[beta]-Cell Differentiation In Vitro - Wiley Online Library

TISSUE-SPECIFIC STEM CELLS Enhanced Oxygenation Promotes ␤-Cell Differentiation In Vitro ´ LVAREZ,a PANAGIOTIS PAPADOPOULOS,a JAIME GIRALDO,a CHRISTOPHER A. FRAKER,a,b SILVIA A b a WEIYONG GU, CAMILLO RICORDI, LUCA INVERARDI,a JUAN DOM´ıNGUEZ-BENDALAa a

Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, Miami, Florida, USA; Department of Biomedical Engineering, University of Miami, Coral Gables, Florida, USA

b

Key Words. Stem cells • Islets • Perfluorocarbon • Oxygenation • ␤ Cells

ABSTRACT Despite progress in our knowledge about pancreatic islet specification, most attempts at differentiating stem/progenitor cells into functional, transplantable ␤ cells have met only with moderate success thus far. A major challenge is the intrinsic simplicity of in vitro culture systems, which cannot approximate the physiological complexity of in vivo microenvironments. Oxygenation is a critical limitation of standard culture methods, and one of special relevance for the development of ␤ cells, known for their high O2 requirements. Based on our understanding of islet physiology, we have tested the hypothesis that enhanced O2 delivery (as provided by novel perfluorocarbon-based culture devices) may result in higher levels of

␤-cell differentiation from progenitor cells in vitro. Using a mouse model of pancreatic development, we demonstrate that a physiological-like mode of O2 delivery results in a very significant upregulation of endocrine differentiation markers (up to 30-fold for insulin one and 2), comparable to relevant in vivo controls. This effect was not observed by merely increasing environmental O2 concentrations in conventional settings. Our findings indicate that O2 plays an important role in the differentiation of ␤ cells from their progenitors and may open the door to more efficient islet differentiation protocols from embryonic and/or adult stem cells. STEM CELLS 2007;25: 3155–3164

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Islet transplantation is a promising therapy, but its prospects as the treatment of choice for type I diabetes will be bleak until the issues of immunosuppression and donor tissue supply are properly addressed [1, 2]. The latter remains arguably the most immediate concern, as the number of potential recipients greatly exceeds that of available pancreata. This problem is further aggravated by a progressive loss of function of the graft over time [3], which may require periodic reinfusions of islets. Therefore, the definition of a self-renewable source of cells with the potential to become ␤ cells would be of great clinical significance. Bone marrow-derived mesenchymal [4, 5], umbilical cord [6, 7], and, more recently, amniotic fluid [8] cells have received considerable attention within the field of adult stem cells. It has also been proposed that the adult pancreas may contain expandable subpopulations of cells with the ability to become endocrine tissue. Among these, ductal [9], acinar [10], or islet-derived sources [11, 12] are commonly cited. Although expansion from the latter was originally thought to happen through epithelial-to-mesenchymal transition [13], recent studies suggest otherwise [14 –16], which raises concerns about their actual potential. Embryonic stem (ES) cells, on the other hand, are known to be pluripotent and immortal under defined conditions [17, 18]. Both mouse [19 –21] and human [22–26] ES cells have been induced to produce insulin under a variety of experimental settings. The first report on “canonical” ES-cell differentiation into ␤ cells [22] came shortly after the description of

the conditions for definitive endoderm differentiation [27, 28]. The resulting ␤ cells, however, were relatively scarce (⬃7%) and did not secrete insulin in response to glucose stimulation. In summary, all attempts to efficiently generate functional ␤ cells from either adult or embryonic stem cells have been largely unsuccessful thus far. Our understanding of islet development has progressed enormously over the last decade, to a point where the most important molecular players appear to have been identified [29, 30]. Not surprisingly, a mainstream goal has been to recapitulate in vitro, to the highest possible extent, the evolving molecular environment of these cells. Little attention has been paid, however, to their physiological environment. Mounting evidence points at physical variables as major determinants of development/cell specification. Among these, some of the most studied are mechanical forces [31, 32], pH and bioelectrical fields [33–35], oxygenation levels [36 –39], and the nature of the substrate/mode of culture [40 – 43]. However, most attempts at differentiating ␤ cells from stem cells have generally disregarded physiological “niche” of islet cells. Growing cells as monolayers may facilitate the even distribution of nutrients, O2, and signaling molecules, but only at the expense of compromising three-dimensional (3D) cell-to-cell interactions deemed essential for the development and maintenance of the ␤-cell phenotype [44 – 47]. Also, adult islets are surrounded by an elaborate network of capillary vessels and connective tissue, critical to their high metabolic activity. In fact, although islets account only for 1%–2% of the total number of cells of the pancreas, they receive nearly 15% of the overall blood flow to

Correspondence: Juan Domıńguez-Bendala, M.Sc., Ph.D., Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, 1450 NW 10th Avenue, Miami, Florida 33136, USA. Telephone: 305-243-4092; Fax: 05-243-4404; e-mail: jdominguez2@med. miami.edu Received June 7, 2007; accepted for publication August 21, 2007; first published online in STEM CELLS EXPRESS August 30, 2007. ©AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2007-0445

