Thyroid Hormone Levels Correlate With the Maturation of Implanted Pancreatic Endoderm Cells in Patients With Type 1 Diabetes.

BACKGROUND: Macroencapsulated pancreatic endoderm cells (PECs) can reverse diabetes in rodents and preclinical studies revealed that thyroid hormones in vitro and in vivo bias PECs to differentiate into insulin-producing cells. In an ongoing clinical trial, PECs implanted in macroencapsulation devices into patients with type 1 diabetes were safe but yielded heterogeneous outcomes. Though most patients developed meal responsive C-peptide, levels were heterogeneous and explanted grafts had variable numbers of surviving cells with variable distribution of endocrine cells. METHODS: We measured circulating triiodothyronine and thyroxine levels in all patients treated at 1 of the 7 sites of the ongoing clinical trial and determined if thyroid hormone levels were associated with the C-peptide or glucagon levels and cell fate of implanted PECs. RESULTS: Both triiodothyronine and thyroxine levels were significantly associated with the proportion of cells that adopted an insulin-producing fate with a mature phenotype. Thyroid hormone levels were inversely correlated to circulating glucagon levels after implantation, suggesting that thyroid hormones lead PECs to favor an insulin-producing fate over a glucagon-producing fate. In mice, hyperthyroidism led to more rapid maturation of PECs into insulin-producing cells similar in phenotype to PECs in euthyroid mice. CONCLUSION: These data highlight the relevance of thyroid hormones in the context of PEC therapy in patients with type 1 diabetes and suggest that a thyroid hormone adjuvant therapy may optimize cell outcomes in some PEC recipients.

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.

Large-scale Generation of Functional and Transplantable Hepatocytes and Cholangiocytes from Human Endoderm Stem Cells.

The ever-increasing therapeutic and pharmaceutical demand for liver cells calls for systems that enable mass production of hepatic cells. Here we describe a large-scale suspension system that uses human endoderm stem cells (hEnSCs) as precursors to generate functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). hEnSC-derived hepatic populations are characterized by single-cell transcriptomic analyses and compared with hESC-derived counterparts, in-vitro-maintained or -expanded primary hepatocytes and adult cells, which reveals that hepatic differentiation of hEnSCs recapitulates in vivo development and that the heterogeneities of the resultant populations can be manipulated by regulating the EGF and MAPK signaling pathways. Functional assessments demonstrate that E-heps and E-chos possess properties comparable with adult counterparts and that, when transplanted intraperitoneally, encapsulated E-heps were able to rescue rats with acute liver failure. Our study lays the foundation for cell-based therapeutic agents and in vitro applications for liver diseases.

Posttransplant Characterization of Long-term Functional hESC-Derived Pancreatic Endoderm Grafts.

The paucity of human donors limits broadened application of ß-cell replacement therapy. Insulin-producing cells derived from human embryonic stem cells (hESCs) have recently been investigated clinically as a feasible surrogate to primary tissue. Herein, we examine the long-term efficacy of hESC-derived pancreatic endoderm cells (PECs) to maintain normoglycemia posttransplant and characterize the phenotype of the PEC grafts. Mice with chemically induced diabetes were transplanted with PECs into the subcutaneous device-less site. Transplant function was assessed through nonfasting blood glucose measurements, intraperitoneal glucose tolerance testing (IPGTT), and human C-peptide secretion for 517 days. Explanted grafts were assessed for ex vivo function and immunohistochemically. All PEC recipients (n = 8) maintained normoglycemia until graft retrieval. IPGTTs at 365 and 517 days posttransplant did not differ (P > 0.05), however, both demonstrated superior glucose clearance compared with nondiabetic and transplant controls (P < 0.001). Serum C-peptide levels demonstrated significant glucose responsiveness (fasted vs. stimulated) (P < 0.01). Small intragraft cysts were palpable in all mice, which resolved but recurred after aspiration. Cysts showed monomorphic neuroendocrine proliferation and lined by ductal epithelium. Explanted grafts demonstrated similar insulin secretory capacity as human islets and stained positively for endocrine cells. Our results demonstrate the ability of PECs to differentiate in vivo and restore glycemic control while confirming minimal proliferation and absence of neoplastic change within the grafts during the time evaluated.

Transplantation of Human Pancreatic Endoderm Cells Reverses Diabetes Post Transplantation in a Prevascularized Subcutaneous Site.

