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.

Although embryonic stem (ES) cells are capable of unlimited proliferation and pluripotent differentiation, effective preparation of neural stem cells from ES cells are not achieved. Here, we have directly generated under the coculture with dissociated primary neurosphere cells in serum-free medium and the same effect was observed when ES cells were cultured with conditioned medium of primary neurosphere culture (CMPNC). ES-neural stem cells (NSCs) could proliferate for more than seven times and differentiate into neurons, astrocytes, and oligodendrocytes in vitro and in vivo. The responsible molecule in CMPNC was confirmed by matrix-assisted laser desorption͞ionization time-of-flight mass spectrometry, which turned out to be cystatin C. Purified cystatin C in place of the CMPNC could generate ES-NSCs efficiently with self-renewal and multidifferentiation potentials. These results reveal the validity of cystatin C for generating NSCs from ES cells.coculture system ͉ conditioned medium of primary neurosphere culture N eurogenesis is considered to be the most complex event of organogenesis during embryonic development and involves a precise signaling, along with cellular interaction cascade, to generate the functional cellular networks. The embryonic organizer allows cells in its vicinity to execute their default neural program by emitting bone morphogenetic protein antagonists (1, 2). However, other works suggest a more complex mechanism (3-5).Neural stem cells (NSCs) are the self-renewal, multipotent cells that generate neurons, astrocytes, and oligodendrocytes (6, 7). They have great potential as a therapeutic tool for the repair of a number of central nervous system (CNS) disorders. Several in vitro systems allowing derivation of neuronal progeny from embryonic stem (ES) cells, which differentiate into all of the cell fates in a developing embryo, have been described. However, attempts to exclusively generate NSCs or neural progenitor cells from ES cells are restricted. It has been shown that neural fates emerge from ES cells in the serum-free conditions (8, 9). Although these procedures are noteworthy, both the quality and the quantity of ES-derived neural cells are not sufficient for further examination or clinical applications. Higher levels of neural differentiation are achieved by treatment of embryoid bodies with retinoic acid (RA) in the presence of FCS (10, 11) or by coculture with particular stromal cell lines (12, 13). The action of RA is pleiotropic (14, 15), whereas the effect of several stromal cell lines is attributed to an undefined neural inducing activity. These factors severely restrict the ability to use cells cultured with FCS or with particular stromal feeder cells for therapeutic treatments.Here, we developed an efficient system for the generation of ES cell-derived NSCs (ES-NSCs) during coculture with dissociated neurosphere cells without a need for FCS or feeder cells, which creates a bottleneck for therapeutic methods. We demonstrated that neurosphere-derived factor (NDF), which induces...

A new member of the serine protease inhibitor (serpin) superfamily with megakaryocyte maturation activity was purified, and its cDNA was cloned and characterized. The predicted amino acid sequence consisting of 380 residues was unique and was 38% identical to the serpin plasminogen activator inhibitor type 2 (PAI-2). The recombinant factor expressed in Chinese hamster ovary cells showed species-specific activity on the induction of megakaryocyte maturation in vitro. When injected into mice, the factor indeed elicited an increase in the number of platelets in plasma. The sequence alignment indicated that the factor possessed a lysine residue at the P 1 position, suggesting that it might function as an inhibitor of Lys-specific proteases. Although we could not show any inhibitory activities toward several known Lys-specific proteases, we detected the activity toward protease activity present in the culture supernatant of COLO 201 cells. These results suggested that the protein might influence the maturation of megakaryocytes via action as a serpin.

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