STEM CELLS 2007;25:3155–3164 www.StemCells.com

Oxygenation Promotes ␤-Cell Differentiation

3156

the organ, using 25% of the pancreatic O2 supply [48, 49]. When removed from their native environment, the islet microvascular network is destroyed and viability decreases dramatically [50]. Although islets in vivo maintain a relatively constant O2 partial pressure throughout their diameter, those cultured in standard conditions are exposed to sharp oxygen partial pressure (pO2) gradients that may range from extreme surface hyperoxia (158 mmHg) to central anoxia (0 mmHg [51]). A thorough review of the state of the art warrants the assertion that standard culture practice is not favorable for long-term islet survival and function [50]. In this context, it is not unreasonable to think that a prerequisite to generating ␤ cells might be to mimic the physiological conditions that they need to survive in the first place. The very same limitations that result in progressive ␤-cell death in culture may also hinder their terminal differentiation from immature progenitors— especially if there is a requirement for reaggregation into 3D structures, which would render them sensitive to hypoxia [45– 47]. The above hypothesis has been difficult to test thus far because of the lack of appropriate culture systems that ensure uniform oxygenation throughout cell clusters. Conventional methods are based on the diffusion of atmospheric O2 through the culture medium to the cells, which rest atop a gas-impermeable plastic bottom. Increasing the concentration of O2 injected into the incubators will not solve this problem, as sharp gradients will still form throughout the tissue. We have designed a novel culture system where tissues receive air both from the top (after diffusion through medium) and the bottom (through direct diffusion across a perfluorohydrocarbon-silicone membrane). Perfluorohydrocarbons (PFCs) are inert compounds made of long carbon chains where hydrogen atoms are replaced with fluorine. This unique feature gives PFCs an unmatched ability to bind and transfer molecular oxygen. Functional studies have shown that O2 solubility of PFCs is approximately 50 times higher than that of culture medium, surpassing even that of hemoglobin at higher environmental pO2 levels [52]. Furthermore, their O2 diffusivity is also 2.5-fold that of water or culture medium [53, 54]. Using a highly reproducible murine model of pancreatic development, we tested the hypothesis that a more physiological mode of O2 delivery, as provided by the above culture system, would result in enhanced rates of endocrine differentiation. Our results indicate that this is indeed the case. We observed a very significant upregulation of all tested pancreatic differentiation genes in the experimental group compared with controls cultured in standard conditions. Endocrine-to-exocrine and ␤-to-␣cell differentiation ratios were also significantly higher, not only compared with in vitro controls but also compared with the corresponding in vivo stage of development. Although high O2 enhanced the proliferation of epithelial cell types both in standard and PFC/silicone (Si) platforms, the positive effect on endocrine differentiation was seen only in the latter. Potential molecular mechanisms underlying these effects are discussed in the context of the definition of strategies to improve the yield and functionality of ␤ cells from stem and/or progenitor cells in vitro.

MATERIALS

AND

METHODS

Tissue Procurement and Culture Pancreatic buds from embryonic day e9.5– e16.5 CBA ⫻ C57BL6 embryos (noon of the day a vaginal plug is found is considered 0.5 days of gestation) were isolated and microdissected as described elsewhere [55]. Culture medium was Glasgow Minimal Essential

Figure 1. Enhanced oxygenation in PFC/Si devices. (A): Schematic representation of the oxygen sandwich principle. In standard culture vessels, atmospheric oxygen can reach the tissue only after diffusion through the culture medium. In PFC/Si devices, the sample rests atop a perfluorocarbon-enriched, air-permeable silicone membrane, which provides additional oxygenation. (B): COMSOL version 3.2 mathematical modeling of oxygen gradients in pancreatic buds immediately after equilibration (day 0) in standard conditions at 21% oxygen (top), standard conditions at 35% oxygen (middle), and PFC/Si devices at 35% oxygen (bottom). Left, oxygen partial pressure scale (mmHg), from blue (Min) to red (Max). White represents areas with ⬍0.1 mmHg oxygen (anoxia). Abbreviations: Max, maximum; Min, minimum; PFC/Si, perfluorohydrocarbon/silicone.

Medium (GMEM, Invitrogen, Carlsbad, CA, http://www. invitrogen.com) supplemented with 0.1 mM Minimal Essential Medium nonessential amino acids (Invitrogen), sodium pyruvate, 5% (vol/vol) newborn bovine serum, 5% (vol/vol) fetal calf serum, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin/100 ␮g/ml streptomycin, and 250 ␮M L-glutamine (Invitrogen). Controls were plated in 12-mm, 0.4 ␮M Millicell, Millipore, Billercia, MA, http://www. millipore.com) inserts (typically two buds per insert) and incubated at 37 0C and 5% CO2 at either 21% or 35% O2. Buds assigned to the experimental group were plated in PFC/Si culture plate inserts (Fig. 1) and incubated at 37°C, 5% CO2, and 35% O2 (manufacture described in supplemental online Methods).

Immunostaining and Image Analysis Explants were grown as above for 3 days and then fixed with 4% paraformaldehyde (30 minutes), washed with phosphate-buffered saline (30 minutes) and frozen in Optimal Cutting Temperature compound (Sakura, Torrance, CA, http://www.sakura.com). Pancreatic rudiments were sectioned in their entirety (5 ␮m) and mounted with 4,6-diamidino-2-phenylindole-Vectashield (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Guinea pig anti-insulin and rabbit anti-glucagon antibodies (ready-to-use solution; BioGenex, San Ramon, CA, http://www.biogenex.com) were used for double staining. 5-bromo-2⬘-deoxyuridine (BrdU) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) was added to the culture medium at a final concentration of 10 ␮M and kept overnight (and added freshly every day) before fixation. Rat anti-BrdU antibody (1:100 dilution; Accurate, Westbury, NY, http:// www.accuratechemical.com) and fluorescein isothiocyanate-conjugated hypoxyprobe (1:100 dilution; Chemicon, Temecula, CA, http://www.chemicon.com) were used for detection of proliferating cells and hypoxia, respectively. Rabbit antibodies against amylase (1:200), carboxypeptidase (1:200), E-cadherin (1:100), and Nestin (1:100) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, http://www.scbt.com), Sigma-Aldrich, Zymed (Invitrogenzymed, Carlsbad, CA, http://www.invitrogen.com), and Covance

´ lvarez, Papadopoulos et al. Fraker, A (Princeton, NJ, http://www.covance.com), respectively. Al secondary antibodies were purchased from Molecular Probes Inc. (Eugene, OR, http://probes.invitrogen.com). Metamorph software was used to quantitate insulin and glucagon staining (details in supplemental online Methods).

