Gillian M. Beattie

Activin A Maintains Pluripotency of Human Embryonic Stem Cells in the Absence of Feeder Layers

To date, all human embryonic stem cells (hESCs) available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Activation of STAT3 by leukemia inhibitory factor is required to maintain “stemness” in mouse embryonic stem cells, but not in hESCs, suggesting the existence of alternate signaling pathways for self‐renewal and pluripotency in human cells. Here we show that activin A is secreted by mouse embryonic feeder layers (mEFs) and that culture medium enriched with activin A is capable of maintaining hESCs in the undifferentiated state for >20 passages without the need for feeder layers, conditioned medium from mEFs, or STAT3 activation. hESCs retained both normal karyotype and markers of undifferentiated cells, including Oct‐4, nanog, and TRA‐1‐60 and remained pluripotent, as shown by the in vivo formation of teratomas.

Maintenance of Pluripotency in Human Embryonic Stem Cells Is STAT3 Independent

The preservation of “stemness” in mouse embryonic stem (mES) cells is maintained through a signal transduction pathway that requires the gp130 receptor, the interleukin‐6 (IL‐6) family of cytokines, and the Janus Kinase‐signal transducer and activator (JAK/STAT) pathway. The factors and signaling pathways that regulate “stemness” in human embryonic stem (hES) cells remain to be elucidated. Here we report that STAT3 activation is not sufficient to block hES cell differentiation when the cells are grown on mouse feeder cells or when they are treated with conditioned media from feedercells. Human ES cells differentiate in the presence of members of the IL‐6 family of cytokines including leukemia inhibitory factor (LIF) and IL‐6 or in the presence of the designer cytokine hyper‐IL‐6, which is a complex of soluble interleukin‐6 receptor (IL‐6R) and IL‐6 with greatly enhanced bio‐activity. Human ES cells express LIF, IL‐6, and gp130 receptors, as well as the downstream signaling molecules. Stimulation of human and mouse ES cells with gp130 cytokines resulted in a robust phosphorylation of downstream ERK1, ERK2, and Akt kinases, as well as the STAT3 transcription factor. Loss of the pluripotency markers Nanog, Oct‐4, and TRA‐1‐60 was observed in hES cells during gp130‐dependent signaling, indicating that signaling through this pathway is insufficient to prevent the onset of differentiation. These data underscore a fundamental difference in requirements of murine versus hES cells. Furthermore, the data demonstrate the existence of an as‐yet‐unidentified factor in the conditioned media of mouse feeder layer cells that acts to maintain hES cell renewal in a STAT3‐independent manner.

Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells.

The effects of nicotinamide (NIC) on human fetal and adult endocrine pancreatic cells were studied in tissue culture. Treatment of the fetal cells with 10 mM NIC resulted in a twofold increase in DNA content and a threefold increase in insulin content. This was associated with the development of beta cell outgrowths from undifferentiated epithelial cell clusters and an increase in the expression of the insulin, glucagon, and somatostatin genes. DNA synthesis was stimulated only in the undifferentiated cells. Half-maximal doses for the insulinotropic and mitogenic effects of NIC were 5-10 and 1-2 mM, respectively. Islet-like cell clusters cultured with NIC responded to glucose stimulation with a biphasic increase in insulin release (fourfold peak), whereas control cells were unresponsive to glucose. Both control and NIC-treated cells developed into functional islet tissue after transplantation into athymic nude mice. As compared with adult islets, the insulinotropic action of NIC could only be demonstrated in the fetal cells. Our results indicate that NIC induces differentiation and maturation of human fetal pancreatic islet cells. This model should be useful for the study of molecular mechanisms involved in beta cell development.

