COBRE: RW HOSP: P2 MYOGENIC POTENTIAL OF BONE MARROW CELLS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。骨髓干细胞最近被证明能够在不同的组织中分化成许多非造血细胞类型，包括心肌细胞、肝细胞、神经细胞、上皮细胞、血管内皮细胞、软骨和骨。绿色荧光蛋白（GFP）阳性骨髓细胞向骨骼肌细胞的转化也已得到证实，这是本项目的重点。我们已经确定供体GFP+骨髓细胞通过在形态学特征的肌纤维中共定位GFP和desmin而转化为肌肉细胞。我们发现，通过改变移植方案的细节，包括细胞数量、移植时间和细胞递送方式（即局部注射vs全身输注或移植细胞的动员），骨髓转化或转分化到骨骼肌细胞的水平增加。我们还发现，转化事件的数量随不同的动员方案而变化，骨髓细胞亚群，即Dexter培养贴壁细胞，特别是谱系阴性细胞，比未分离的骨髓细胞具有更高的转化率。骨骼肌损伤的性质，辐射或直接心脏毒素注射，也是决定骨髓向肌肉细胞转移水平的关键。这些研究与可能的临床应用的相关性取决于转换的“稳健性”。在没有伤害的环境中或在我们的一些实验模型中，转化水平相当低，为0.01%的水平。然而，在对先前移植的小鼠进行心脏毒素肌肉损伤的初步研究中，结合辐射和直接注射不同群体的骨髓细胞，我们获得了高达12%的转化率，即12%的肌纤维是GFP+骨骼肌细胞。据我们所知，这是已发表文献中骨髓转化为肌肉细胞的最高速率，可以认为是相对“健壮”的，是向具有临床意义的“健壮”迈出的重要一步。在这些相同的研究中，我们还首次观察到胫骨前肌中GFP+肌肉细胞的菌落。我们目前的建议是继续评估导致骨髓向肌肉高水平转化的肌肉损伤的具体情况，并探索哪种特定的细胞类型可以以最高的频率产生肌肉，适合这种转化的移植时间以及获得显著肌肉转化所需的细胞数量。我们还将测试不同的“肌肉活性”细胞因子（HGF、LIF、FGF6和IGF1）对骨髓向肌肉细胞分化的影响。然后，我们将把我们的优化系统应用于小鼠肌肉萎缩症的相关模型。我们也特别感兴趣的是，在项目3中，siRNA的工作可能会开辟阻断特定造血干细胞分化途径并将其转移到肌生成途径的可能性。因此，我们计划在PU上与项目1合作。1阻断及其对骨髓向肌转化的影响。随着这项工作的进行，我们当然会评估其他转录调控阻滞。在这项资助中获得的知识可用于制定治疗肌肉萎缩症和其他遗传疾病的临床方案。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Marrow stem cells have recently been shown capable of differentiating into a number of nonhematopoietic cell types in different tissues including cardiac myocytes, hepatocytes, neuronal cells, epithelial cells, vascular endothelial cells, cartilage and bone. Conversion of green-fluorescent protein (GFP) positive marrow cells to skeletal muscle cells has also been demonstrated and is the focus of this project. We have determined that donor GFP+ marrow cells have converted to muscle cells by colocalizing GFP and desmin in morphologically characteristic muscle fibers. We have found increasing levels of marrow conversion or transdifferentiation to skeletal muscle cells by altering the specifics of the transplant regimen including cell number, timing of transplant and mode of cell delivery (i.e. local injection vs systemic infusion or mobilization of transplanted cells). We have also found that the number of conversion events changes with different mobilization regimens and that subsets of marrow cells, i.e. Dexter culture adherent cells and especially lineage negative cells, give higher rates of conversion than unseparated marrow cells. The nature of the skeletal muscle injury, radiation or direct cardiotoxin injection, is also critical in determining the level of transidfferentiation of marrow to muscle cells seen in vivo. The relevance of these studies to possible clinical application depends upon the "robustness" of the conversions. In settings without injury or in some of our experimental models the levels of conversion have been quite low, at the 0.01% level. However, in preliminary studies using a cardiotoxin muscle injury in previously transplanted mice, combined with radiation and direct injection of different populations of marrow cells we have obtained conversion rates up to12%, i.e. 12% of the muscle fibers were GFP+ skeletal muscle cells. This, to our knowledge, is the highest rate of marrow conversion to muscle cells in the published literature, and can be considered as relatively "robust" and a major step to clinically significant "robustness". In these same studies, we have also observed, for the first time, colonies of GFP+ muscle cells in the anterior tibialis muscle. Our present proposal is to continue to evaluate the specifics of muscle injury which will lead to high-level conversion of marrow to muscle and to explore which particular cell type can give rise to muscle at the highest frequency, the timing of transplant suitable for such conversions and the number of cells necessary to obtain significant muscle conversion. We will also test the effect of different "muscle active" cytokines (HGF, LIF, FGF6, and IGF1 ) on the differentiation of marrow to muscle cells. We will then apply our optimized system to relevant models of muscular dystrophy in mice. We are also particularly interested in the possibility that work in project three on siRNA may open up possibilities blocking specific hematopoietic stem cell differentiation pathways and diverting them to a myogneic pathway. Thus we plan to work with project 1 on PU. 1 blockade and its effect of marrow to muscle conversions. As this work proceeds we would of course evaluate other transcriptional regulatory blockades. The knowledge gained in this grant can be applied for the development of clinical protocols for treatment of muscular dystrophy and other genetic diseases.