Real-Time Quantitative Reverse TranscriptionPolymerase Chain Reaction Total RNA was purified using Qiagen kits (QIAShredder, RNeasy, and DNase-free; Qiagen, Hilden, Germany, http://www.qiagen. com). The First-Strand system (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) was used to generate cDNA (random oligomers). Relative gene expression was calculated using TaqMan assays in a 7500 Fast Real Time polymerase chain reaction (PCR) cycler (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). The ⌬Ct method for relative quantification was deemed optimal for this application after discussion with Applied Biosystems researchers. All assays (Applied Biosystems) were designed to span exon-exon junctions, thus eliminating the possibility of genomic DNA contamination. Quantitative reverse transcription (qRT)-PCR results are the average of several independent experiments, as indicated in Results. In addition, in each experiment each marker was analyzed in triplicates. Specific assay numbers are provided as supplementary online information. Gene expression was normalized against 18S ribosomal RNA. This endogenous control has been validated in our system and proven extremely stable and more accurate over varying O2 concentrations than other standards (data not shown).

Genomic DNA Quantitation DNA was measured using the Quant-iT pico Green kit (Invitrogen), and cell number was inferred using the biological constant of ⬃7 pg of DNA per nucleus.

Statistical Analysis Analysis of variance (ANOVA) tests were used to analyze variance between the means of the different groups (p ⬍ .05). When more than two groups are compared, paired Student’s t tests usually result in a higher probability of type I errors (i.e., when the null hypothesis is rejected even though it is true), hence the use of ANOVA. SEM was used for all our analyses.

O2 Diffusion Modeling 3D diffusion/reaction theoretical modeling was performed on permutations of control and experimental culture systems using COMSOL version 3.2 finite element analysis software. Iterative solutions for concentration profiles were determined using the time-dependent solver. The modeling allowed us to examine the effect of multiple variables, including medium height, tissue proximity, perfluorocarbon volume fraction, and external O2 concentration. Our calculations were based on published diffusion coefficients for tissue and culture medium, as well as measured dimensions and O2 consumption rates of mouse pancreatic buds (supplemental online Methods). The validity of the models was confirmed by direct O2 readings of pancreatic buds in vitro, using polarographic microelectrode-based techniques (supplemental online Methods).

Multiplex Analyses Basic protein kinase profiles were obtained using a Bioplex instrument from Bio-Rad (Hercules, CA, http://www.bio-rad.com) and manufacturer’s assays, as described previously [56].

Insulin Measurement Total protein was extracted from groups of six to eight pancreatic buds, and insulin content was measured using Mercodia (Uppsala, Sweden, http://www.mercodia.com) enzyme-linked immunosorbent assay kits.

www.StemCells.com

3157

RESULTS PFC/Si Devices Ensure High Oxygenation Levels in e13.5 Pancreatic Buds We used dorsal pancreatic buds explanted at e13.5 as our primary model of in vitro differentiation. This point is within the period of maximal ␤-cell specification in the developing mouse [57], and the effect of interventions on their in vitro spontaneous development can be readily quantified [55]. Thirty dorsal pancreatic buds pooled from e13.5 embryos (six pregnant C57BL6 females) were microdissected and measured. Their average dimensions were 449.7 ⫾ 78.5 ␮m ⫻ 649.8 ⫾ 79.3 ␮m ⫻ 450.1 ⫾ 79.1 ␮m. Average O2 consumption rate was 8.95 ⫾ 0.75 ⫻ 10⫺3 mol/m3 䡠 second⫺1. Variability between samples was ⬍10%, despite using tissue from different harvests. Traditional culture of pancreatic buds is done in inserts where the tissue rests atop a permeable membrane, barely bathed with the medium beneath. These “medium/air interface” conditions ensure acceptable oxygenation levels, but nutrient diffusion is suboptimal, growth is limited, and medium needs to be replenished often to prevent desiccation. This is a cumbersome and very specialized culture system with few uses, if any, outside the area of developmental studies of small explants. Stem cell differentiation, particularly for large-scale therapeutic applications, will require standard expansion systems where cells are properly immersed in culture medium. To replicate the air transfer restrictions of standard stem cell culture methods, while accommodating the special needs of our biological model (which requires a basal membrane to maintain the morphological integrity of the buds), we have modified the basal control conditions so that pancreatic buds sit on top of a permeable membrane but culture medium bathes them all around (up to 1 mm above the cells; Fig. 1). As it could be argued that the level of oxygenation in this “hybrid” system would be lower than in conventional settings, we added a second control where O2 concentration is increased to 35% in the incubator. Mathematical modeling and direct in vitro measurements demonstrate that such adjustment results in oxygenation levels comparable to those of the medium/air interface conditions (data not shown), while maintaining an adequate medium height to support nutrition, expansion, and differentiation. Thus, we modeled three culture platforms: (a) standard control (Millipore [Billerica, MA, http://www.millipore.com] Teflon inserts cultured in conventional O2 concentration in a 95% room air/5% CO2 incubator); (b) high O2 control (the above system cultured in the presence of 35% O2); and (c) PFC/Si (where the bottom of the Millipore inserts was replaced by a 450-␮m-thick silicone/20% perfluorocarbon membrane), also cultured at 35% O2. In both controls, the samples rest at the top of liquid-permeable Teflon membranes that suspend them above the plastic bottom, so that both their apical and basal regions are bathed with culture medium (Fig. 1A, top). PFC/Si membranes, in contrast, are liquid-impermeable. By resting atop these membranes, samples are bathed by medium just from the top but are directly exposed to environmental O2 from the bottom (Fig. 1A, bottom). The mass transfer rate of O2 to the cells is greatly enhanced in this system, as it is not limited by diffusivity through the culture medium. Finite element modeling of the diffusion/reaction parameters of each culture environment was performed using COMSOL version 3.2 software. These calculations were validated by experimental O2 measurements throughout the tissue (supplemental online Methods). At the beginning of the culture (day 0), pancreatic buds cultured in standard control conditions (Fig. 1B, top) had

3158


overall oxygenation levels below 100 mmHg, with large areas (depicted in white) below 0.1 mmHg. This is generally considered the threshold of anoxia [58]. Increasing the O2 concentration from 21% to 35% in the standard culture system (Fig. 1B, middle) prevented anoxia and improved oxygenation, although not as much as in PFC/Si devices (Fig. 1B, bottom).

