Mouse Pluripotent Stem Cell Differentiation Under Physiological Oxygen Reduces Residual Teratomas.

Introduction: Residual pluripotent stem cells (PSC) within differentiated populations are problematic because of their potential to form tumors. Simple methods to reduce their occurrence are needed.

Methods: Here, we demonstrate that control of the oxygen partial pressure (pO) to physiological levels typical of the developing embryo, enabled by culture on a highly oxygen permeable substrate, reduces the fraction of PSC within and the tumorigenic potential of differentiated populations.

Long-term viability and function of transplanted islets macroencapsulated at high density are achieved by enhanced oxygen supply.

Transplantation of encapsulated islets can cure diabetes without immunosuppression, but oxygen supply limitations can cause failure. We investigated a retrievable macroencapsulation device wherein islets are encapsulated in a planar alginate slab and supplied with exogenous oxygen from a replenishable gas chamber. Translation to clinically-useful devices entails reduction of device size by increasing islet surface density, which requires increased gas chamber pO Here we show that islet surface density can be substantially increased safely by increasing gas chamber pO to a supraphysiological level that maintains all islets viable and functional.

Favorable outcome of experimental islet xenotransplantation without immunosuppression in a nonhuman primate model of diabetes.

Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with critical metabolic lability resulting from the need for immunosuppression and the shortage of donor organs. To overcome these barriers, we developed a strategy to macroencapsulate islets from different sources that allow their survival and function without immunosuppression. Here we report successful and safe transplantation of porcine islets with a bioartificial pancreas device in diabetic primates without any immune suppression.

Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation.

Background: Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR) in predicting clinical islet autotransplant (IAT) insulin independence (II). IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity.

Oxygen supply to encapsulated therapeutic cells.

Therapeutic cells encapsulated in immunobarrier devices have promise for treatment of a variety of human diseases without immunosuppression. The absence of sufficient oxygen supply to maintain viability and function of encapsulated tissue has been the most critical impediment to progress. Within the framework of oxygen supply limitations, we review the major issues related to development of these devices, primarily in the context of encapsulated islets of Langerhans for treating diabetes, including device designs and materials, supply of tissue, protection from immune rejection, and maintenance of cell viability and function.

Transplantation of human islets without immunosuppression.

Transplantation of pancreatic islets is emerging as a successful treatment for type-1 diabetes. Its current stringent restriction to patients with critical metabolic lability is justified by the long-term need for immunosuppression and a persistent shortage of donor organs. We developed an oxygenated chamber system composed of immune-isolating alginate and polymembrane covers that allows for survival and function of islets without immunosuppression.

The efficacy of an immunoisolating membrane system for islet xenotransplantation in minipigs.

Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets.

Enhanced oxygen supply improves islet viability in a new bioartificial pancreas.

The current epidemic of diabetes with its overwhelming burden on our healthcare system requires better therapeutic strategies. Here we present a promising novel approach for a curative strategy that may be accessible for all insulin-dependent diabetes patients. We designed a subcutaneous implantable bioartificial pancreas (BAP)-the "β-Air"-that is able to overcome critical challenges in current clinical islet transplantation protocols: adequate oxygen supply to the graft and protection of donor islets against the host immune system.

Improvement of islet function in a bioartificial pancreas by enhanced oxygen supply and growth hormone releasing hormone agonist.

Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients.

Quantitative assessment of islets of Langerhans encapsulated in alginate.

Improved methods have recently been developed for assessing islet viability and quantity in human islet preparations for transplantation, and these measurements have proven useful for predicting transplantation outcome. The objectives of this study were to adapt these methods for use with microencapsulated islets, to verify that they provide meaningful quantitative measurements, and to test them with two model systems: (1) barium alginate and (2) barium alginate containing a 70% (w/v) perfluorocarbon (PFC) emulsion, which presents challenges to use of these assays and is of interest in its own right as a means for reducing oxygen supply limitations to encapsulated tissue. Mitochondrial function was assessed by oxygen consumption rate measurements, and the analysis of data was modified to account for the increased solubility of oxygen in the PFC-alginate capsules.

Quantitative analysis of cell composition and purity of human pancreatic islet preparations.

Despite improvements in outcomes for human islet transplantation, characterization of islet preparations remains poorly defined. This study used both light microscopy (LM) and electron microscopy (EM) to characterize 33 islet preparations used for clinical transplants. EM allowed an accurate identification and quantification of cell types with measured cell number fractions (mean±s.

Enumeration of islets by nuclei counting and light microscopic analysis.

