Characteristics of Human Nasal Turbinate Stem Cells under Hypoxic Conditions

Abstract

This study investigated the influence of hypoxic culture conditions on human nasal inferior turbinate-derived stem cells (hNTSCs), a subtype of mesenchymal stem cells (MSCs). It aimed to discern how hypoxia affected hNTSC characteristics, proliferation, and differentiation potential compared to hNTSCs cultured under normal oxygen levels. After obtaining hNTSCs from five patients, the samples were divided into hypoxic and normoxic groups. The investigation utilized fluorescence-activated cell sorting (FACS) for surface marker analysis, cell counting kit-8 assays for proliferation assessment, and multiplex immunoassays for cytokine secretion study. Differentiation potential-osteogenic, chondrogenic, and adipogenic-was evaluated via histological examination and gene expression analysis. Results indicated that hNTSCs under hypoxic conditions preserved their characteristic MSC phenotype, as confirmed by FACS analysis demonstrating the absence of hematopoietic markers and presence of MSC markers. Proliferation of hNTSCs remained unaffected by hypoxia. Cytokine expression showed similarity between hypoxic and normoxic groups throughout cultivation. Nevertheless, hypoxic conditions reduced the osteogenic and promoted adipogenic differentiation potential, while chondrogenic differentiation was relatively unchanged. These insights contribute to understanding hNTSC behavior in hypoxic environments, advancing the development of protocols for stem cell therapies and tissue engineering.

Keywords: hypoxia; stem cells; turbinates.

Figure 1

Schematic of the hypoxic cultivation set up. hNTSCs and a gas-controlling agent were placed in a gas-barrier bag, which was sealed with a clip to lower the oxygen saturation within the bag. The oxygen meter showed an initial oxygen level of 13.4% (left). After 10~20 min, the oxygen meter showed that the desired oxygen level of 5% was achieved (middle). Once reaching 5% oxygen, the gas-controlling agent was isolated from the hypoxic cell cultures using another clip (right).

Figure 2

Cell morphology after primary explant culture (A) and fluorescence-activated cell sorting analysis (B) of hNTSCs cultured under hypoxic and normoxic conditions (sample, n = 1). Cells in both groups adhered to the culture dish and displayed a similar spindle-shaped, fibroblast-like morphology (100× magnification). Scale bar: 10 μm (A). Flow cytometry analysis after three passages confirmed that hNTSCs from both groups were positive for CD29, CD73, and CD90, and negative for CD14, CD34, and HLA-DR (B).

Figure 2

Cell morphology after primary explant culture (A) and fluorescence-activated cell sorting analysis (B) of hNTSCs cultured under hypoxic and normoxic conditions (sample, n = 1). Cells in both groups adhered to the culture dish and displayed a similar spindle-shaped, fibroblast-like morphology (100× magnification). Scale bar: 10 μm (A). Flow cytometry analysis after three passages confirmed that hNTSCs from both groups were positive for CD29, CD73, and CD90, and negative for CD14, CD34, and HLA-DR (B).

Figure 3

Effects of serum-free cultivation on cytokine and chemokine secretion by hNTSCs (sample, n = 5). The supernatants of hNTSC cultures under normoxic and hypoxic conditions were evaluated via enzyme-linked immunosorbent assay for the secretion of cytokines and chemokines IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IP-10 (CXCL10), RANTES (CCL5), TNF-α, GM-CSF, and IFN-γ. In both groups, the levels of GM-CSF, IL-6, IL-8, IP-10 (CXCL10), RANTES, and TNF-α increased during the cultivation period, whereas the IFN-γ level decreased during the cultivation period. These cytokine and chemokine secretion patterns were similar to those of hNTSCs from normoxic cultivation. Error bars are standard errors. t-test was used for statistical analysis.

Figure 4

Comparison of hNTSC proliferation between hypoxic and normoxic cultivation (sample, n = 5). The cellular proliferation assay was performed over a 7-day period. hNTSCs from normoxic cultivation exhibited rapid proliferation after day 1 through day 7. The proliferation patterns were similar to those of MSCs grown under hypoxic cultivation. Error bars are standard errors. t-test was used for statistical analysis.

Figure 5

Comparison of the osteogenic differentiation potential of hNTSCs cultivated under hypoxic and normoxic conditions (sample, n = 5). hNTSCs cultured in osteogenic induction medium showed similar levels of alkaline phosphatase staining by visual assessment (200× magnification) between hypoxic ((A); right) and normoxic ((A); left) culture conditions (Scale bar: 10 μm). However, mRNA expression of the osteogenic differentiation markers Runx2, type I collagen (early stage of osteogenic differentiation), and osteocalcin (late markers of osteogenic differentiation), detected with RT-PCR, was higher in hNTSCs under normoxic cultivation than hypoxic cultivation (B). Error bars are standard errors. t-test was used for statistical analysis.

Figure 6

Comparison of the adipogenic differentiation potential of hNTSCs cultivated under hypoxic and normoxic conditions (sample, n = 5). hNTSCs cultured in adipogenic induction medium showed similar levels of intracytoplasmic lipid droplet staining with Oil Red O by visual assessment (200× magnification) between hypoxic ((A); right) and normoxic ((A); left) culture conditions (Scale bar: 10 μm). Panel show graphs the area ratio (%) of Oil Red O staining as determined by ImageJ. Error bars are standard deviations (B). In mRNA expression by RT-PCR, ACCS2 was significantly higher in control, but PPARγ and C/EBPα were significantly higher on day 14 of hypoxic condition. Error bars are standard errors (C). t-test was used for statistical analysis.

Figure 7

Comparison of the chondrogenic differentiation potential of hNTSCs cultivated under hypoxic and normoxic conditions (sample, n = 5). hNTSCs cultured in chondrogenic induction medium showed similar levels of Alcian blue staining by visual assessment (200× magnification) between hypoxic ((A); right) and normoxic ((A); left) culture conditions (Scale bar: 10 μm). hNTSCs under both culture conditions exhibited a similar pattern of increasing mRNA expression of the chondrogenic differentiation markers SOX9 and aggrecan over culture time based on RT-PCR analysis (B). Error bars are standard errors. t-test was used for statistical analysis.