High O2 Promotes the Growth of Pancreatic Buds in PFC/Si Settings Without Hypoxia or Oxygen-Related Stress e13.5 dorsal pancreatic buds were explanted and cultured in normal conditions (standard control; n ⫽ 13), 35% O2 (high O2 control; n ⫽ 14) and PFC/Si membranes at 35% O2 (experimental group; n ⫽ 14). Maximal growth of the explants was observed at 72 hours of culture. Longer periods (up to 1 week) did not result in additional expansion (data not shown). Buds in the experimental group almost tripled their volume over the 3-day culture period. Explants kept at high O2 alone also expanded, but not as much (twofold). Buds in standard conditions, finally, showed the least increase in size (1.1-fold) (Fig. 2A; Fig. 2B, left column). DNA quantitation experiments (n ⫽ 3 buds per group) were subsequently performed to calculate overall number of cells using the Quant-iT pico Green kit (Invitrogen). Freshly explanted buds had an average of 413,483 cells; after 3 days, these values were as follows: 567,654 in buds cultured in control conditions, 585,233 in high O2 controls, and 910,985 in PFC/Si-grown explants. qRT-PCR measurements showed a 4.4fold increase in PCNA expression in the PFC/Si-cultured buds over both controls (n ⫽ 7 experiments, 2–3 buds per group). Our theoretical calculations, adjusted for the increased size of the buds at 72 hours, indicate that this growth was accompanied by a decrease in O2 diffusion across the tissue (data not shown). This was confirmed by the use of a histological probe for hypoxia in samples fixated after 3 days in culture. The hypoxyprobe binds to protein adducts formed when tissue is exposed to O2 partial pressures of ⬍10 mmHg [59]. Midpoint sections of representative samples of each group show large hypoxic areas in buds cultured both in standard and high O2 conditions but not in those plated in PFC/Si devices (Fig. 2B, right column). Taken together, the above observations strongly suggest that the volumetric increase observed in the PFC/Si group is due mostly to proliferation, whereas changes in the high O2 control might be due to just a decrease in tissue compaction (probably due to cell death in the core of the buds after an initial burst of proliferation). We also conducted a basic multiplex kinase profile analysis (Bio-Rad) to test whether enhanced oxygenation, as provided by PFC/Si devices, might lead to oxygen-induced stress. Preliminary experiments (n ⫽ 2 ⫻ 10 pooled buds, cultured for 3 days) showed differential activation of the survival signal AKT (phosphorylated/total protein ratio: Control (C) ⫽ 1.07 ⫾ 0.162; PFC/Si ⫽ 1.67 ⫾ 0.004) and suppression of the stress-activated kinases c-jun (ratio: C ⫽ 0.825 ⫾ 0.003; PFC/Si ⫽ 0.72 ⫾ 0.018), NF-␬B (ratio: C ⫽ 0.26 ⫾ 0.024; PFC/Si ⫽ 0.12 ⫾ 0.012), and ERK (ratio: C ⫽ 6.2 ⫻ 10⫺2 ⫾ 0.003; PFC/Si ⫽ 3.6 ⫻ 10⫺2 ⫾ 0.001) in the PFC/Si group compared with the regular O2 control. In conclusion, our studies confirm that PFC/Si platforms prevent hypoxia and ensure high oxygenation levels in our test system without inducing oxygenrelated stress.

PFC/Si-Induced Growth Is Due to Replication of Undifferentiated Epithelial Cells To determine the nature of the cells responsible for the proliferation observed during culture, we added BrdU to the culture medium for the entire length of the experiment (72 hours) and

Figure 2. PFC/Si devices induce higher proliferation rates. (A): Volume of pancreatic buds (m3) after 3 d of culture in each condition (standard control, high-oxygen control, and PFC/Si), showing a favorable effect of oxygen on cell proliferation. The baseline represents the average volume of buds immediately after harvesting (d 0). Error bars, SE for each group. (B): Left column, microphotographs of buds cultured for 3 d in each condition. Scale bar ⫽ 400 ␮m. Right column, immunofluorescent analysis of pancreatic buds (d 3 of culture) with a hypoxyprobe (Chemicon), which detects areas at ⬍10 mmHg (hypoxia) (green). Blue, 4,6-diamidino-2-phenylindole nuclear counterstaining. Scale bar ⫽ 500 ␮m. Abbreviations: d, day; PFC/Si, perfluorohydrocarbon/silicone.

then fixed, sectioned, and immunostained the samples. Coexpression of BrdU and markers of terminal differentiation (insulin, glucagon, amylase, and carboxypeptidase A) was rare in all the groups, as shown in Figure 3 (top row). This observation indicates that there is no significant replication of mature epithelial cells. It is known that mesenchymal E-cadherin⫺/nestin⫹ cells are intermingled with epithelial cells at this stage of development [60]. These cells, however, turned out to be scarce and largely BrdU-negative in all the groups (data not shown). Most of the BrdU incorporation was found to be localized in E-cadherin⫹/differentiation marker⫺ cells (Fig. 3, bottom row). In summary, our results indicate that proliferation occurs preferentially in undifferentiated epithelial cells that do not mature during the course of the experiment.

´ lvarez, Papadopoulos et al. Fraker, A

3159

Figure 3. Perfluorohydrocarbon/silicone (PFC/Si)-induced growth is due to replication of undifferentiated epithelial cells. Confocal immunofluorescent analysis of pancreatic buds cultured for 3 days in PFC/Si devices in the presence of BrdU (green). Blue, DAPI nuclear counterstaining. Top row: in red, clockwise from the left, Glu, Ins, CPA, and Amy. Few terminally differentiated cells had BrdU costaining. Bottom row: BrdU (green), DAPI (blue), and E-cad (red) staining of PFC/Si-cultured buds, shown as two-channel combinations (microphotographs 1–3) and three-channel combination (microphotograph 4). As expected, most of the cells in the bud are E-cad⫹ (epithelial). Proliferation occurs preferentially within the E-cad⫹-undifferentiated population. Scale bars ⫽ 50 ␮m (Glu, Ins, CPA) and 75 ␮m (Amy, E-cad). Abbreviations: Amy, amylase; BrdU, bromodeoxyuridine; CPA, carboxypeptidase A; DAPI, 4,6-diamidino-2-phenylindole; E-cad, E-cadherin; Glu, glucagon; Ins, insulin.

