Acquisition of Dynamic Function in Human Stem Cell-Derived β Cells

Velazco-Cruz, Leonardo; Song, Ji‐Won; Maxwell, Kristina G.; Goedegebuure, Madeleine M.; Augsornworawat, Punn; Hogrebe, Nathaniel J.; Millman, Jeffrey R.

doi:10.1016/j.stemcr.2018.12.012

Cited by 301 publications

(437 citation statements)

References 56 publications

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“…Substantial progress has been made to differentiate pluripotent stem cells to insulin-producing cells [46][47][48][49] . In common to these studies is the activation or inhibition of developmental signaling pathways to promote differentiation to specific cell types.…”

Section: Discussionmentioning

confidence: 99%

Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells

Sui

Egli

Xin

et al. 2020

Preprint

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Limitations in cell proliferation are a key barrier to reprogramming differentiated cells to pluripotent stem cells, and conversely, acquiring these limitations may be important to establish the differentiated state. The pancreas, and beta cells in particular have a low proliferative potential, which limits regeneration, but how these limitations are established is largely unknown.Understanding proliferation potential is important for the safty of cell replacement therapy with 2 cell products made from pluripotent stem cell which have unlimited proliferative potential. Here we test a novel hypothesis, that these limitations are established through limitations in S-phase progression. We used a stem cell-based system to expose differentiating stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing Sphase entry, or S-phase completion. Upon release from these molecules, we determined growth potential, differentiation and function of insulin-producing endocrine cells both in vitro and after grafting in vivo. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells, improved the stability of the differentiated state, and protected mice from diabetes without the formation of cystic growths. Therefore, a compromised ability to enter S-phase and replicate the genome is a functionally important property of pancreatic endocrine differentiation, and can be exploited to generate insulin-producing organoids with predictable growth potential after transplantation.

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Section: Discussionmentioning

confidence: 99%

Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells

Sui

Egli

Xin

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…The size of transplanted islets has been previously reported to impact insulin secretion and viability[50–52]. Small islet clusters can exhibit low amounts of insulin secretion, which has been attributed to limited cell-cell contact, while excessively large clusters are considered to have limitations from nutrient availability[53]. Based on these results with islets, the influence of pore size, which would determine the hPSC-derived β-cell cluster size, was investigated.…”

Section: Discussionmentioning

confidence: 99%

Microporous scaffolds drive assembly and maturation of progenitors into β-cell clusters

et al. 2019

Preprint

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Human pluripotent stem cells (hPSCs) represent a promising cell source for the development of β -cells for use in therapies for type 1 diabetes. Current culture approaches provide the signals to drive differentiation towards β -cells, with the cells spontaneously assembling into clusters. Herein, we adapted the current culture systems to cells seeded on microporous biomaterials, with the hypothesis that the pores can guide the assembly into β -cell clusters of defined size that can enhance maturation. The microporous scaffold culture allows hPSC-derived pancreatic progenitors to form clusters at a consistent size as cells undergo differentiation to immature β -cells. By modulating the scaffold pore sizes, we observed 250-425 µm pore size scaffolds significantly enhance insulin expression and key β -cell maturation markers compared to suspension cultures. Furthermore, when compared to suspension cultures, the scaffold culture showed increased insulin secretion in response to glucose stimulus indicating the development of functional β -cells. In addition, scaffolds facilitated cell-cell interactions enabled by the scaffold design and cell-mediated matrix deposition of extracellular matrix (ECM) proteins associated with the basement membrane of islet cells. We further investigated the influence of ECM on cell development by incorporating an ECM matrix on the scaffold prior to cell seeding; however, their presence did not further enhance maturation. These results suggest the microporous scaffold culture facilitates 3D cluster formation, supports cellcell interactions, and provides a matrix similar to a basement membrane to drive in vitro hPSCderived β -cell maturation and demonstrates the feasibility of these scaffolds as a biomanufacturing platform.

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“…The SC-β cells work similarly to β cells from human islets in sensing changing glucose concentration 9 . Insulin secretion response is typically characterized to have two phases 24 , the first phase being a large spike occurring within minutes after glucose stimulation, attributed to the exocytosis of readily releasable insulin granules close to the plasma membrane which release insulin in response to nutrient and non-nutrient secretagogues 25 , followed by the second phase of insulin release, where the insulin quantity reduces to a suprabasal level and continues to increase gradually as long as external glucose stimulation persists.…”

Section: Discussionmentioning

confidence: 99%

“…The goal for this study was to characterize the in vitro function of SC-β cells generated with our recent differentiation protocol 9 . This protocol uses six stages with defined, time-specific combinations of small molecule and protein factors to recapitulate pancreatic development to generate functional SC-β cells (Fig.…”

Section: Generation Of Sc-β Cells For Assessmentmentioning

confidence: 99%

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

Srivatsava¹,

Shahan²,

Gutgesell³

et al. 2019

Preprint

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Insulin-producing human embryonic stem cell-derived β (SC-β) cells are a promising cell source for diabetes cell replacement therapy. We have recently reported a differentiation strategy that produces SC-β 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-β cells in vitro. We utilized a Seahorse extracellular flux analyzer to measure mitochondrial respiration of SC-β cells at low and high glucose. We also used photolithography to fabricate a microfluidic device containing microwells to immobilize SC-β 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-β cells have increased cellular respiration and cytoplasmic calcium ion concentration in response to a high glucose stimulation. Our results indicate that SC-β 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.3 Diabetes Mellitus (DM) is a group of metabolic disorders that leads to the inability of the body to regulate blood glucose levels. Type 1 diabetes (T1D) involves the autoimmunemediated destruction of the insulin-producing pancreatic β cells located in the islets of Langerhans, leading to insulin deficiency and hyperglycemia. T1D is typically managed by injection of exogenous insulin. However, this clinical intervention does not emulate the normal behavior of the native β cells, making patients at risk for many long term complications 1 . An alternative therapy for T1D is the transplantation of cadaveric human islets, with the hope of reducing hyper-and hypoglycemic episodes. Some patients who were transplanted with islets have remained insulin independent for several years 2 . A major limitation of this approach, however, is the scarcity and quality of islets sourced from cadavers, limiting the widespread application of this therapy. Differentiation of human embryonic stem cells (hESCs) to insulinproducing β (SC-β) cells in islet organoids could serve as an unlimited supply of cells to treat millions of patients 3 , particularly if combined with transplantation strategies that vascularization, allow retrievability, and/or protect the cells from immune attack 4-8 .We recently reported a strategy for making large numbers of SC-β cells from hESCs 9 . This protocol is highly efficient, generating an almost pure population of pancreatic endocrine, of which most cells express insulin. These cells are highly functional, capable of undergoing glucose-stimulated insulin secretion. These cells were capable of restoring glucose tolerance when transplanted into streptozotocin-treated mice. Of particular note was the ability of these cells to display first and second phase insulin s...

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Acquisition of Dynamic Function in Human Stem Cell-Derived β Cells

Cited by 301 publications

References 56 publications

Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells

Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells

Microporous scaffolds drive assembly and maturation of progenitors into β-cell clusters

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

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