Increased proliferation and analysis of differential gene expression in human Wharton's jelly-derived mesenchymal stromal cells under hypoxia

Abstract

Multipotent mesenchymal stromal cells (MSCs) from Wharton's jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.

Keywords: Cell proliferation.; Hypoxia; Mesenchymal stem cells (MSCs); Transcription; Transformation markers; Wharton's jelly.

Fig 1

Growth kinetics and senescence in WJ-MSCs cultured under normoxia and hypoxia. (A) Growth curves of WJ-MSCs cultured under normoxia and hypoxia are demonstrated (B) Comparison of cumulative population doubling (PD) at each passage between different culture conditions: Normoxia and Hypoxia. Significant differences were observed between normoxia and hypoxia at later passages (p < 0.05). (C) Analysis of mean PD time in hour±SEM of WJ MSCs cultured under hypoxia and normoxia. (D) Senescence associated β- galactosidase staining of WJ-MSCs at passage 10 under normoxia and hypoxia. (E) Histogram of WJ-MSC area when cultured under hypoxia and normoxia at early (P2-P5) and late passages (P10). Area of individual cells was quantified via automated image analysis from bright-field images. Results represent the average of at least three culture replicates (n=3) with SEM. Scale bar=50µm. Abbreviation: WJ-MSCs, Wharton's jelly-derived mesenchymal stem cells; SEM- standard error mean.

Fig S1

Morphology and multilineage differentiation potential of WJ-MSCs cultured under hypoxic and normoxic condition till late passages. WJ-MSCs (P10-P13) were investigated for in vitro multilineage differentiation capacity when cultured under hypoxia and normoxia. Adipogenesis was confirmed by neutral oil droplet formation stained with Oil Red O at day 18 (B & C). Formation of mineralized matrix was detected by von Kossa staining at day 21 (E & F). Chondrogenic differentiation (n=2) was demonstrated by Alcian blue staining (H & I), 4x objective. Phase contrast images of adipogenesis (J), osteogenesis (K) and chondrogenesis (L). Non-induced control cultures in growth medium without adipogenic (A), osteogenic (D) or chondrogenic differentiation (G) stimuli are shown. Morphology of WJ-MSCs, cultured under normoxia and hypoxia, as observed at passage 9 (M & N). Representative results of three independent experiments are shown. Abbreviation: WJ-MSCs- Wharton's Jelly derived mesenchymal stem cells. (Scale bar for A, B, C =25µm; D-F and J-N=50µm).

Fig 2

Detection of surface marker expression of WJ-MSCs cultured under hypoxia and normoxia by flow cytometry analysis. WJ-MSCs were cultured for ten passages under hypoxia and normoxia, labeled with the indicated antibodies and analyzed by flow cytometry. An open area represents an antibody isotype control for background fluorescence and a shaded area shows signal from MSC surface marker antibodies. Representative histograms are depicted. Abbreviations: WJ-MSC- Wharton's Jelly derived mesenchymal stem cells.

Fig 3

Karyotype and RT-PCR analysis of transformation markers and notch pathway genes and validation of array data for WJ-MSCs cultured under hypoxia and normoxia. (A) Reverse transcription polymerase chain reaction (RT-PCR) analysis of DNA repair, tumor suppressor and oncogenes of WJ-MSCs cultured under hypoxic and normoxic conditions for 10 passages. Expression of genes RAD51, ERCC3, XRCC4, p21 and p53 were analyzed by real-time RT-PCR using SYBR green reagent and values are normalised to expression of 18s ribosomal RNA. Bars represent the mean ± SE of the ratio of message expressed under hypoxia as compared to that under normoxia for three independent experiments performed in duplicate. (B) Expression p16 and c-Myc using semi-quantitative RT-PCR. Band densities were quantified and plotted. (C) Karyotype analysis of WJ-MSCs using standard Giemsa-banding procedure represents Normal 46, XY karyotype of WJ-MSCs cultured under hypoxic and normoxic conditions. A representative analysis of three independent experiments is shown (E) Abbreviations: WJ-MSCs - Wharton's Jelly derived mesenchymal stem cells; SE- standard error.

Fig 4

(A) RT-PCR based comparison of HIFs and Notch pathway genes expressed by WJ-MSCs when exposed to hypoxia, with normoxia as control, for 48 hrs and for ten passages. Results are representative of at least three independent experiments. RT-PCR analysis of select differentially expressed genes to validate PCR array data. cDNA was pooled from three different samples each of hypoxic and normoxic WJ-MSCs at passage 11. (B-E) Expression of FLT1, DESMIN, IFITM1 and OCT4 was analyzed by real-time PCR using SYBR green reagent and values were normalized to 18s rRNA. (F & G) Semi-quantitative RT-PCR analysis of GATA6 and SOX17 with intensity of each band being measured by densitometry and plotted. 18S ribosomal RNA was used as an internal control for all semi-quantitative RT-PCRs. Abbreviations: WJ-MSCs - Wharton's Jelly derived mesenchymal stem cells; SE- standard error.