Isolation and characterization of bone marrow-derived mesenchymal progenitor cells with myogenic and neuronal properties

Abstract

Sphere formation has been utilized as a way to isolate multipotent stem/progenitor cells from various tissues. However, very few studies on bone marrow-derived spheres have been published and assessed their multipotentiality. In this study, multipotent marrow cell populations were isolated using a three-step method. First, after elimination of hematopoietic cells, murine marrow-derived adherent cells were cultured in plastic dishes until small cells gradually appeared and multiplied. Cells were then cultured under non-adherent conditions and formed spheres that were immunopositive for a neural precursor marker, nestin. RT-PCR analysis also revealed that the spheres were positive for nestin in addition to PPARgamma, osf2, SOX9, and myoD, which are markers of precursors of adipocytic, osteoblastic, chondrocytic, and skeletal myeloblastic lineages, respectively. Finally, spheres were dissociated into single cells and expanded in adherent cultures. Under appropriate induction conditions, the sphere-derived cells acquired the phenotypic properties in vitro of neurons, skeletal myoblasts, and beating cardiomyocytes, as well as adipocytes, osteoblasts, and chondrocytes. Next, sphere-derived cells were transplanted into murine myocardial infarction models. One month later, they had become engrafted as cardiomyocytes, and cardiac catheterization showed significant functional improvements. Thus, sphere-derived cells represent a new approach to enhance the multi-differentiation potential of murine bone marrow.

CD45-CD14+CD34+ Murine Bone Marrow Low-Adherent Mesenchymal Primitive Cells Preserve Multilineage Differentiation Potential in Long-Term In Vitro Culture

“…Recent study by Shiota and co-workers showed the impact of sphere formation on following differentiation of murine MSC towards several lineages, but not into endothelium (Shiota et al, 2007). Similarly, significant change in differentiation potential of low-adherent MSC after sphere formation was observed in the current study, which, however, included also endothelial differentiation.…”

“…The sphere formation has been initially described as a feature of neural stem cells (Reynolds and Weiss, 1992), but other reports indicated this phenomenon as appropriate method for isolation of multipotent stem cells from a multitude of sources and differentiating them to various cell lineages (Shiota et al, 2007). Since the CD14 +…”

Pax3 activation promotes the differentiation of mesenchymal stem cells toward the myogenic lineage

“…Unlike other mesenchymal lineages (such as adipocytes or osteoblasts), skeletal myoblasts have not been easily differentiated from MSCs. Initial induction protocols to differentiate MSCs into myogenic progenitors have employed 5-azacytidine [11], a strong demethylating agent associated with muscle gene activation, in particular MyoD [12][13][14]. The drawback of this non-physiological method is excessive cell death, which results in virtually none [15] or very few surviving cells that are then endowed with myogenic features (not shown).…”

Mesenchymal Stem Cells in CNS Regeneration

“…Other methods to induce MSCs into cells with neural characteristics include: transfection of MSCs with Noggin and Notch transcription factors (Dezawa et al, 2004;Kohyama et al, 2001); manipulation with surface proteins of culture substrate (Qian & Saltzman, 2004); co-culturing MSCs with NSCs or neural cells (Alexanian, 2005;Chu, Yu, Zhang, & Yu, 2008;Krampera et al, 2007;Wislet-Gendebien et al, 2005;Y. Q. Zhang et al, 2010); and growing MSCs as spheres in cultures (Shiota et al, 2007), transfection of MSCs with microRNA-9 (Jing et al, 2011). In several other studies, MSCs were turned into multipotent stage and then induced into neural cell lineages, by exposing them to appropriate neural differentiation conditions (Alexanian, 2007;Kohyama et al, 2001;Qu et al, 2004).…”