(a) RT-PCR analysis of gene expression profiles related to stem cells (and 0

(a) RT-PCR analysis of gene expression profiles related to stem cells (and 0.005. by immunofluorescence staining for these antigens. Osteogenic Differentiation For osteogenic induction in vitro, SSPCs (1.25 105 per dish) were seeded on 35-mm dishes (Corning, Corning, NY) and cultured in the growth medium until the cells reached confluence. To induce osteogenic differentiation, the medium was changed to an osteogenic medium consisting of l-ascorbic acid phosphate and -glycerophosphate. One week after osteogenic induction, total RNA was extracted and analyzed for osteogenic markers by RT-PCR. After 8 weeks, calcium deposits created by osteoblast on the dishes were recognized by staining with 2% alizarin reddish S (pH 4.2; Sigma-Aldrich).19,20 Circulation Cytometric Analysis of SSPCs Passage 2 stem cells were cultured under the growth medium. Single-cell suspensions (2 105/100 L each marker) were incubated with R-phycoerythrin (PE) or fluorescein isothiocyanate (FITC) conjugated mouse monoclonal antibodies specific to cell surface markers (1 g/100 L each) for 30 minutes on snow. As negative settings, isotype-matched mouse immunoglobulins were incubated instead of the main antibodies. We analyzed the samples and calculated the data using circulation cytometry (FACSCalibur; BD Biosciences). Semiquantitative RT-PCR Primers were designed with Primer-BLAST software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/, National Center for Biotechnology Info [NCBI], Bethesda, MD). Total RNA was isolated from your cultures (TRIzol Reagent; Invitrogen), according to the manufacturer’s protocols. The cDNA HA6116 was synthesized from 500 ng of total RNA (Superscript III; Invitrogen). PCR was then performed with gene specific primers Itraconazole (Sporanox) and PCR supermix (Platinum; Invitrogen). The primers used are demonstrated in Table 1. The amplified PCR products were subjected to 2% agarose gels which contain ethidium bromide and visualized under UV light. Band intensity was measured by using NIH image-J software and normalized to GAPDH (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). Table 1. Primers for Reverse Transcription-Polymerase Chain Reaction 0.005. (c) Populace doublings of multicolony-derived mouse SSPCs and BMMSCs. Data are mean SD of three samples. * 0.05 SSPCs versus BMMSCs. Next, we compared the gene manifestation profile of SSPCs to that of BMMSCs to identify putative SSPC-specific markers. Semiquantitative reverse transcription (RT)-PCR analysis showed that SSPCs communicate high levels of stem cell markers (neurogenic and ocular developing marker), vimentin (mesenchymal cell marker), (-clean muscle actin, cellular activation, and myogenic marker), and type I collagen (major composition protein of sclera), but failed to communicate cytokeratin 12 (corneal epithelium marker), cytokeratin 19 (corneal limbal stem cell Itraconazole (Sporanox) specific marker), and desmin (mesoderm stem cell marker) transcripts. This implies the isolated cells were Itraconazole (Sporanox) not from a corneal epithelial, corneal limbal stem cell, or muscular source. Immunocytochemical staining further confirmed that all SSPCs indicated high levels of type I collagen and -SMA compared with BMMSCs (Fig. 2b). Open in a separate window Number 2. Phenotype for SSPCs. (a) RT-PCR analysis of gene manifestation profiles related to stem cells (and 0.005. RT-PCR ( em right /em ) showed gene manifestation profiles related to adipogenesis in SSPCs compared to BMMSCs. (b) Chondrogenic differentiation of SSPCs and BMMSCs. Chondrogenic differentiation was assessed by Alcian blue staining for proteoglycans and the manifestation of aggrecan, type II collagen, and SOX9 ( em arrows /em : positive nuclear stain). Pub, 100 m. (c) Neurodifferentiation of SSPCs. Immunofluorescent staining showed SSPCs expressing 3-tubulin, Nestin, and NFM. RT-PCR ( em right /em ) confirmed that gene manifestation profiles of SSPCs indicated neural markers as explained above. After 4 weeks of tradition in the presence of B27 product, bFGF (40 ng/mL), and epidermal growth element (20 ng/mL; Neural Diff.+), manifestation levels of NFM and 3-tubulin were upregulated when compared with levels in regular tradition conditions (Neural Diff.?). However, manifestation levels of Nestin remained the same after treatment. When cultured under neurogenesis conditions, SSPCs lost their fibroblastic morphology, developed multicytoplasmic Itraconazole (Sporanox) processes, and created a bipolar axon neuronlike morphology (Fig. 3c). To elucidate the neural-differentiation potential of SSPCs, we examined the manifestation of neural markers in SSPCs. We found that cultured SSPCs indicated Itraconazole (Sporanox) several neural cell markers including 3-tubulin, nestin, and NFM, as measured by immunocytochemical staining and RT-PCR analysis. After 4 weeks of neural inductive tradition, manifestation levels of nestin remained the same, but 3-tubulin and NFM improved (Fig. 3c). To induce osteogenic differentiation, confluent cells were cultured in osteo-inductive medium for 4 weeks. SSPCs failed to form mineralized nodules, as observed in BMMSCs by alizarin reddish staining (Fig. 4a, remaining). To further confirm that SSPCs lack osteogenic.