Differentiation Is Greatly Enhanced in PFC/Si Devices Compared with Standard and High O2 Controls e13.5 dorsal pancreatic buds were harvested and cultured as described above. At day 3, the explants were lysed for RNA isolation and protein extraction or fixed for immunohistochemical analysis. A panel of genes involved in the progression of pancreatic differentiation and ␤-cell maturation was tested by qRT-PCR in seven independent experiments (2–3 buds per group). As shown in Figure 4A, all genes examined are upregulated in the PFC/Si group compared with the standard control: insulin 1 (30-fold), insulin 2 (34-fold), glucagon (8-fold), Pdx1 (2-fold), Ngn3 (8-fold), Isl1 (4-fold), Pax4 (14-fold), Pax6 (3.5fold), Arx (15-fold), P48 (4.5-fold), carboxypeptidase A (9fold), amylase (5-fold), and Glut-2 (4-fold). Surprisingly, the values observed in the high O2 control were barely above those of the standard control and in some cases even lower. Differences between the PFC/Si group and the controls were statistically significant for insulin 1 (p ⫽ .025) and 2 (p ⫽ .025), glucagon (p ⫽ .020), Ngn3 (p ⫽ .017), Pax4 (p ⫽ .039), Pax6 (p ⫽ .037), and Glut-2 (p ⫽ .025) but not for the pancreatic endocrine markers Pdx1 (p ⫽ .07), Isl1 (p ⫽ .06), and Arx (p ⫽ .09) or the exocrine markers P48 (p ⫽ .45), carboxypeptidase A (p ⫽ .43) and amylase (p ⫽ .34). Metamorph analysis (supplemental online Methods) of immunofluorescence-labeled buds (n ⫽ 5 independent experiments, three buds per group) confirmed the upregulation of insulin and glucagon, the two major endocrine hormones observed at this point of development. As represented in Figure 4B, insulin⫹ staining in PFC/Si buds is nearly 10-fold that of standard controls (2.5-fold that of high O2 controls). Similarly, glucagon⫹ signal was 5 times more abundant in the experimental group than in conventional settings (threefold that of high O2 controls). ANOVA tests show that differences between the PFC/Si and the standard control group (p ⫽ .01 and 0.001 for insulin and glucagon, respectively), but not those between the two control groups, are statistically significant. Representative sections of buds cultured in each conwww.StemCells.com

dition, shown in Figure 4C, show that clusters of insulin- and glucagon-producing cells were thicker and denser in PFC/Si than in control buds. Scattered somatostatin⫹ cells could be seen in PFC/Si-cultured buds but not in any of the control groups. This was confirmed by qRT-PCR analysis in two independent samples (n ⫽ 3 buds/condition), which showed an average 18-fold somatostatin signal increase in the PFC/Si group versus the standard control (in which, given the absence of signal, a Ct of 40 was used for calculations). As expected of the natural pattern of development of pancreatic polypeptide cells (which do not arise until e18 [61]), no PP signal could be detected in any of the groups by either immunolabeling or qRT-PCR. Since the majority of these new endocrine and exocrine cells that differentiated during in vitro culture arose from postmitotic progenitor cells (Fig. 3), it could be concluded that the increased rates of growth and differentiation are two distinct effects of culture in PFC/Si devices. We sought additional proof by examining PFC/Si-induced differentiation in the absence of proliferation. Prior to culture in each condition, we treated freshly microdissected e13.5 buds with mitomycin C, a potent inhibitor of cell division. Cell growth arrest was confirmed by genomic DNA quantitation in buds (n ⫽ 3) before and after treatment. Although the difference was less dramatic than before, differentiation was still significantly higher in the experimental group compared with both controls (twofold to sixfold for endocrine cell markers; data not shown). This observation is consistent with the hypothesis that the enhancement of endocrine differentiation in PFC/Si-cultured buds is independent from the parallel increase in their proliferative capacity.

In Vitro Maturation in PFC/Si Devices Closely Mimics In Vivo Development To assess the extent to which buds cultured in PFC/Si approximate in vivo levels of differentiation, pancreatic buds obtained at e16.5 (a time point corresponding to e13.5 buds ⫹ 3 days of ex vivo development) were lysed for RNA and protein extrac-

3160

Figure 4. Culture in PFC/Si platforms promotes endocrine differentiation. (A): Relative quantitative reverse transcription polymerase chain reaction analysis of pancreatic buds cultured in standard conditions (black columns), high oxygen (dark gray columns), and PFC/Si devices (light gray columns). Values are represented as fold increase over the control (control ⫽ 1). Error bars, SE (seven independent experiments). P values lower than 0.05 are indicated with asterisks. All values were normalized against 18S RNA (as described in Materials and Methods). (B): Metamorph analysis of Ins (left) and Glu (right) signal in immunostained buds cultured in standard (black columns), high oxygen (dark gray columns), and PFC/Si (light gray columns) settings. y-axis, fold increase over standard control (control ⫽ 1). Error bars, SE (five independent experiments). (C): Confocal microphotographs of representative sections from embryonic day 13.5 buds cultured for 3 days in standard conditions (left), high oxygen (middle), and PFC/Si platforms (right). Ins-positive cells are stained in red and Glu-positive cells in green. Blue, 4,6-diamidino-2-phenylindole nuclear counterstaining. A white dotted line has been added to highlight the contour of the samples. Scale bar ⫽ 100 ␮m. Abbreviations: CPA, carboxypeptidase A; Glu, glucagon; Ins, insulin; PFC/Si, perfluorohydrocarbon/silicone, Standard C, standard control.

tion (n ⫽ 4 independent experiments). These values were compared with those previously obtained from PFC/Si-cultured buds. Total insulin content of buds cultured for 3 days in PFC/Si was nearly 80% that of freshly explanted e16.5 buds, compared with 30% and 15% in the high O2 and standard control groups, respectively (Fig. 5A). This observation was confirmed and further expanded by qRT-PCR, where the above panel of genes was run in the e16.5 harvests and compared with the 7 PFC/Si in vitro experiments. Figure 5B presents gene expression profiles of the PFC/Si group expressed as a percentage of that determined for e16.5. Most of the differences between the PFC/Si group and e16.5 are statistically insignificant (p ⬎ .05), suggesting that in vitro maturation occurred at in vivo rates.