Trehalose: A Cryoprotectant That Enhances Recovery and Preserves Function of Human Pancreatic Islets After Long-Term Storage

The scarcity of available tissue for transplantation in diabetes and the need for multiple donors make it mandatory to use an optimal cryopreservation method that allows maximal recovery and preservation of β-cell function. We have developed a method to cryopreserve islets with excellent survival of endocrine cells. Current methods use DMSO as cryoprotectant. Our method involves introducing both DMSO and the disaccharide trehalose into the cells during cooling. Uptake and release of trehalose occurred during the thermotropic lipid-phase transition measured in pancreatic endocrine cells between 5° and 9°C, using [14C]trehalose. Recovery of adult islets after cryopreservation with 300 mmol/l trehalose was 92 vs. 58% using DMSO alone. In vitro function, in terms of insulin content and release in response to secretagogues, was indistinguishable from fresh islets. Grafts from islets cryopreserved with trehalose contained 14-fold more insulin than grafts from islets cryopreserved without trehalose. Results with human fetal islet-like cell clusters (ICCs) were more pronounced: recovery from cryopreservation was 94%, compared with 42% without trehalose. Complete functionality of fetal cells was also restored; tritiated thymidine incorporation and insulin content and release were similar to fresh tissue. After transplantation in nude mice, there was a 15-fold increase in insulin content of grafts from ICCs cryopreserved with trehalose compared with ICCs cryopreserved without trehalose. Thus, the addition of trehalose to cryopreservation protocols leads to previously unobtainable survival rates of human pancreatic endocrine tissue.

Microfabricated immunoisolating biocapsules

A microfabricated silicon-based biocapsule for the immunoisolation of cell transplants is presented. The biocapsule-forming process employs bulk micromachining to define cell-containing chambers within single crystalline silicon wafers. These chambers interface with the surrounding biological environment through polycrystalline silicon filter membranes. The membranes are surface micromachined to present a high density of uniform pores, thus affording sufficient permeability to oxygen, glucose, and insulin. The pore dimensions, as small as 20 nm, are designed to impede the passage of immune molecules and graft-borne viruses. The underlying filter-membrane nanotechnology has been successfully applied in controlled cell culture systems (Ferrari et al., 1995), and is under study for viral elimination in plasma fractionation protocols. Here we report the encouraging results of in vitro experiments investigating the biocompatibility of the microfabricated biocapsule, and demonstrate that encapsulated rat neonatal pancreatic islets significantly outlive and outperform controls in terms of insulin-secretion capability over periods of several weeks. These results appear to warrant further investigations on the potential of cell xenografts encapsulated within microfabricated, immunoisolating environments for the treatment of insulin-dependent diabetes.

Regulation of Proliferation and Differentiation of Human Fetal Pancreatic Islet Cells by Extracellular Matrix, Hepatocyte Growth Factor, and Cell-Cell Contact

Ex vivo expansion of human fetal pancreatic endocrine cells is important for biological studies and as a potential tissue source for transplantation in insulin-deficient states. In tissue culture experiments involving the use of hepatocyte growth factor/scatter factor and selected extracellular matrices, we obtained a 30-fold increase in cell number of human fetal pancreatic epithelial cells. This proliferation in monolayer culture was associated with marked downregulation of insulin and glucagon gene expression. However, gene expression increased when the cells were combined into three-dimensional aggregates, suggesting that cell-cell contact mediated mechanisms regulate the transcription of islet-specific genes, a process enhanced by nicotinamide (NIC). After transplantation into nude mice, either as cell suspensions or aggregates, only the cell aggregates treated with NIC developed into mature functional islet-like structures. These are the first experiments to describe the interactions of specific matrices and growth factors in the ex vivo expansion of human fetal pancreatic cells, and they also show the importance of cell aggregates in the context of cellular and molecular events that might positively influence islet cell transplantation.

Growth Factor/Matrix-Induced Proliferation of Human Adult β-Cells

Proliferation of human β-cells in vitro is desirable for both transplantation and biological studies. In this study, human pancreatic islets obtained from cadavers were kept in tissue culture plates that favored cell attachment. When the cells attached to the matrix produced by the rat-bladder carcinoma cell line 804G, 5′-bromo-2′-deoxyuridine (BrdU) labeling increased from 4.7 ± 2.5 to 13.2 ± 2.2%, while cells simultaneously labeled for insulin and BrdU increased from 0 to 32%. Addition of the growth factor hepatocyte growth factor/scatter (HGF/SF) increased BrdU labeling to 17.5 ± 1.8 and the percentage of double positive (BrdU + insulin) cells to 69%. This is the first in vitro demonstration that human β-cells grown in monolayer culture are able to replicate when exposed to selected matrices and growth factors. These experiments add further evidence that HGF/SF is an important mitogenic agent for human β-cells.