Beta-cell replacement therapy is an effective means to restore glucose homeostasis in select humans with autoimmune diabetes. The scarcity of "healthy" human donor pancreata restricts the broader application of this effective curative therapy. "ß-Like" cells derived from human embryonic stem cells (hESC), with the capacity to secrete insulin in a glucose-regulated manner, have been developed in vitro, with limitless capacity for expansion. Here we report long-term diabetes correction in mice transplanted with hESC-derived pancreatic endoderm cells (PECs) in a prevascularized subcutaneous site. This advancement mitigates chronic foreign-body response, utilizes a device- and growth factor-free approach, facilitates in vivo differentiation of PECs into glucose-responsive insulin-producing cells, and reliably restores glycemic control. Basal and stimulated human C-peptide secretion was detected throughout the study, which was abolished upon graft removal. Recipient mice demonstrated physiological clearance of glucose in response to metabolic challenge and safely retrieved grafts contained viable glucose regulatory cells.

Embryonic stem cell-derived pancreatic endoderm transplant with MCT1-suppressing miR-495 attenuates type II diabetes in mice.

Type 2 diabetes mellitus (T2D) is a chronic metabolic disorder resulting from defects in both insulin secretion and insulin activity. The deficit and dysfunction of insulin secreting ß-cells are signature symptoms of T2D. Additionally, in pancreatic ß-cells, a small group of genes that are abundantly expressed in most other tissues is highly selectively repressed. Monocarboxylate transporter 1 (MCT1) is one of these genes. In this study, we identified an MCT1-suppressing microRNA (hsa-miR-495) and used this microRNA together with human embryonic stem cell (hESC) derived pancreatic endoderm (PE) cells transplanted into a high-fat diet induced T2D mouse model. Glucose metabolism significantly improved and other symptoms of T2D were attenuated after the procedure. Our findings support the potential for T2D treatment using the combination of microRNA and hESC differentiated PE cells.

A Combination of Human Embryonic Stem Cell-Derived Pancreatic Endoderm Transplant with LDHA-Repressing miRNA Can Attenuate High-Fat Diet Induced Type II Diabetes in Mice.

Type II diabetes mellitus (T2D) is a chronic metabolic disorder that results from defects in both insulin secretion and insulin action. The deficit and dysfunction of insulin secreting ß-cell are signature symptom for T2D. Additionally, in pancreatic ß-cell, a small group of genes which are abundantly expressed in most other tissues are highly selectively repressed. Lactate dehydrogenase A (LDHA) is one of such genes. Upregulation of LDHA is found in both human T2D and rodent T2D models. In this study, we identified a LDHA-suppressing microRNA (hsa-miR-590-3p) and used it together with human embryonic stem cell (hESC) derived pancreatic endoderm (PE) transplantation into a high-fat diet induced T2D mouse model. The procedure significantly improved glucose metabolism and other symptoms of T2D. Our findings support the potential T2D treatment using the combination of microRNA and hESC-differentiated PE cells.

Engraftment potential of spheroid-forming hepatic endoderm derived from human embryonic stem cells.

Transplantation and drug discovery programs for liver diseases are hampered by the shortage of donor tissue. While recent studies have shown that hepatic cells can be derived from human embryonic stem cells (hESCs), few cases have shown selective enrichment of hESC-derived hepatocytes and their integration into host liver tissues. Here we demonstrate that the dissociation and reaggregation procedure after an endodermal differentiation of hESC produces spheroids mainly consisted of cells showing hepatic phenotypes in vitro and in vivo. A combined treatment with Wnt3a and bone morphogenic protein 4 efficiently differentiated hESCs into definitive endoderm in an adherent culture. Dissociation followed by reaggregation of these cells in a nonadherent condition lead to the isolation of spheroid-forming cells that preferentially expressed early hepatic markers from the adherent cell population. Further differentiation of these spheroid cells in the presence of the hepatocyte growth factor, oncostatin M, and dexamethasone produced a highly enriched population of cells exhibiting characteristics of early hepatocytes, including glycogen storage, indocyanine green uptake, and synthesis of urea and albumin. Furthermore, we show that grafted spheroid cells express hepatic features and attenuate the serum aspartate aminotransferase level in a model of acute liver injury. These data suggest that hepatic progenitor cells can be enriched by the spheroid formation of differentiating hESCs and that these cells have engraftment potential to replace damaged liver tissues.