Figure 5. Insulin content and gene expression profiles of PFC/Sicultured e13.5 buds approximate those of e16.5 buds. (A): Total insulin content in each group (standard control, high-oxygen control, and PFC/ Si), represented as the proportion of that found in dorsal pancreatic buds obtained from e16.5 embryos (⫽ 1). Error bars, SE for each group (n ⫽ 4). (B): Gene expression profile of PFC/Si-cultured e13.5 buds, expressed as a percentage of that of freshly isolated e16.5 buds. A dotted line highlights 100% of e16.5 expression. Error bars, SE for each group (n ⫽ 4 independent harvests for e16.5 and 7 independent experiments for PFC/Si). ⴱ, P values equal to or lower than 0.05. Abbreviations: CPA, carboxypeptidase A; e, embryonic day; N.S., not statistically significant; PFC/Si, perfluorohydrocarbon/silicone.

PFC/Si Promotes Endocrine over Exocrine and ␤- over ␣-Cell Differentiation One possible interpretation of the above results is that a better method of O2 delivery simply promotes overall differentiation. We hypothesized, however, that our method for enhanced oxygenation would preferentially induce the specification of endocrine cells, as their O2 demands exceed greatly those of exocrine tissue [48, 49]. To test this hypothesis, and based on the qRTPCR data presented above, we calculated the ratios of endocrine-to-exocrine and ␤-to-␣-cell differentiation. As shown in Figure 6, these ratios are consistently higher in the PFC/Si group than in the two in vitro controls (up to 30-fold for endocrine/ exocrine and 2–3-fold for ␤-to-␣-cell ratios) and even the e16.5 pancreatic buds (2–3-fold for both types of ratios). As it could be argued that the varying O2 levels could be affecting gene expression without significantly altering cell fate choices, we sought additional confirmation of our hypothesis by doing a ratiometric analysis of the values previously obtained by Metamorph quantitation of immunofluorescently labeled buds (n ⫽ 5). The ␤-to-␣-cell ratio in the PFC/Si group was almost twofold higher than that of the standard control (1.5-fold higher than the high O2 control). Although further immunolabeling studies would be necessary to fully confirm this observation for other

´ lvarez, Papadopoulos et al. Fraker, A

Figure 6. PFC/silicone (Si) induces preferential endocrine over exocrine and ␤-cell over ␣-cell differentiation. Ratiometric analysis of gene expression in the PFC/Si group compared with e16.5 buds (dark gray columns), as well as standard (light gray columns) and high-oxygen (dark gray columns) controls. Values are represented as fold increase of the net ratio for each pair of genes in the PFC/Si group over that of the other three groups (supplemental online Methods). (A): Endocrine-toexocrine ratiometric analysis: INS1/Amy, Ins1/p48, and Ins1/CPA. The same ratios were calculated for INS2. (B): ␤-to-␣-cell ratiometric analysis: Pax4/Arx, INS1/GLU, INS2/GLU, and Pax4/Pax6. Error bars, SE for each group. ⴱ, P values equal to or lower than 0.05. Abbreviations: AMY, amylase; C, control; CPA, carboxypeptidase A; e, embryonic day; GLU, glucagon; INS, insulin; PFC, perfluorohydrocarbon.

cell types, our results are consistent with a preferential differentiation toward endocrine/␤-cell fates.

DISCUSSION Success at efficiently differentiating pancreatic endocrine tissue from a renewable source of cells could have immediate therapeutic applications for the treatment of type I diabetes. However, current methods for the in vitro specification of ␤ cells are still inefficient. We hypothesized that a better recapitulation of the physiological environment of ␤ cells (of which oxygenation is a key component) may be conducive to higher differentiation yields. Our results confirmed the above hypothesis in a mouse model of pancreatic development, but only when air was delivered in a basal-apical fashion. Increasing the concentration of O2 in the incubator in standard culture conditions did not result in any significant upregulation of endocrine differentiation. Although the “oxygen sandwich” effect could also be achieved with membranes made of silicone alone, those that included PFC in their composition proved superior in preliminary experiments. Expression of endocrine markers (glucagon, insulin 1, insulin 2, and Pax4) was between twofold and fivefold higher in PFC/Si than in the silicone alone group, with no significant www.StemCells.com

