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Velazco-Cruz, L.

Publications and source records attributed to Velazco-Cruz, L..

3 recordsLinked to original sources

Multiomic profiling defines cell fate plasticity of in vitro-derived islets

Transplantation of insulin-secreting {beta}-cells differentiated from human pluripotent stem cells holds great potential as a cell therapy for treating insulin-dependent diabetes. While these stem cell-derived islets (SC-islets) are able to reverse diabetes in animal models, they are not fully equivalent to their in vivo counterparts. To better define the state of the cell types generated within these SC-islets and provide a resource for identifying deficiencies in lineage specification, we used single-cell multiomic sequencing to simultaneously measure the chromatin accessibility and transcriptional profiles of SC-islets at multiple time points as well as primary human islets. The integrated analysis of both the transcriptional and chromatin landscape for each cell provided greater resolution for defining cell identity, allowing us to derive novel gene lists for identifying each islet cell type. Furthermore, this multiomic analysis revealed that the difference between SC-{beta} cells and enterochromaffin-like cells, which are a major off-target from in vitro differentiation, is a gradient of progressive cell states rather than a stark difference in identity. The chromatin landscape of primary human islets was much more restricted, suggesting that stem cell-derived cells are not fully locked into their cell fate. While long term culture of SC-islets both in vitro and in vivo does close overall chromatin state, only in vivo transplantation directs cells toward their correct identities. Collectively, our multiomic analysis demonstrates that both the chromatin and transcriptional landscapes play significant roles in islet cell identity, and these data can be used as a resource to identify specific deficiencies in the chromatin and transcriptional state of SC-islet cell types.

cell biology↗

Assessment of the in vitro function of human stem cell-derived β cells

Insulin-producing human embryonic stem cell-derived {beta} (SC-{beta}) cells are a promising cell source for diabetes cell replacement therapy. We have recently reported a differentiation strategy that produces SC-{beta} cells in islet organoids that not only undergo glucose-stimulated insulin secretion but also have an islet-like dynamic insulin release profile, displaying both first and second phase insulin secretion. The goal of this study was to further characterize the functional profile of these SC-{beta} cells in vitro. We utilized a Seahorse extracellular flux analyzer to measure mitochondrial respiration of SC-{beta} cells at low and high glucose. We also used photolithography to fabricate a microfluidic device containing microwells to immobilize SC-{beta} cells for perfusional analysis, monitoring cytoplasmic calcium using Fluo-4 AM at low and high glucose. Here we find that in addition to increased insulin secretion, SC-{beta} cells have increased cellular respiration and cytoplasmic calcium ion concentration in response to a high glucose stimulation. Our results indicate that SC-{beta} cells have similar function to that reported for islets, providing further performance characterization that could help with eventual development for diabetes cell therapy and drug screening.

bioengineering↗

Hydrogel platform for in vitro three-dimensional assembly of human stem cell-derived β cells and endothelial cells

Differentiation of stem cells into functional replacement cells and tissues is a major goal of the regenerative medicine field. However, one limitation has been organization of differentiated cells into multi-cellular, three-dimensional assemblies. The islets of Langerhans contain many endocrine and non-endocrine cell types, such as insulin-producing {beta} cells and endothelial cells. Transplantation of exogenous islets into diabetic patients can serve as a cell replacement therapy, replacing the need for patients to inject themselves with insulin, but the number of available islets from cadaveric donors is low. We have developed a strategy of assembling human embryonic stem cell-derived {beta} cells with endothelial cells into three-dimensional aggregates on a hydrogel. The resulting islet organoids express {beta} cell markers and are functional, capable of undergoing glucose-stimulated insulin secretion. These results provide a platform for evaluating the effects of the islet tissue microenvironment on human embryonic stem cell-derived {beta} cells and other islet endocrine cells to develop tissue engineered islets.

bioengineering↗