Evaluation of characteristic of human turbinate derived mesenchymal stem cells cultured in the serum free media

<div><p>We evaluated the effect of serum-free and xeno-cultivation (SFXFM) on the characterization, proliferation, and differentiation properties of human nasal stem cells (airway tissue; hTMSCs). hTMSCs were isolated from 10 patients, after which patient samples were separated into two groups, an SFXFM group and a control group. The control group was treated with bovine serum-containing medium. FACS analysis revealed that SFXFM-cultured hTMSCs maintained a characteristic mesenchymal stem cell phenotype. hTMSC proliferation was not influenced by SFXFM. In addition, upregulation of IL-8 and GM-CSF and downregulation of RANTES expression were shown in response to SFXFM. Moreover, two-lineage differentiation properties (osteocyte and adipocyte) of hTMSCs were enhanced under SFXFM. Finally, the genetic stability of SFXFM-cultured hTMSCs was demonstrated by normal karyotype results. SFXFM enables good expansion, multipotentiality, and normal genotype maintenance of MSCs. Moreover, this approach serves as a substitute to conventional media for the cultivation of capable MSCs for upcoming medical applications.</p></div

Comparison of the osteogenic differentiation potentials of hTMSCs cultured under serum-free medium versus those of hTMSCs cultured under serum-containing medium.

<p>Under osteogenic conditions, alkaline phosphatase staining (x 400) of hTMSCs cultured in serum-free medium (left) and serum-containing medium (right) demonstrated similar levels of alkaline phosphatase expression, as assessed visually. The mRNA expression levels of osteocalcin, Runt-related transcription factor 2, and type I collagen in hTMSCs (B) were perceived by RT-PCR. hTMSCs cultured under serum-free medium showed higher expression of osteogenic differentiation markers versus hTMSCs cultured under serum-containing medium.</p

Karyotype analysis of hTMSCs cultured in serum-free medium.

<p>To check whether cells derived from serum-free cultivation showed the chromosomal stability or not, the cytogenetic karyotypes of cells at passage 3 (left) and 6 (right) were analyzed. All sex chromosomes were XY. No chromosome eliminations, displacements, or imbalances were observed.</p

Comparison of proliferation of hTMSCs cultured in serum-free medium versus that of hTMSCs cultured under serum-containing medium.

<p>A cellular proliferation assay was conducted for 7 days. hTMSCs cultured in serum-free medium showed rapid proliferation from 3 to 4 days. The proliferation patterns resembled those observed in MSCs cultured under serum-containing medium. Additionally, culture under serum-free medium did not affect the proliferation of hTMSCs.</p

Comparison of the adipogenic differentiation potentials of hTMSCs cultured in serum-free medium versus those of hTMSCs cultured in serum-containing medium.

<p>Under adipogenic conditions, adipogenesis was perceived after 2 weeks of culture by staining of intracytoplasmic microvacuole with Oil Red O. Visual assessment revealed similar levels of intracytoplasmic Oil Red O staining (x 400) in hTMSCs cultured under serum-free medium (left) and in hTMSCs cultured under serum-containing medium (right). The mRNA expression levels of peroxisome proliferator activated receptor r (PPARr) and AcylCoA synthetase (ACS) in hTMSCs (B) were detected by RT-PCR. hTMSCs cultured under serum-free medium exhibited higher expression of adipogenic differentiation markers compared with hTMSCs cultured under serum-containing medium.</p

Comparison of the chondrogenic differentiation potentials of hTMSCs cultured in serum-free medium versus those of hTMSCs cultured in serum-containing medium.

<p>Under chondrogenic conditions (A), visual assessment revealed similar levels of toluidine blue staining (x 400) in hTMSCs cultured under serum-free medium (left) and hTMSCs cultured under serum-containing medium (right). However, RT-PCR analysis of type II collagen and aggrecan mRNA expression in hTMSCs cultured under serum-free medium showed consistently increased expression, but much lower expression of chondrogenic differentiation markers compared with the levels in hTMSCs cultured under serum-containing medium.</p

Effects of serum-free cultivation on cytokine and chemokine release by hTMSCs.

<p>Release 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] from hTMSCs cultured under serum-free medium compared to serum-containing medium was evaluated with an enzyme-linked immunosorbent assay. hTMSCs cultured in serum-free medium exhibited upregulation of IL-8 and GM-CSF, but downregulation of IL-1β, IL-10, IL-12, RANTES, and TNF-α versus the control group. These patterns of cytokine and chemokine secretion were different from those of hTMSCs cultured under serum-containing medium.</p

Morphology (A) after primary explant culture and fluorescence-activated cell sorting analysis of human nasal inferior turbinate derived mesenchymal stem cells (hTMSCs) cultured under serum-free medium (B) and serum-containing medium (C).

<p>Cells in both groups attached to the culture dish and displayed a similar spindle-shaped, fibroblast-like shape (magnification x 100) (A). Flow cytometry analysis revealed that hTMSCs from both groups were positive for MSC markers (CD73, CD90, and CD105) and negative for hematopoietic cell markers (CD14, CD19, CD34, and HLA-DR) (B and C).</p