Histological study provided evidence of cartilage and bone-like tissue formation. inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-1-F2 and rhBMP-2, achieve skeletogenic potential and growth and promote their osteogenic maturation remain poorly defined, it is now well established that members of the transforming growth factor-beta (TGF-) family play a prominent role in the development, growth and maintenance of the vertebrate skeleton [13,14]. The effect of TGF-1 around the proliferation and osteoblastic differentiation of MSCs < 0.05 and < 0.01, respectively) when compared with controls. In rhTGF-1-F2-treated cultures induced also with a pulse of rhBMP-2 on day 14 slightly lower cell numbers were obtained, only significantly different compared to control group (< 0.05 at day 16). Open in a separate window Physique 1 Quantification of DNA content as indication of cell replication. Values represent the PRDM1 means SD, = 3; * < 0.05, ** < 0.01. Under a phase contrast microscope, these cells cultured with rhTGF-1-F2 (in absence or presence of rhBMP-2) increased in size and formed well-defined colonies at the end of differentiation period (Physique 2). BM-derived cells cultured under control conditions never formed colonies and presented a different morphology. Open in a separate window Physique 2 Cell morphology in 3D (three-dimensional) collagen matrix at the end of differentiation period (day 16). (a) Control cultures in the absence of growth factor; (b) Cell cultures in presence of rhTGF-1-F2; and (c) Cell cultures in presence of rhTGF (recombinant human transforming growth factor)-1-F2 and the pulse of rhBMP (recombinant human bone morphogenetic protein)-2. Bars, 200 m. 2.2. Flow Cytometry Analysis of Cells CBL-0137 ADH (Adherent culture) culture showed the immunophenotype described for MSCs, where most of the cells expressed CD29, CD105, CD166, CD271 and STRO-1, and were unfavorable for CD34, CD45 (data not shown). However, the profiles of 3D culture revealed a heterogeneous cell populace, where positive cells for hematopoietic (CD34, CD45), mesenchymal (CD29, CD105, CD166, CD271, STRO-1), and endothelial markers (CD34, CD133) were found CBL-0137 (Physique 3). Comparing 3D cultures at days 10 and 16, CD166+ population decreased CBL-0137 over time (17.5%, < 0.05), while there was an increase in the expression of some mesenchymal cell markers such as CD29, CD271 and STRO-1 (+29.2%, +25.1%, +39.3%, < 0.001, respectively). In addition, the endothelial progenitor cells (EPCs)-like populace (CD34+/CD133+/CD45?) decreased significantly (?14.8%, < 0.001). Open in a separate window Physique 3 Immunophenotype profiles of 3D culture cells for hematopoietic, mesenchymal and endothelial markers. Representative FACS (fluorescence-activated cell sorting) analysis at days 10 (a) and 16 (b). 2.3. Quantitative Real Time RT-PCR (RT-qPCR) Analysis Pluripotency of embryonic stem cells is usually controlled by defined transcription factors, such as Nanog, Oct4 and Sox2. We analyzed the expression of mRNA for these pluripotent specific markers by RT-qPCR at days 10 and 14, and the signal was compared to ADH culture (Physique 4). Nanog, Oct4 and Sox2 were highly expressed in both 3D cultures at days 10 and 14 but was absent in ADH cells (< 0.001). In addition, we evaluated the osteoblastic potential of 3D culture cells at the end of the differentiation period (day 16), analyzing the expression of mRNA Bsp (Bone sialoprotein), Osx (Osterix) and Oc (Osteocalcin) (Physique 4). Bsp and Osx mRNA displayed the highest expression levels in 3D cultures (< 0.001 ADH). Oc mRNA was also detected in 3D culture cells, but its expression level was significantly lower than that found in ADH cells (< 0.001). Comparison within the 3D culture showed that Osx and Oc mRNA levels were significantly different with or without the pulse of rhBMP-2.