Metabolic switching, growth kinetics and cell yields in the scalable manufacture of stem cell-derived insulin-producing cells.

BACKGROUND: Diabetes is a disease affecting over 500 million people globally due to insulin insufficiency or insensitivity. For individuals with type 1 diabetes, pancreatic islet transplantation can help regulate their blood glucose levels. However, the scarcity of cadaveric donor islets limits the number of people that could receive this therapy. To address this issue, human pluripotent stem cells offer a potentially unlimited source for generating insulin-producing cells through directed differentiation. Several protocols have been developed to make stem cell-derived insulin-producing cells. However, there is a lack of knowledge regarding the bioprocess parameters associated with these differentiation protocols and how they can be utilized to increase the cell yield. METHODS: We investigated various bioprocess parameters and quality target product profiles that may influence the differentiation pipeline using a seven-stage protocol in a scalable manner with CellSTACKs and vertical wheel bioreactors (PBS-Minis). RESULTS: Cells maintained > 80% viability through all stages of differentiation and appropriately expressed stage-specific markers. During the initial four stages leading up to the development of pancreatic progenitors, there was an increase in cell numbers. Following pancreatic progenitor stage, there was a gradual decrease in the percentage of proliferative cells, as determined by Ki67 positivity, and a significant loss of cells during the period of endocrine differentiation. By minimizing the occurrence of aggregate fusion, we were able to enhance cell yield during the later stages of differentiation. We suggest that glucose utilization and lactate production are cell quality attributes that should be considered during the characterization of insulin-producing cells derived from stem cells. Our findings also revealed a gradual metabolic shift from glycolysis, during the initial four stages of pancreatic progenitor formation, to oxidative phosphorylation later on during endocrine differentiation. Furthermore, the resulting insulin-producing cells exhibited a response to several secretagogues, including high glucose. CONCLUSION: This study demonstrates process parameters such as glucose consumption and lactate production rates that may be used to facilitate the scalable manufacture of stem cell-derived insulin-producing cells.

Prospects of self-powering leadless pacemakers using piezoelectric energy harvesting technology by heart kinetic motion.

This paper studies the possibility of heart kinetic motion for designing a self-powered intracardiac leadless pacemaker by piezoelectric energy harvesting. A Doppler laser displacement sensor measures in vivo heart kinetic motion. Cantilevered and four-point bending piezoelectric harvesters are studied under the measured in vivo heart kinetic motion. The heart movement is above 15 mm. The cantilevered and four-point bending harvesters generate a maximum voltage of ~ 0.28 V and 0.8 V, respectively with the measured heart motion with a heart rate of 168 beats per minute. Two DC/DC converters, LTC3588 and MAX17220, combined with full-bridge rectifiers and their start-up performance are tested.Clinical Relevance-This paper analyzed the heart kinetic motion and establishes the piezoelectric energy harvesting for a new era of self-powered leadless pacemakers.

The Impact of Different Implantation Sites and Sex on the Differentiation of Human Pancreatic Endoderm Cells Into Insulin-Secreting Cells In Vivo.

Few studies have examined the differentiation of human embryonic stem cell (hESC)-derived pancreatic endoderm cells (PECs) in different implantation sites. Here, we investigate the influence of implantation site and recipient sex on the differentiation of hESC-derived PECs in vivo. Male and female mice were implanted with 5 × 106 hESC-derived PECs under the kidney capsule, in the gonadal fat pad, or subcutaneously within macroencapsulation (TheraCyte) devices. PECs implanted within TheraCyte devices developed glucose-stimulated human C-peptide secretion faster than cells implanted under the kidney capsule or in the gonadal fat pad. Interestingly, hESC-derived PECs implanted under the kidney capsule in females developed glucose-stimulated human C-peptide faster than in males and secreted higher levels of arginine-stimulated glucagon and glucagon-like peptide 1 than other implantation sites. Furthermore, hESC-derived grafts collected from the kidney capsule and gonadal fat pad sites displayed a mix of endocrine and ductal cells as well as contained cysts, whereas TheraCyte device grafts displayed mostly endocrine cells and cysts were not observed. Here we demonstrate that the macroencapsulated subcutaneous site and the female recipient can promote faster differentiation of hESC-derived PECs to endocrine cells in mice. ARTICLE HIGHLIGHTS: Few studies have directly compared the differentiation of human embryonic stem cell-derived progenitors in different implantation sites in male and female recipients. We investigated whether the site of implantation and/or the sex of the recipient influenced the differentiation of pancreatic progenitors in vivo in mice. Mice implanted with cells in macroencapsulation devices contained fewer off-target structures and developed stimulated insulin release faster than other implant sites, while females implanted with cells under the kidney capsule developed stimulated insulin release before males. Macroencapsulation devices reduced the formation of off-target cells from human embryonic stem cell-derived progenitors, a useful characteristic for clinical applications.