Hepatocyte Growth Factor/Scatter Factor Has Insulinotropic Activity in Human Fetal Pancreatic Cells

Fetal mesenchyme-derived factors are likely to play an important role in pancreatic islet development and growth. We have used primary cultures of human fetal pancreatic tissue to identify growth factors that have morphogenic, mitogenic, and insulinotropic activity. The formation of islet-like cell clusters (ICCs) during a 6-day culture was stimulated two- to threefold by hepatocyte growth factor/scatter factor (HGF/SF) basic fibroblast growth factor (FGF)-2, and to a lesser extent by keratinocyte growth factor (FGF-7) and insulin-like growth factor-II (IGF-II). In contrast, transforming growth factor-β (TGF-β) had a strong inhibitory effect. The ICCs formed during HGF/SF stimulation consisted mainly of epithelial cells, whereas FGF-2-induced ICCs were predominantly nonepithelial. Furthermore, although both FGF-2 and HGF/SF increased the total insulin content of the cultures, only HGF/SF increased the insulin content per DNA. Quantitatively, HGF/SF stimulated a 2.3-fold increase in the proportion of insulin-positive cells and a 3-fold higher number of replicating β-cells. Blocking of the IGF-I receptor inhibited ICC formation but did not affect their insulin content. Immunoneutralizing TGF-β resulted in increased cell growth and insulin content, indicating the presence of an endogenous inhibitory TGF-β activity in the model system. Our results suggest that HGF/SF may be an important component of the fetal mesenchyme-derived factors responsible for pancreatic islet development. HGF/SF also may prove valuable for supporting the in vitro growth of islet cells.

An in vivo model for study of the angiogenic effects of basic fibroblast growth factor

We have investigated the angiogenic effects of basic fibroblast growth factor following its implantation in slow release beads under the kidney capsule. The presence of basic fibroblast growth factor in the subcapsular space induced a marked angiogenic response maximal at 1 microgram dose per kidney. Histological examination at the site of treatment failed to reveal evidence of an inflammatory response, thus supporting the observation that basic fibroblast growth factor alone can stimulate in vivo neovascularization. Beads pretreated with saline or with human growth hormone had no angiogenic effect. Because of the readily accessible location in the retroperitoneal space, the ease of drug delivery, and the marked vascular proliferation seen in response to FGF, our results suggest that the kidney capsule is an excellent model for study of the physiological role played by FGF and related peptides in promoting angiogenesis in vivo.

Limited Capacity of Human Adult Islets Expanded In Vitro to Redifferentiate Into Insulin-Producing β-Cells

Limited organ availability is an obstacle to the widespread use of islet transplantation in type 1 diabetic patients. To address this problem, many studies have explored methods for expanding functional human islets in vitro for diabetes cell therapy. We previously showed that islet cells replicate after monolayer formation under the influence of hepatocyte growth factor and selected extracellular matrices. However, under these conditions, senescence and loss of insulin expression occur after >15 doublings. In contrast, other groups have reported that islet cells expanded in monolayers for months progressed through a reversible epithelial-to-mesenchymal transition, and that on removal of serum from the cultures, islet-like structures producing insulin were formed (1). The aim of the current study was to compare the two methods for islet expansion using immunostaining, real-time quantitative PCR, and microarrays at the following time points: on arrival, after monolayer expansion, and after 1 week in serum-free media. At this time, cell aliquots were grafted into nude mice to study in vivo function. The two methods showed similar results in islet cell expansion. Attempts at cell differentiation after expansion by both methods failed to consistently recover a β-cell phenotype. Redifferentiation of β-cells after expansion is still a challenge in need of a solution.