3161

differences in exocrine gene differentiation or proliferation markers (data not shown). The advantages of PFC/Si over silicone alone in terms of O2 diffusion were also confirmed by direct measurements using noninvasive optical O2 biosensors (Presens GmbH, Munich, Germany, http://www.presens.de; data not shown). Hence, we opted for PFC/Si devices for all subsequent experiments. Our data show an unequivocal enhancement of endocrine differentiation, with insulin 1 and 2 expression levels exceeding 30-fold those of buds cultured in standard conditions (21% or 35% O2). All markers of endocrine differentiation were also upregulated, including Ngn3 (a marker of proendocrine cell types), glucagon and Pax-6 (␣ cells), Isl1 (endocrine cells), Pax4 (pro-␤ cells), Glut-2 and Pdx1 (␤ cells), and Pax6 (pro-␣ cells). Although the increase in Pdx1 levels observed in PFC/Si-cultured buds over the standard control was a seemingly modest twofold (sixfold over high O2 control), it must be noted that, other than throughout the duct epithelium, expression of this gene is just starting to reappear around this time in arising ␤ cells. Our results could be interpreted as a reflection of better culture conditions, rather than a preferential effect of enhanced oxygenation on endocrine differentiation. However, the observed upregulation of exocrine markers in PFC/Sicultured buds was statistically insignificant. The above hypothesis was further disproved by calculating the quotients between endocrine (insulin 1 and 2) and exocrine (carboxypeptidase A, amylase, and p48) markers. Within endocrine cells, we also determined insulin/glucagon, Pax4/Pax6, and Pax4/Arx ratios. The latter has been shown to be important at the crossroads between ␣- and ␤-cell segregation from a common progenitor cell (excess of Pax4 over Arx will result in ␤-cell differentiation, whereas the opposite will lead toward the generation of ␣ cells [62, 63]). Similarly, expression of Pax6, but not Pax4, is normally associated with ␣-cell specification at this stage of development [64]. In all the experiments conducted, the PFC/Si group showed invariably higher endocrine-to-exocrine and ␤-to-␣-cell differentiation ratios than the two in vitro controls. Surprisingly, they were even higher than in freshly explanted e16.5 pancreatic buds, which represent a valuable control of in vivo differentiation. Although we cannot entirely rule out the possibility that all these effects occur only at the gene expression level, ␤-to␣-cell differentiation ratios calculated from immunofluorescently labeled buds are indeed higher in the PFC/Si group than in the controls. It is certainly possible that ␤ cells differentiated in PFC/Si dishes not only are more numerous but also express higher amounts of insulin. This would be consistent with the observation that the ratios obtained by immunofluorescence are slightly lower than those calculated from the qRT-PCR values. This slight disparity, however, could be simply a reflection of the variability expected of two different methodologies. An enhanced growth rate was also observed when using PFC/Si devices. BrdU incorporation studies show that the majority of the newly generated cells were epithelial (Ecadherin⫹). BrdU, however, was very rarely seen in terminally differentiated cells (amylase⫹, carboxypeptidase⫹, insulin⫹, or glucagon⫹). This suggests that most of the newly differentiated endocrine or exocrine cells arose from progenitors that were already quiescent at the beginning of the experiment. Our data, therefore, support the notion that PFC/Si matrices have two distinct effects on e13.5 pancreatic buds: (a) enhanced proliferation of epithelial cells that do not differentiate during the course of the experiment, and (b) enhanced endocrine differentiation of a postmitotic subpopulation of progenitor cells. The independence between prolif-

3162


Figure 7. A hypothetical model to explain the role of O2 in pancreatic development. According to the hypothesis presented in the main text, hypoxic conditions present in the pancreas prior to the initiation of blood flow would favor the HIF-1␣-mediated activation of Notch in endocrine progenitor cells, promoting their self-renewal but largely preventing their differentiation. HIF-1␣ stabilization in hypoxic conditions also induces VEGF-mediated angiogenesis, which will eventually lead to the initiation of blood flow at approximately e13.5. Enhanced oxygenation of pancreatic tissues at that point will destabilize HIF-1␣, resulting in Notch downregulation and differentiation of endocrine cell types. Exocrine progenitor cells, in contrast, may react to higher O2 levels by activating the Wnt/␤-catenin pathway via NRX sequestration by ROS. This would promote their expansion throughout the rest of embryonic development. Abbreviations: Dvl, Dishevelled; e, embryonic day; HIF, hypoxiainducible factor; NRX, nucleoredoxin; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.

eration and differentiation was further confirmed by experiments where, despite a mitomycin C-induced arrest of proliferation, endocrine differentiation was still enhanced. We did not observe any evidence of PFC/Si-enhanced mesenchymal cell proliferation, which may have had some influence in the fate of epithelial progenitors. At this point, we can only speculate about the molecular mechanisms by which enhanced oxygenation exerts the observed influence on pancreatic development. Among the differentiation pathways directly influenced by O2 tension, Notch is of utmost relevance for pancreatic development [65]. This pathway is generally involved in the maintenance of an undifferentiated state, and its downregulation is key for the initiation of the endocrine differentiation cascade [66 –71]. Under hypoxic conditions, the hypoxia-inducible factor 1␣ (HIF-1␣) is stabilized and interacts with the intracellular domain of Notch, activating this signaling cascade [36, 39, 72–74]. In this context, it follows that higher oxygenation would destabilize HIF-1␣, which in turn would inhibit Notch signaling, promoting endocrine differentiation. This would be consistent with the observation that the second and most significant wave of ␤-cell specification (secondary transition [61]) is concurrent with the initiation of blood flow within the pancreatic buds, long after endothelial cells first appear in the tissue [75]. However (as we also see in our in vitro model), higher oxygenation can also induce proliferation of nonendocrine cell types, a behavior that cannot be explained by the HIF-1/Notch pathway. This typically happens through the generation of reactive oxygen species (ROS), which have been shown to participate as signal transducers in numerous biological processes [76 –79]. Recently, Funato et al. demonstrated that the redoxregulating protein nucleoredoxin (NRX) blocks the Wnt/␤-catenin pathway by binding to Dishevelled (Dvl), a signal transducer in the process [80]. ROS-mediated oxidation of NRX results in its dissociation from Dvl. This results in activation of the Wnt/ ␤-catenin pathway, which has been associated with the maintenance of undifferentiated proliferating exocrine progenitor cells [81].

A model that fits the experimental evidence is presented in Figure 7. Hypoxic conditions present until e13.5 would theoretically favor the Notch-dependent proliferation of pancreatic progenitors. Since these conditions also promote angiogenesis in a HIF-1-dependent manner, the initiation of blood flow and subsequent oxygenation would (a) arrest Notch signaling in endocrine progenitor cells (thus allowing a massive wave of endocrine differentiation around that time), and (b) activate Wnt/␤-catenin signaling in exocrine progenitor cells (thus triggering sustained acinar cell proliferation). According to this hypothesis, PFC/Si devices (but not hypoxic control conditions) would, to some extent, mimic the two independent effects of angiogenesis in different cell subsets of the developing pancreas. The testing of this hypothesis is presently the subject of several lines of research in our laboratory. The observation that high O2 concentrations per se did not enhance differentiation over basal levels demonstrates that standard methods of air delivery are ill-suited to sustain growth and differentiation of cell aggregates. After 3 days in culture, buds cultured in high O2 had a very significant incidence of hypoxia, which was not observed in PFC/Si-cultured explants. The detection of ␦ cells in PFC/Si-cultured buds offers additional evidence that these platforms are close to reproducing in vivo development conditions. Notably, improved O2 delivery was not accompanied by oxygen-induced stress, as shown by preliminary multiplex analyses.