A Century-long Journey From the Discovery of Insulin to the Implantation of Stem Cell-derived Islets.

For the past century, insulin injections have saved millions of lives, but glycemic instability is still a persistent challenge for people with diabetes, leading to tremendous morbidity and premature mortality. Research in the field of islet transplantation has demonstrated that replacing insulin-producing ß cells can restore euglycemia comparable to individuals without diabetes. However, a short supply of cadaveric islet donors, the technically challenging process of isolating islets, and the requirement for chronic immune suppression have impeded widespread clinical adoption. Rather than relying on cadaveric cells, pluripotent stem cells could serve as a virtually unlimited supply of insulin-producing ß cells. Protocols have been developed that mimic the normal in vivo development of the human pancreas to generate pancreatic progenitor cells in vitro. Ongoing investigations have yielded progressively more mature ß-like cells in vitro that produce insulin but do not yet fully mimic healthy mature ß cells. Alongside development of differentiation protocols, other work has provided insight into potential implantation sites for stem cell-derived islet cells including the subcutaneous space, portal vein, and omentum. To optimize implanted cell survival and function, development of immune modulation therapies is ongoing, including selection of immunomodulatory medications and genetic modification of implanted cells to evade immune responses. Further, macroencapsulation or microencapsulation devices could be used to contain and/or immunoprotect implanted cells from the immune response including by using 3-dimensional bioprinting to facilitate the process. Remarkably, ongoing clinical trials have now yielded the first patient relying on differentiated stem cells rather than syringes as their insulin replacement therapy.

Online Condition Monitoring of Rotating Machines by Self-Powered Piezoelectric Transducer from Real-Time Experimental Investigations.

This paper investigates self-powering online condition monitoring for rotating machines by the piezoelectric transducer as an energy harvester and sensor. The method is devised for real-time working motors and relies on self-powered wireless data transfer where the data comes from the piezoelectric transducer's output. Energy harvesting by Piezoceramic is studied under real-time motor excitations, followed by power optimization schemes. The maximum power and root mean square power generation from the motor excitation are 13.43 mW/g2 and 5.9 mW/g2, which can be enough for providing autonomous wireless data transfer. The piezoelectric transducer sensitivity to the fault is experimentally investigated, showing the considerable fault sensitivity of piezoelectric transducer output to the fault. For instance, the piezoelectric transducer output under a shaft-misalignment fault is more than 200% higher than the healthy working conditions. This outcome indicates that the monitoring of rotating machines can be achieved by using a self-powered system of the piezoelectric harvesters. Finally, a discussion on the feasible self-powered online condition monitoring is presented.

Energy Harvesting from a Thermoelectric Zinc Antimonide Thin Film under Steady and Unsteady Operating Conditions.

In practice, there are some considerations to study stability, reliability, and output power optimization of a thermoelectric thin film operating dynamically. In this study stability and performance of a zinc antimonide thin film thermoelectric (TE) specimen is evaluated under transient with thermal and electrical load conditions. Thermoelectric behavior of the specimen and captured energy in each part of a thermal cycle are investigated. Glass is used as the substrate of the thin film, where the heat flow is parallel to the length of the thermoelectric element. In this work, the thermoelectric specimen is fixed between a heat sink exposed to the ambient temperature and a heater block. The specimen is tested under various electrical load cycles during a wide range of thermal cycles. The thermal cycles are provided for five different aimed temperatures at the hot junction, from 160 to 350 °C. The results show that the specimen generates approximately 30% of its total electrical energy during the cooling stage and 70% during the heating stage. The thin film generates maximum power of 8.78, 15.73, 27.81, 42.13, and 60.74 kW per unit volume of the thermoelectric material (kW/m³), excluding the substrate, corresponding to hot side temperature of 160, 200, 250, 300, and 350 °C, respectively. Furthermore, the results indicate that the thin film has high reliability after about one thousand thermal and electrical cycles, whereas there is no performance degradation.

Report of the Key Opinion Leaders Meeting on Stem Cell-derived Beta Cells.

