Generating multipotent stem cells from primary human adipocytes for tissue repair

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

Immune response to COVID‐19 vaccination in patients with Waldenström macroglobulinaemia who pause their BTKi therapy

Abstract Patients with Waldenström macroglobulinaemia (WM) are at increased risk of severe COVID‐19 infection and have poor immune responses to COVID‐19 vaccination. This study assessed whether a closely monitored pause in Bruton's Tyrosine Kinase inhibitor (BTKi) therapy might result in an improved humoral response to a 3rd COVID‐19 vaccine dose. Improved response was observed in WM patients who paused their BTKi, compared to a group who did not pause their BTKi. However, the response was attenuated after BTKi recommencement. This data contributes to our understanding of vaccination strategies in this patient group and may help inform consensus approaches in the future

Pdgf-ab and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineagecommitted cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.Link_to_subscribed_fulltex