Highly efficient generation of definitive endoderm lineage from human induced pluripotent stem cells.

BACKGROUND: Although hepatocytes can be an option for liver transplantation, the shortage of donor organs continues to worsen. Since the development of induced pluripotent stem (iPS) cell technology, it is eagerly anticipated to produce functional elements from pluripotent stem cells. These functional cells differentiated from iPS cells could be used for transplantation, drug screening, and in vitro toxicology. METHODS: Human iPS cells are maintained on Mitomycin C-treated mouse embryonic fibroblast layers in DMEM-Ham F12-based medium supplemented with Knockout Serum Replacement, nonessential amino acids, 2-mercaptoethanol, and Glutamax. Differentiation of human iPS cells into a definitive endodermal lineage was induced with PRMI 1640 medium supplemented with B27 and 100 ng/mL human activin A. Two B27 supplements were examined with and without insulin. Furthermore, the PI3 kinase inhibitor LY294002 was used to examine the effect of inhibiting insulin signaling. RESULTS AND DISCUSSION: We established efficient induction of definitive endodermal differentiation from iPS cells. Quantitative analysis revealed efficient (93.03 ± 2.74%) differentiation of human iPS cells into definitive endoderm cells using B27 minus insulin. This protocol may contribute as a fundamental technique to promote human iPS studies to develop cellular sources for transplantation.

Spontaneous in vivo differentiation of embryonic stem cell-derived pancreatic endoderm-like cells corrects hyperglycemia in diabetic mice.

BACKGROUND: Whole pancreas and islet transplantation are currently used for the treatment of type 1 diabetes. However, the major limitations of this potentially curative approach are an inadequate supply of cadaveric pancreata, lifelong immunosuppression, and chronic graft rejection. Therefore, there is an urgent need to develop new sources of insulin-producing cells (IPCs). Here, we investigated whether embryonic stem (ES) cells can be exploited for the derivation of IPCs, and whether their transplantation can correct hyperglycemia in diabetic mice. METHODS: ES cells engineered to express pancreatic and duodenal homeobox 1 (Pdx1), a critical pancreatic transcription factor, were differentiated into pancreatic endoderm-like cells (PELCs) and evaluated for their potential to correct hyperglycemia after transplantation in diabetic mice. RESULTS: After systemic injection, PELCs localized to the pancreas, liver, and kidney. They then spontaneously differentiated into IPCs that corrected hyperglycemia in diabetic mice. When transplanted under the kidney capsule, PELC-derived IPCs were equally efficient at correcting hyperglycemia. Real-time noninvasive in vivo bioluminescence imaging (BLI) of rat insulin promoter (RIP)-driven luciferase was used to monitor the fate of the transplanted PELCs. To confirm that the transplanted cells were responsible for the correction of hyperglycemia, kidneys containing the transplanted cells were nephrectomized, causing rapid hyperglycemia. Interestingly, none of the animals transplanted with PELCs developed tumors, a potential consequence of the differentiation and purification procedures. CONCLUSIONS: Our data suggest that Pdx1-expressing PELCs are capable of spontaneously undergoing differentiation in vivo into IPCs and leading to a sustained correction of hyperglycemia in diabetic mice.

Transplantation of embryonic stem cell-derived endodermal cells into mice with induced lethal liver damage.

ESCs are a potential cell source for cell therapy. However, there is no evidence that cell transplantation using ESC-derived hepatocytes is therapeutically effective. The main objective of this study was to assess the therapeutic efficacy of the transplantation of ESC-derived endodermal cells into a liver injury model. The beta-galactosidase-labeled mouse ESCs were differentiated into alpha-fetoprotein (AFP)-producing endodermal cells. AFP-producing cells or ESCs were transplanted into transgenic mice that expressed diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. Selective damage was induced in the recipient hepatocytes by the administration of DT. Although the transplanted AFP-producing cells had repopulated only 3.4% of the total liver mass 7 days after cell transplantation, they replaced 32.8% of the liver by day 35. However, these engrafted cells decreased (18.3% at day 40 and 7.9% at day 50) after the cessation of DT administration, and few donor cells were observed by days 60-90. The survival rate of the AFP-producing cell-transplanted group (66.7%) was significantly higher in comparison with that of the sham-operated group (17.6%). No tumors were detected by day 50 in the AFP-producing cell-transplanted group; however, splenic teratomas did form 60 days or more after transplantation. ESC transplantation had no effect on survival rates; furthermore, there was a high frequency of tumors in the ESC-transplanted group 35 days after transplantation. In conclusion, this study demonstrates, for the first time, that ESC-derived endodermal cells improve the survival rates after transplantation into mice with induced hepatocellular injury. Disclosure of potential conflicts of interest is found at the end of this article.