Beta cell replacement has the potential to restore euglycemia in patients with insulin-dependent diabetes. Although great progress has been made in establishing allogeneic islet transplantation from deceased donors as the standard of care for those with the most labile diabetes, it is also clear that the deceased donor organ supply cannot possibly treat all those who could benefit from restoration of a normal beta cell mass, especially if immunosuppression were not required. Against this background, the International Pancreas and Islet Transplant Association in collaboration with the Harvard Stem Cell Institute, the Juvenile Diabetes Research Foundation (JDRF), and the Helmsley Foundation held a 2-day Key Opinion Leaders Meeting in Boston in 2016 to bring together experts in generating and transplanting beta cells derived from stem cells. The following summary highlights current technology, recent significant breakthroughs, unmet needs and roadblocks to stem cell-derived beta cell therapies, with the aim of spurring future preclinical collaborative investigations and progress toward the clinical application of stem cell-derived beta cells.

Sex Differences in Maturation of Human Embryonic Stem Cell-Derived ß Cells in Mice.

Pancreatic progenitors derived from human embryonic stem cells (hESCs) are now in clinical trials for insulin replacement in patients with type 1 diabetes. Animal studies indicate that pancreatic progenitor cells can mature into a mixed population of endocrine cells, including glucose-responsive ß cells several months after implantion. However, it remains unclear how conditions in the recipient may influence the maturation and ultimately the function of these hESC-derived cells. Here, we investigated the effects of (1) pregnancy on the maturation of human stage 4 (S4) pancreatic progenitor cells and (2) the impact of host sex on both S4 cells and more mature stage 7 (S7) pancreatic endocrine cells implanted under the kidney capsule of immunodeficient SCID-beige mice. Pregnancy led to increased proliferation of endogenous pancreatic ß cells, but did not appear to affect proliferation or maturation of S4 cells at midgestation. Interestingly, S4 and S7 cells both acquired glucose-stimulated C-peptide secretion in females before males. Moreover, S4 cells lowered fasting blood glucose levels in females sooner than in males, whereas the responses with S7 cells were similar. These data indicate that the host sex may impact the maturation of hESC-derived cells in vivo and that this effect can be minimized by more advanced differentiation of the cells before implantation.

Hypothyroidism Impairs Human Stem Cell-Derived Pancreatic Progenitor Cell Maturation in Mice.

Pancreatic progenitors derived from human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating diabetes and are currently being tested in clinical trials. Yet, how the milieu of pancreatic progenitor cells, including exposure to different factors after transplant, may influence their maturation remains unclear. Here, we examined the effect of thyroid dysregulation on the development of hESC-derived progenitor cells in vivo. Hypothyroidism was generated in SCID-beige mice using an iodine-deficient diet containing 0.15% propyl-2-thiouracil, and hyperthyroidism was generated by addition of L-thyroxine (T4) to drinking water. All mice received macroencapsulated hESC-derived progenitor cells, and thyroid dysfunction was maintained for the duration of the study ("chronic") or for 4 weeks posttransplant ("acute"). Acute hyperthyroidism did not affect graft function, but acute hypothyroidism transiently impaired human C-peptide secretion at 16 weeks posttransplant. Chronic hypothyroidism resulted in severely blunted basal human C-peptide secretion, impaired glucose-stimulated insulin secretion, and elevated plasma glucagon levels. Grafts from chronic hypothyroid mice contained fewer ß-cells, heterogenous MAFA expression, and increased glucagon(+) and ghrelin(+) cells compared to grafts from euthyroid mice. Taken together, these data suggest that long-term thyroid hormone deficiency may drive the differentiation of human pancreatic progenitor cells toward α- and ε-cell lineages at the expense of ß-cell formation.

Replacing and safeguarding pancreatic ß cells for diabetes.

Pluripotent stem cells are a scalable source of pancreatic cells for transplantation into patients with diabetes. Here, we describe how the field is gaining momentum toward a ß cell replacement therapy.

Accelerated Maturation of Human Stem Cell-Derived Pancreatic Progenitor Cells into Insulin-Secreting Cells in Immunodeficient Rats Relative to Mice.

Pluripotent human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating patients with diabetes. To investigate the impact of the host recipient on hESC-derived pancreatic progenitor cell maturation, cells were transplanted into immunodeficient SCID-beige mice or nude rats. Following the transplant, basal human C-peptide levels were consistently higher in mice compared with rats, but only rats showed robust meal- and glucose-responsive human C-peptide secretion by 19-21 weeks. Grafts from rats contained a higher proportion of insulin:glucagon immunoreactivity, fewer exocrine cells, and improved expression of mature ß cell markers compared with mice. Moreover, ECM-related genes were enriched, the collagen network was denser, and blood vessels were more intricately integrated into the engrafted endocrine tissue in rats relative to mice. Overall, hESC-derived pancreatic progenitor cells matured faster in nude rats compared with SCID-beige mice, indicating that the host recipient can greatly influence the fate of immature pancreatic progenitor cells post-transplantation.

