Ana D. Lopez

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.

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.

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.

Effective Detection of Human Leukocyte Antigen Risk Alleles in Celiac Disease Using Tag Single Nucleotide Polymorphisms

Background The HLA genes, located in the MHC region on chromosome 6p21.3, play an important role in many autoimmune disorders, such as celiac disease (CD), type 1 diabetes (T1D), rheumatoid arthritis, multiple sclerosis, psoriasis and others. Known HLA variants that confer risk to CD, for example, include DQA1*05/DQB1*02 (DQ2.5) and DQA1*03/DQB1*0302 (DQ8). To diagnose the majority of CD patients and to study disease susceptibility and progression, typing these strongly associated HLA risk factors is of utmost importance. However, current genotyping methods for HLA risk factors involve many reactions, and are complicated and expensive. We sought a simple experimental approach using tagging SNPs that predict the CD-associated HLA risk factors. Methodology Our tagging approach exploits linkage disequilibrium between single nucleotide polymorphism (SNPs) and the CD-associated HLA risk factors DQ2.5 and DQ8 that indicate direct risk, and DQA1*0201/DQB1*0202 (DQ2.2) and DQA1*0505/DQB1*0301 (DQ7) that attribute to the risk of DQ2.5 to CD. To evaluate the predictive power of this approach, we performed an empirical comparison of the predicted DQ types, based on these six tag SNPs, with those executed with current validated laboratory typing methods of the HLA-DQA1 and -DQB1 genes in three large cohorts. The results were validated in three European celiac populations. Conclusion Using this method, only six SNPs were needed to predict the risk types carried by >95% of CD patients. We determined that for this tagging approach the sensitivity was >0.991, specificity >0.996 and the predictive value >0.948. Our results show that this tag SNP method is very accurate and provides an excellent basis for population screening for CD. This method is broadly applicable in European populations.

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.

Isolation and characterization of a cell line from the epithelial cells of the human fetal pancreas

Pancreatic cell lines are useful for basic studies of pancreatic biology and for possible application to cell transplantation therapies for diabetes. A retroviral vector expressing simian virus 40 (SV40) T antigen and H-rasval12 was used to infect a monolayer culture of epithelial cells from an 18-wk human fetal pancreas. Infected cells gave rise to a clonal epithelial cell line, designated TRM-1. This cell line expresses epithelial markers as well as gult2 and small amounts of insulin and glucagon. TRM-1 is the first cell line to be generated from the human fetal pancreas and also the first cell line derived directly from the fetal pancreas of any species. The approach that we have used to develop TRM-1 should be applicable to isolating cell lines from other stages of human pancreatic development.

Functional β-Cell Mass After Transplantation of Human Fetal Pancreatic Cells: Differentiation or Proliferation?

The scarcity of human adult islets available for transplantation in IDDM makes the use of human fetal pancreatic cells desirable. Human fetal pancreatic cells grow and differentiate after transplantation in nude mice. It is unclear whether proliferation of preexisting endocrine cells or differentiation of precursor cells is mainly responsible for the increased islet mass and if β-cell enrichment before transplantation enhances the functional outcome of the graft. To answer these questions, we transplanted purified human fetal islets, isletlike cell clusters (ICCs), and fresh tissue under the kidney capsule of nude mice. Insulin content was highest in the fresh tissue but fell rapidly during culture as either fetal islets or ICCs. Although fetal islets contained fourfold more insulin than ICCs before transplantation, the insulin content of the resulting grafts was the same after 3 months in vivo. The degree of stimulation after glucose challenge was comparable; however, more tissue was needed to generate the fetal islets. Grafts of fresh tissue also had similar total insulin contents, but when normalized to DNA, insulin concentration was significantly higher in the grafts from cultured tissue. Moreover, there were distinct morphological differences; the grafts from fresh tissue were more fibrous, with prominent ductal and cystic elements. Grafts from cultured tissue were two- to threefold enriched in endocrine tissue when compared with grafts originating from fresh tissue. These results suggest that islet cells identified in the grafted ICCs are mainly derived through differentiation of endocrine precursors and that cultured ICCs are more preferable than either fetal islets or uncultured tissue for transplantation.