Characterization of human intestinal stromal cell lines: response to cytokines and interactions with epithelial cells.

The maintenance of the physiological homeostasis of the gut mucosa characterized by continuous proliferation and differentiation processes results from epithelial-mesenchymal cell cross-talk. To set out stable and homogeneous models for the study of the (dys)regulation of various morphofunctional aspects, we established and characterized three clonal cell lines (C9, C11, and C20) derived from human duodenal mucosal connective tissue. We defined the expression of (i) cytoskeletal proteins; (ii) basement membrane molecules (laminins, collagen IV, nidogen) which have been shown formerly to be deposited at the epithelial/mesenchymal interface in situ by the mesenchymal compartment; and (iii) soluble factors, HGF, and TGFbeta1. The three cell lines display common but also specific proliferative responses to cytokines (IL1beta, IL2, IL8, TNFalpha, IFNgamma, TGFbeta1, and HGF). When cocultured with embryonic intestinal endoderms or with human colonic Caco2 or HT29 cancer cells, C9 versus C11 and C20 cell lines induced limited versus extensive growth of the associated epithelial cells. In addition C20 cells allowed spreading of HT29 cells with the formation of a basement membrane at the heterologous interface. Morphogenesis obtained by intracoelomic grafts of associations comprising the mesenchymal cell lines and intestinal endoderms was also different among those composed of C9 cells or of C11 or C20 cells. In conclusion, these data indicate that the mucosal connective tissue is heterogeneous and comprises several phenotypically different mesenchyme-derived cells whose equilibrium may be important in the gut homeostasis. These cells can now be used to define tissue-specific factors which may be involved in the physiopathology of the intestinal epithelium.

Specification of the zebrafish nervous system by nonaxial signals.

The organizer of the amphibian gastrula provides the neurectoderm with both neuralizing and posteriorizing (transforming) signals. In zebrafish, transplantations show that a spatially distinct transformer signal emanates from tissues other than the organizer. Cells of the germring (nonaxial mesendoderm) posteriorized forebrain progenitors when grafted nearby, resulting in an ectopic hindbrain-like structure; in contrast, cells of the organizer (axial mesendoderm) caused no posterior transformation. Local application of basic fibroblast growth factor, a candidate transformer in Xenopus, caused malformation but not hindbrain transformation in the forebrain. Thus, the zebrafish gastrula may integrate spatially distinct signals from the organizer and the germring to pattern the neural axis.

Para-aortic splanchnopleura from early mouse embryos contains B1a cell progenitors.

Definitive erythropoiesis in birds originates from stem cells that emerge in the splanchnopleural mesoderm near the embryonic aorta. The yolk sac is still generally held to be the unique provider of haematopoietic stem cells during mammalian ontogeny, although there may be an alternative intraembryonic source of stem cells in the mouse fetus. Here we search for a possible non-yolk-sac source of stem cells by grafting intraembryonic splanchnopleura from 10- to 18-somite mouse embryos into adult immunodeficient SCID mice. We find significant amounts of donor-derived serum IgM, normal numbers of IgM-secreting plasma cells, and the B1a (IgM(a)brightB220dullCD5+) cell subset to be fully reconstituted by donor progenitors 3 to 6 months after engraftment. The haematogenic capacity revealed in our experiments is present in a previously unrecognized site, the earliest described in the embryo, 12 hours before fetal liver colonization.

Emergence of endothelial and hemopoietic cells in the avian embryo.

