BIOLOGY OF NON-HUMAN PRIMATE MARROW STROMAL CELLS

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. The data indicate that MSCs isolated from rhesus bone marrow (rBMSCs) and human adipose tissue (hASCs) had more similar biologic properties than MSCs of rhesus adipose tissue (rASCs) and human bone marrow MSCs (hBMSCs). Analyses of in vitro growth kinetics revealed shorter doubling time for rBMSCs and hASCs. rBMSCs and hASCs underwent significantly more population doublings than the other MSCs. MSCs from all sources showed a marked decrease in telomerase activity over extended culture; however they maintained their mean telomere length. All of the MSCs expressed embryonic stem cell markers, Oct-4, Rex-1 and Sox-2 for at least 10 passages. Early populations of MSCs types showed similar multilineage differentiation capability. However, only the rBMSCs and hASCs retain greater differentiation efficiency at higher passages. 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. 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 mice, using stereotaxic delivery. We are currently assessing engraftment and differentiation of these cells in immune deficient mice and nonhuman primate 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.