Functional characterization of human mesenchymal stem cells that maintain osteochondral fates

Abstract

Adult stem cells are essential for tissue renewal, regeneration and repair, and their expansion in defined culture medium is on focus for regenerative medicine and genetic pathologies. The bone marrow has been shown to be very rich is pluripotent mesenchymal stem cells (MSCs) capable of forming bone, cartilage and also may give rise, to neurons and astrocytes in vivo and in vitro. MSCs can be isolated and expanded in culture, but human cells cannot be verified for a cartilage or a bone fate by transfer experiments. Accordingly, here we used different approaches to characterize hMSCs osteoblastic differentiation in vitro. hMSCs grown in culture in the presence of fetal bovine serum (FBS) expressed the bone-specific transcription factor Runx2/AML3. When cells were incubated in osteoblastic differentiation medium, cells expressed transcripts belonging to the signaling of Indian HH-PTHrP axis, GLI transcription factors, and bone target genes including osteopontin. The HH pathway proved to be functional since it induced cells to grow. Cells growing or differentiating to osteoblasts presented the Runx2/AML3 transcription factor, its partner CBFB, and Smad2/3 at the nuclei associated with the nuclear matrix. Furthermore, Runx2/AML3 was observed to co-localize with SC35 to the nuclear intermediary filaments. These data support the notion that hMSCs isolated from human bone are or become bone progenitor cells upon culture. In the absence of FBS and in the presence of insulin or prolactin, cells show cytoskeletal organization and an AP-1 transcription site activity resembling proliferative osteochondrocytes while cells in the presence of dexamethasone and added prolactin or TGF-beta resembled differentiated osteoblasts. These specific cellular conditions match those observed during endochondral bone formation.

Signaling and biological effects of glucagon-like peptide 1 on the differentiation of mesenchymal stem cells from human bone marrow

“…The characteristics of the same batches of hMSCs used here have been reported in previous studies (31) and confirmed by internal control experiments showing the appearance of osteocytic and chondriocytic phenotypes in response to standard protocols (31). The mRNA and protein of the GLP-1R were identified in hMSCs, which is consistent with the actions of its agonists on the processes we have described.…”

“…All procedures were carried out according to the Declaration of Helsinki II after permission had been obtained from the Hospital Universitario La Princesa Ethics Committee together with the volunteers' consent. The isolation and expansion of hMSCs from 36 healthy donors (between 24 and 52 yr old) were carried out as described previously (31). Cells were grown in a standard culture medium consisting of Dulbecco's modified Eagle's medium-low glucose (DMEM-LG; Life Technologies), 15% fetal bovine serum (FBS) from selected batches (Hyclone, Madrid, Spain), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM glutamine (BioWhittaker, Madrid, Spain) at 37°C and 5% CO 2.…”

Surface modification, characterization and biofunctionality of pegylated titanate films obtained by the sol‐gel method

“…High TIPT insertion in the hybrid films (PEG60) induced a fibroblastic-like morphology with the characteristics described in proliferating cells. [8] We observe slightly Y-shaped cells with a homogeneous distribution on the surface. In parallel, a huge increase in cell density is observed with respect to the pure PEG film.…”

Monolithic 3D labs- and organs-on-chips obtained by lithography-based ceramic manufacture

“…After cell culture, samples were processed as previously described [24,25]. Briefly, the monolithic ceramic microsystems were rinsed twice with ice-cold PBS and fixed in 3.7% formaldehyde in PBS for 30 min at room temperature (RT), then washed and in PBS and either used or kept at 4°C.…”