Islet enumeration in impure preparations by conventional dithizone staining and visual counting is inaccurate and operator dependent. We examined nuclei counting for measuring the total number of cells in islet preparations, and we combined it with morphological analysis by light microscopy (LM) for estimating the volume fraction of islets in impure preparations. Cells and islets were disrupted with lysis solution and shear, and accuracy of counting successively diluted nuclei suspensions was verified with (1) visual counting in a hemocytometer after staining with crystal violet, and automatic counting by (2) aperture electrical resistance measurement and (3) flow cytometer measurement after staining with 7-aminoactinomycin-D.

Prediction of marginal mass required for successful islet transplantation.

Islet quality assessment methods for predicting diabetes reversal (DR) following transplantation are needed. We investigated two islet parameters, oxygen consumption rate (OCR) and OCR per DNA content, to predict transplantation outcome and explored the impact of islet quality on marginal islet mass for DR. Outcomes in immunosuppressed diabetic mice were evaluated by transplanting mixtures of healthy and purposely damaged rat islets for systematic variation of OCR/DNA over a wide range.

Accurate control of oxygen level in cells during culture on silicone rubber membranes with application to stem cell differentiation.

Oxygen level in mammalian cell culture is often controlled by placing culture vessels in humidified incubators with a defined gas phase partial pressure of oxygen (pO(2gas)). Because the cells are consuming oxygen supplied by diffusion, a difference between pO(2gas) and that experienced by the cells (pO(2cell)) arises, which is maximal when cells are cultured in vessels with little or no oxygen permeability. Here, we demonstrate theoretically that highly oxygen-permeable silicone rubber membranes can be used to control pO(2cell) during culture of cells in monolayers and aggregates much more accurately and can achieve more rapid transient response following a disturbance than on polystyrene and fluorinated ethylene-propylene copolymer membranes.

Islet assessment for transplantation.

Purpose Of Review: There is a critical need for meaningful viability and potency assays that characterize islet preparations for release prior to clinical islet cell transplantation. Development, testing, and validation of such assays have been the subject of intense investigation for the last decade. These efforts are reviewed, highlighting the most recent results while focusing on the most promising assays.

The effects of low oxygen on self-renewal and differentiation of embryonic stem cells.

Purpose Of Review: To summarize recent reports on the effects of low oxygen on the undifferentiated phenotype and differentiation of embryonic stem cells (ESCs).

Recent Findings: The oxygen level to which ESCs are exposed is an important environmental parameter. Under conditions maintaining the undifferentiated phenotype, low oxygen reduces spontaneous differentiation of human ESCs but reduces pluripotency gene expression in mouse ESCs, although reports are conflicting.

Engineering microenvironments for embryonic stem cell differentiation to cardiomyocytes.

Embryonic stem cells and induced pluripotent stem cells have the potential to be a renewable source of cardiomyocytes for use in myocardial cell replacement strategies. Although progress has been made towards differentiating stem cells to specific cell lineages, the efficiency is often poor and the number of cells generated is not suitable for therapeutic usage. Recent studies demonstrated that controlling the stem cell microenvironment can influence differentiation.

Effects of oxygen on mouse embryonic stem cell growth, phenotype retention, and cellular energetics.

Most embryonic stem (ES) cell research is performed with a gas phase oxygen partial pressure (pO(2)) of 142 mmHg, whereas embryonic cells in early development are exposed to pO(2) values of 0-30 mmHg. To understand effects of these differences, we studied murine ES (mES) growth, maintenance of stem cell phenotype, and cell energetics over a pO(2) range of 0-285 mmHg, in the presence or absence of differentiation-suppressing leukemia inhibitory factor (LIF). With LIF, growth rate was sensitive to pO(2) but constant with time, and expression of self-renewal transcription factors decreased at extremes of pO(2).

A stirred microchamber for oxygen consumption rate measurements with pancreatic islets.

Improvements in pancreatic islet transplantation for treatment of diabetes are hindered by the absence of meaningful islet quality assessment methods. Oxygen consumption rate (OCR) has previously been used to assess the quality of organs and primary tissue for transplantation. In this study, we describe and characterize a stirred microchamber for measuring OCR with small quantities of islets.

Long-term normoglycemia in rats receiving transplants with encapsulated islets.

Background: To follow up on previously successful transplantation of encapsulated islets in mice, the present study was performed in rats to determine the effects of several factors, including alginate composition and concentration of cross-linking agent and capsule size on the effectiveness of encapsulated islets.

Methods: Highly purified alginate of either high guluronic acid or high mannuronic acid (M) with low endotoxin content was used. Regular-size (0.