Generating multipotent stem cells from primary human adipocytes for tissue repair

Abstract

Current trends in regenerative medicine for tissue repair focus on generating tissue-specific stem cells. However, given the complexity of most tissues, the ideal stem cell would be one that could undergo multilineage context-dependent differentiation to bring about holistic repair of the injured tissue.

This thesis describes application of a vector- and transcription factor-free method to reprogram human somatic cells into induced Multipotent Stem (iMS) cells utilizing the combination of 5-Azacytidine and recombinant human Platelet Derived Growth Factor-AB. I optimized xenofree conditions for this Demethylation Cytokine-induced (DCi) reprogramming technique that yielded autologous iMS cells at high efficiency from human adipocytes harvested from subjects aged 18-80 years. Human iMS cells display in vitro colony forming and serial re-plating ability, multilineage differentiation capacity and maintain a stable karyotype over several months. They express MSC markers but not markers of the blood lineage. iMS cells can be expanded long-term in medium containing autologous/allogeneic human serum. They have a transcriptional profile distinct to adipocytes or tissue-derived mesenchymal stem cells. IPA analysis revealed activation of genes associated with embryonic stem cells, EMT, PDGF signaling and downstream JAK/STAT, PI3K/AKT/mTOR pathways in iMS cells compared to adipocytes. Although iMS cells expressed pluripotency factors (OCT4, Nanog, SOX2 and SSEA4) they lacked spontaneous teratogenicity characteristic of pluripotent cells. When transplanted into injured intervertebral disc of NOD/SCID mice, human iMS cells were retained at transplant site for the duration of assessment (1 year) with no evidence of malignant transformation. iMS cells displayed in vivo plasticity and directly contributed to formation of new blood vessels, bone, cartilage and smooth muscle at the site of injury. To assess the specificity of cell plasticity, human iMS cells were also injected into cardiotoxin injured tibialis anterior muscle of SCID/beige mice. Donor iMS cells contributed to hCD56 expressing muscle satellite cells and hSpectrin expressing myofibres without heterotopic transformation or aberrant differentiation.

Together these findings demonstrate the feasibility and utility of DCi reprogramming for generation of safe, therapeutically relevant autologous iMS cells, and provide a solid foundation to evaluate their tissue regenerative potential in controlled clinical trials.