Treating diet-induced diabetes and obesity with human embryonic stem cell-derived pancreatic progenitor cells and antidiabetic drugs.

Human embryonic stem cell (hESC)-derived pancreatic progenitor cells effectively reverse hyperglycemia in rodent models of type 1 diabetes, but their capacity to treat type 2 diabetes has not been reported. An immunodeficient model of type 2 diabetes was generated by high-fat diet (HFD) feeding in SCID-beige mice. Exposure to HFDs did not impact the maturation of macroencapsulated pancreatic progenitor cells into glucose-responsive insulin-secreting cells following transplantation, and the cell therapy improved glucose tolerance in HFD-fed transplant recipients after 24 weeks. However, since diet-induced hyperglycemia and obesity were not fully ameliorated by transplantation alone, a second cohort of HFD-fed mice was treated with pancreatic progenitor cells combined with one of three antidiabetic drugs. All combination therapies rapidly improved body weight and co-treatment with either sitagliptin or metformin improved hyperglycemia after only 12 weeks. Therefore, a stem cell-based therapy may be effective for treating type 2 diabetes, particularly in combination with antidiabetic drugs.

Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.

Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.

Differentiated human stem cells resemble fetal, not adult, ß cells.

Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic ß cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal ß cells than adult ß cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true ß cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional ß cells.

Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells.

Human embryonic stem cells (hESCs) were used as a model system of human pancreas development to study characteristics of the polyhormonal cells that arise during fetal pancreas development. HESCs were differentiated into fetal-like pancreatic cells in vitro using a 33-day, 7-stage protocol. Cultures were ~90-95% PDX1-positive by day (d) 11 and 70-75% NKX6.1-positive by d17. Polyhormonal cells were scattered at d17, but developed into islet-like clusters that expressed key transcription factors by d33. Human C-peptide and glucagon secretion were first detected at d17 and increased thereafter in parallel with INS and GCG transcript levels. HESC-derived cells were responsive to KCl and arginine, but not glucose in perifusion studies. Compared to adult human islets, hESC-derived cells expressed ~10-fold higher levels of glucose transporter 1 (GLUT1) mRNA, but similar levels of glucokinase (GCK). In situ hybridization confirmed the presence of GLUT1 transcript within endocrine cells. However, GLUT1 protein was excluded from this population and was instead observed predominantly in non-endocrine cells, whereas GCK was co-expressed in insulin-positive cells. In rubidium efflux assays, hESC-derived cells displayed mild potassium channel activity, but no responsiveness to glucose, metabolic inhibitors or glibenclamide. Western blotting experiments revealed that the higher molecular weight SUR1 band was absent in hESC-derived cells, suggesting a lack of functional KATP channels at the cell surface. In addition, KATP channel subunit transcript levels were not at a 1:1 ratio, as would be expected (SUR1 levels were ~5-fold lower than KIR6.2). Various ratios of SUR1:KIR6.2 plasmids were transfected into COSM6 cells and rubidium efflux was found to be particularly sensitive to a reduction in SUR1. These data suggest that an impaired ratio of SUR1:KIR6.2 may contribute to the observed KATP channel defects in hESC-derived islet endocrine cells, and along with lack of GLUT1, may explain the absence of glucose-stimulated insulin secretion.

The effect of endothelial cells on hESC-derived pancreatic progenitors in a 3D environment.