During organogenesis, endothelial cells develop through two different mechanisms: differentiation of intrinsic precursors in organ rudiments constituted of mesoderm associated with endoderm, and colonization by extrinsic precursors in organs constituted of mesoderm associated with ectoderm (Pardanaud et al. 1989). On the other hand, both types of rudiment are colonized by extrinsic hemopoietic stem cells. In the present work we extend our former study by investigating the hemangioblastic (i.e. hemopoietic and angioblastic) potentialities of primordial germ layers in the area pellucida during the morphogenetic period. By means of interspecific grafts between quail and chick embryos, we show that splanchnopleural mesoderm gives rise to abundant endothelial cells, and to numerous hemopoietic cells in a permissive microenvironment, while somatopleural mesoderm produces very few cells belonging to these lineages, or none. Thus we confirm that the angioblastic capacities of the mesoderm differ radically, depending on its association with ectoderm or endoderm. Furthermore, at this embryonic period, both endothelial and hemopoietic potentialities are displayed by splanchnopleural mesoderm. However the site of emergence of intraembryonic hemopoietic stem cells appears spatially restricted by comparison to more widespread angioblastic capacities.

A nude mouse xenograft model of fetal intestine development and differentiation.

This report describes a novel in vivo model of intestinal differentiation. Fourteen day, undifferentiated fetal rat small intestine, stripped of the major part of its mesenchyme, suspended in a type I collagen gel and then xenografted into a nude mouse, undergoes small intestinal morphogenesis and cytodifferentiation. All four major epithelial lineages, namely Paneth, goblet, columnar and endocrine are present. Double-label nonisotopic in situ hybridization, employing biotinylated and digoxigenin-labelled whole rat DNA and whole mouse DNA probes, was performed to distinguish donor cells from host cell types. The outer longitudinal smooth muscle layer, and the major part of the lamina propria, including pericryptal fibroblasts, are of host mouse origin; the inner circular smooth muscle layer is of donor rat origin. Cells of the muscularis propria and lamina propria acquired smooth muscle alpha-actin, presumably under the influence of the donor endoderm. Furthermore, this xenograft develops a host vascular network, and cells with the morphological appearance of lymphocytes are present within the intestinal epithelium. The production of chemotactic factors by the endoderm is postulated because grafting of collagen gel alone results in a minimal invasion by stromal cells which do not express smooth muscle alpha-actin.

Formation of ectopic neurepithelium in chick blastoderms: age-related capacities for induction and self-differentiation following transplantation of quail Hensen's nodes.

Hensen's node, regarded as the avian and mammalian homologue of Spemann's neural inducer (i.e., the amphibian dorsal blastoporal lip), has been transplanted in many previous studies to the germinal crescent of avian blastoderms to examine ectopic neural induction. All these studies have suffered from one or more major shortcomings, the most significant of which has been the lack of a reliable cell marker to determine the contributions of graft cells to ectopic embryos. In the absence of such marker, induced (i.e., derived from the host) and self-differentiated (i.e., derived from the graft) neurepithelium cannot be distinguished from one another with certainty. We have transplanted quail Hensen's nodes to chick host blastoderms and have subsequently used the quail nucleolar heterochromatin marker to identify graft cells unequivocally. We systematically varied both donor and host ages (i.e., stages 3-8 and 3-5, respectively) to examine the effects of age on ectopic neural induction and self-differentiation. Our results demonstrate that the age of the donor is more critical than that of the host over the stages examined. With advancing donor age, the frequency of host induction decreases, while the frequency of graft self-differentiation increases. Previous studies not using cell markers have concluded that the craniocaudal level of the induced neuraxis is determined by the age of the donor, that is, young donors induce cranial neuraxial levels, whereas old donors induce caudal levels. By contrast, we found that with grafts from older donors, neurepithelium was more commonly self-differentiated rather than induced and that progressively more caudal levels of the neuraxis self-differentiated with advancing donor age. Induction of caudal neuraxial levels never occurred in the absence of induced cranial levels. The frequency of neural induction was inversely correlated with the age of the donor and directly correlated with the quantity of graft endodermal cells contributed to the ectopic embryo, supporting a previous assertion that in avian embryos, the earliest and principal source of neural inducer lies within the endoderm rather than mesoderm. From our results, we propose that the role of neural induction is to produce neurepithelium of unspecified regional character, and that the formation of regional character depends on subsequent morphogenetic events.

Peptide immunoreactivity in developing entero-endocrine cells in endodermal grafts.

Presumptive endoderm was excised from 19-h chick or quail embryos and grafted, together with a fragment of notochord from a 48-h host chick embryo. After 14 days the graft showed differentiation towards several digestive and respiratory tract structures. Entero-endocrine cells were demonstrated in these structures. Peptide immunoreactivity in these structures was similar to some extent to the peptide spectrum in corresponding regions during normal development, though the number of demonstrable peptides per region under the conditions of grafting appeared to be higher than in control conditions.