and apoptosis. Hypoxia inducible factors (HIFs) are major regulators of gene expression during hypoxia. HIF-1α and HIF-2α are the two main subunits of HIF that play a role in hypoxic conditions in humans and are activated differently depending on the duration of hypoxia. HIF-1α plays a role during acute hypoxia and HIF-2α plays a role during acute hypoxia. HIF-1α expression was significantly increased under hypoxic conditions within 24 hours and then decreased. Meanwhile, HIF-2α expression increased under hypoxic conditions for > 24 hours (Saxena et al., 2019; Zhi et al., 2018). HIF-2α acts as a Nanog regulator that contributes to the formation of multiprotein complexes to increase Nanog expression. In hESC culture under hypoxic conditions, endogenous HIF-2α binds directly to HRE (Hypoxic Response Element) on the proximal promoter of Nanog (Petruzzelli et al., 2014). This is a possibility that causes a
decrease in the relative expression of Nanog at 5% hypoxic conditions for 24 hours because at this duration there has not been an increase in HIF-2α expression which has resulted in the unexpression of Nanog.