Generating transplantable ß-like-cells from human embryonic stem cells (hESC) could serve as an ideal cell-based therapy for treatment of type 1 diabetes, which is characterized by the destruction of insulin-secreting pancreatic ß-cells. There are several protocols for differentiating hESCs into pancreatic or endocrine precursors. However, so far, production of mature, functional ß-like-cells has been achieved mainly by transplanting hESC derived pancreatic progenitors (PPs) and allowing several months for maturation to occur in vivo. One approach, believed to have potential in promoting differentiation into ß-like-cells prior to transplantation, is culturing PPs alongside blood vessels. Endothelium and blood vessels have been shown to direct pancreatic development during embryogenesis and also induce endocrine differentiation in vitro. Here we designed a three-dimensional (3D) construct utilizing highly porous polymeric scaffolds that mimic natural conditions and provide cells with mechanical support, and used it in the differentiation protocol. Clusters of hESC derived pancreatic precursor cells were embedded within the scaffolds along with human endothelial cells (ECs) and fibroblasts forming vessel-like networks. Culturing these clusters with ECs for one week significantly increased the population of PPs, characterized by co-expression of the pancreatic markers Pdx1 and Nkx6.1 and also highly induced Ngn3 expression which indicates commitment to endocrine fate. The presence of fibroblasts, however, reduced this cell population. Three months upon implantation of constructs containing clusters and ECs or clusters alone, implanted mice retained normal blood glucose levels after treatment with STZ, while un-implanted mice became diabetic. These findings may lay the foundation for creating an optimal tissue-construct that will support PPs' maturation in vitro and enhance graft function upon implantation.

Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.

Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs, composed of mainly pancreatic endoderm (PDX1/NKX6.1-positive), endocrine precursors (NKX2.2/synaptophysin-positive, hormone/NKX6.1-negative), and polyhormonal cells (insulin/glucagon-positive, NKX6.1-negative). However, the relative contributions of NKX6.1-negative versus NKX6.1-positive cell fractions to the maturation of functional ß-cells remained unclear. To address this question, we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant, both populations contained a high proportion of PDX1-expressing cells (~85%-90%) but were distinguished by their relatively high (~80%) or low (~25%) expression of NKX6.1. NKX6.1-high and NKX6.1-low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1-low cells remained hyperglycemic throughout the 5-month post-transplant period whereas diabetes was reversed in NKX6.1-high recipients within 3 months. Fasting human C-peptide levels were similar between groups throughout the study, but only NKX6.1-high grafts displayed robust meal-, glucose- and arginine-responsive insulin secretion as early as 3 months post-transplant. NKX6.1-low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue, but NKX6.1-high grafts contained a greater proportion of insulin-positive and somatostatin-positive cells, whereas NKX6.1-low grafts contained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high, but not NKX6.1-low grafts expressed nuclear MAFA. Collectively, this study demonstrates that a pancreatic endoderm-enriched population can mature into highly functional ß-cells with only a minor contribution from the endocrine subpopulation.

Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice.

AIMS/HYPOTHESIS: Islet transplantation is a promising cell therapy for patients with diabetes, but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However, the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore, we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo. METHODS: The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte, Laguna Hills, CA, USA) devices into diabetic mice. RESULTS: Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing >80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices, despite lacking direct contact with the host environment, and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells. CONCLUSIONS/INTERPRETATION: Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device, yielding glucose-responsive insulin-producing cells capable of reversing diabetes.

A simple tool to improve pluripotent stem cell differentiation.

We describe a method to help overcome restrictions on the differentiation propensities of human pluripotent stem cells. Culturing pluripotent stem cells in dimethylsulfoxide (DMSO) activates the retinoblastoma protein, increases the proportion of cells in the early G1 phase of the cell cycle and, in more than 25 embryonic and induced pluripotent stem cell lines, improves directed differentiation into multiple lineages. DMSO treatment also improves differentiation into terminal cell types in several cell lines.

Generating ß-cells in vitro: progress towards a Holy Grail.

PURPOSE OF REVIEW: Diabetes is a debilitating disease characterized by a chronic inability to normalize blood glucose levels. Transplanting cadaveric pancreata or isolated pancreatic islets can restore glucose homeostasis, but organ demand outstrips supply. Consequently, there is significant interest in alternative tissue sources. This review summarizes state-of-the-art efforts to generate scalable, functional ß-cells to treat diabetes. RECENT FINDINGS: Applying knowledge gleaned from developmental biology, human pluripotent stem cells can be treated stepwise with combinations of small molecules, developmentally relevant growth factors, and morphogens to generate pancreatic progenitor cells (PPCs) in vitro. Transplanted PPCs can then further mature in vivo into functional islet-like tissues containing all of the endocrine hormone cells present in adult islets and can reverse hyperglycemia in a diabetic animal model. Recent publications demonstrate that skin, liver, and other cell lineages may also be reprogrammed to functional ß-like cells. SUMMARY: Although generation of fully functional ß-cells in vitro has not yet been achieved, possible intermediate approaches to treat diabetes include using PPCs or reprogramming adult cells to ß-like cells. A cell therapy with either approach will require isolation from the host immune response. Ongoing efforts are addressing this need through the use of immune-isolation devices to avoid immunosuppressive drugs.