BIOLOGY OF NON-HUMAN PRIMATE MARROW STROMAL CELLS

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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. The overall aim of the project is to develop procedures whereby adult stem cells from the bone marrow stroma can be used for trials of gene therapy in non-human primates. The adult stem cells, referred to as mesenchymal stem cells or marrow stromal cells (MSCs), are of interest for cell and gene therapy because they can readily be obtained from a patient, expanded in culture, genetically engineered with or without the use of viruses, and then returned for therapy of the same patient. They are also of interest because they home to damaged tissues and differentiate to replace the damaged cells in the tissues. The cells are currently being tested in many small animal models of human diseases and several promising clinical trials with the cells have been initiated in rare diseases in children. However, extensive trials of the cells in non-human primates are clearly essential for some of the currently proposed applications to common diseases such as osteoporosis, cardiac failure, Parkinsonism, leukodystrophies, and Alzheimer's disease. The goals of the proposal are: Specific Aim 1. Isolate and expand primate MSCs with the improved protocol our laboratory has recently developed to isolate and expand cultures of human MSCs. We have successfully isolated rhesus MSCs from both the bone marrow and adipose tissue. We have done an extensive characterization of the in vitro biologic properties of the stem cell populations. Our data indicate that the MSCs from these two tissues share many characteristics. Human ASCs (hASCs) and rhesus BMSCs (rBMSCs) undergo significantly more total population doublings than human BMSCs (hBMSCs) and rhesus ASCs (rASCs). The cell cycle profile of all MSCs is altered as cultures aged in vitro. hMSCs underwent an increase in the frequency of cells in the S phase at P20 and P30. However, rhesus MSCs from both sources developed a distinct polyploid population of cells at P20, which progressed to aneuploidy by P30. Karyotype analysis of MSCs revealed the development of tetraploid or aneuploid karyotypes in the rhesus cells at P20 or P30. Analysis of the transcriptome of the MSCs from early and late passages revealed significant alterations in the patterns of gene expression (8.8% of the genes were differentially expressed in hBMSCs vs. hASCs; and 5.5% in rBMSCs vs. rASCs). Gene expression changes were much less evident within the same cell type as aging occurred (0.7% in hMSCs and 0.9% in rMSCs). Gene ontology analysis showed that functions involved in protein catabolism and regulation of pol II transcription were over-represented in rASCs, while the regulation of I¿B/NF-¿B cascade were over-represented in hBMSCs. Functional analysis of genes that were differentially expressed in rASCs and hBMSCs revealed that pathways involved in cell-cycle, cell cycle checkpoints, protein-ubiquitination, and apoptosis were altered. Overall in vitro characterization of MSCs from these two species and tissue sources revealed a high level of common biologic properties. However, the results demonstrate clear biologic distinctions, as well. Specific Aim 2. Compare the primate MSCs in culture with human MSCs in their ability to expand rapidly and to differentiate into osteoblasts, chondrocytes, adipocytes, and neural cells. We have found that the rhesus cells efficiently undergo differentiation along osteogenic, chondrogenic and adipogenic lineages. The efficiency between the human and rhesus MSCs is virtually indistinguishable. In terms of neural differentiation, we have found that rhesus ASCs differentiate along this lineage with greater efficiency in vitro than human or bone marrow derived cells. We are presently attempting to develop culture conditions that mediate oligodendrocyte lineage differentiation in these cell populations. Specific Aim 3. Compare the primate MSCs to human MSCs in vivo in their ability to engraft into multiple tissues after systemic or intracranial infusion into immunodeficient mice. These studies are currently ongoing. We have injected human and rhesus bone marrow and adipose tissue derived MSCs into the CNS of NIHIII AND Twitcher (Krabbe-affected) mice, using stereotaxic delivery. We are currently assessing engraftment and differentiation of these cells in the CNS. Data from the immune deficient mice indicate the cells engraft, persist for as long as 180 days and undergo moderate differentiation along neural lineages. Direct injection of MSCs (derived from bone marrow and adipose tissue) into the lateral ventricles of the brains of Twitcher mice is currently being collected. To date, 40 mice have been injected and mice are being sacrificed at predetermined dates to assess engraftment and differentiation of MSCs.