CONCLUSIONS To date, most attempts at differentiating islets from stem cells have focused only on their molecular environment. Our results emphasize the importance of providing developing progenitor cells with the right physiological environment, opening a new avenue of research that timely complements parallel advances in the field. In short, this work (a) presents evidence of a direct relationship between oxygenation and pancreatic endocrine cell

´ lvarez, Papadopoulos et al. Fraker, A differentiation, whose molecular mechanism is presently under study; and (b) describes a novel cell culture tool designed to deliver O2 in a physiological-like fashion, which could be of immediate use in the development of more efficient islet differentiation protocols from adult and/or embryonic stem cells.

3163

of kinase profiles; and Ricardo Pastori, Elsie Zahr, Herman Cheung, Armando Meńdez, Sonia Rocha, Felipe Echeverri, and Ramoń Poo for advice and helpful discussions. This work was funded by the American Diabetes Association, the Wallace H. Coulter Center for Translational Research, and the Diabetes Research Institute Foundation.

ACKNOWLEDGMENTS We thank Kevin Johnson and Brigitte Shaw from the University of Miami Histology and Imaging Core for help with sample histological processing and confocal microphotography; Alessia Fornoni and Thor Tejada for assistance in preliminary analyses

DISCLOSURE

23

2 3 4 5 6

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Ricordi C, Strom TB. Clinical islet transplantation: Advances and immunological challenges. Nat Rev Immunol 2004;4:259 –268. Shapiro AM, Lakey JR, Ryan EA et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000;343:230 –238. Ryan EA, Paty BW, Senior PA et al. Five-year follow-up after clinical islet transplantation. Diabetes 2005;54:2060 –2069. Li Y, Zhang R, Qiao H et al. Generation of insulin-producing cells from PDX-1 gene-modified human mesenchymal stem cells. J Cell Physiol 2007;211:36 – 44. D’Ippolito G, Howard GA, Roos BA et al. Isolation and characterization of marrow-isolated adult multilineage inducible (MIAMI) cells. Exp Hematol 2006;34:1608 –1610. Sun B, Roh KH, Lee SR et al. Induction of human umbilical cord blood-derived stem cells with embryonic stem cell phenotypes into insulin producing islet-like structure. Biochem Biophys Res Commun 2007;354:919 –923. Zhao Y, Wang H, Mazzone T. Identification of stem cells from human umbilical cord blood with embryonic and hematopoietic characteristics. Exp Cell Res 2006;312:2454 –2464. De Coppi P, Bartsch G Jr, Siddiqui MM et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:100 –106. Bonner-Weir S, Toschi E, Inada A et al. The pancreatic ductal epithelium serves as a potential pool of progenitor cells. Pediatr Diabetes 2004; 5(suppl 2):16 –22. Hao E, Tyrberg B, Itkin-Ansari P et al. Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas. Nat Med 2006;12:310 –316. Ouziel-Yahalom L, Zalzman M, Anker-Kitai L et al. Expansion and redifferentiation of adult human pancreatic islet cells. Biochem Biophys Res Commun 2006;341:291–298. Gershengorn MC, Hardikar AA, Wei C et al. Epithelial-to-mesenchymal transition generates proliferative human islet precursor cells. Science 2004;306:2261–2264. Gershengorn MC, Geras-Raaka E, Hardikar AA et al. Are better islet cell precursors generated by epithelial-to-mesenchymal transition? Cell Cycle 2005;4:380 –382. Chase LG, Ulloa-Montoya F, Kidder BL et al. Islet-derived fibroblastlike cells are not derived via epithelial-mesenchymal transition from Pdx-1 or insulin-positive cells. Diabetes 2007;56:3–7. Kayali AG, Flores LE, Lopez AD et al. Limited capacity of human adult islets expanded in vitro to redifferentiate into insulin-producing betacells. Diabetes 2007;56:703–708. Atouf F, Park CH, Pechhold K et al. No evidence for mouse pancreatic beta-cell epithelial-mesenchymal transition in vitro. Diabetes 2007;56: 699 –702. Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines. STEM CELLS 2001;19:193–204. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147. Lumelsky N, Blondel O, Laeng P et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001;292:1389 –1394. Soria B, Roche E, Berna G et al. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000;49:157–162. Hori Y, Rulifson IC, Tsai BC et al. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. Proc Natl Acad Sci U S A 2002;99:16105–16110. D’Amour KA, Bang AG, Eliazer S et al. Production of pancreatic

www.StemCells.com

CONFLICTS

The authors indicate no potential conflicts of interest.

REFERENCES 1

OF POTENTIAL OF INTEREST

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

45

hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 2006;24:1392–1401. Assady S, Maor G, Amit M et al. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691–1697. Segev H, Fishman B, Ziskind A et al. Differentiation of human embryonic stem cells into insulin-producing clusters. STEM CELLS 2004;22: 265–274. Kwon YD, Oh SK, Kim HS et al. Cellular manipulation of human embryonic stem cells by TAT-PDX1 protein transduction. Mol Ther 2005;12:28 –32. Jiang J, Au M, Lu K et al. Generation of insulin-producing islet-like clusters from human embryonic stem cells. STEM CELLS 2007;25: 1940 –1953. Kubo A, Shinozaki K, Shannon JM et al. Development of definitive endoderm from embryonic stem cells in culture. Development 2004;131: 1651–1662. D’Amour KA, Agulnick AD, Eliazer S et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 2005;23:1534 –1541. Edlund H. Pancreatic organogenesis– developmental mechanisms and implications for therapy. Nat Rev Genet 2002;3:524 –532. Kumar M, Melton D. Pancreas specification: A budding question. Curr Opin Genet Dev 2003;13:401– 407. Chen C. Using microenvironment to engineer stem cell function. Conf Proc IEEE Eng Med Biol Soc 2004;7:4964. Nelson CM, Jean RP, Tan JL et al. Emergent patterns of growth controlled by multicellular form and mechanics. Proc Natl Acad Sci U S A 2005;102:11594 –11599. Levin M. Bioelectromagnetics in morphogenesis. Bioelectromagnetics 2003;24:295–315. Robinson KR, Messerli MA. Left/right, up/down: The role of endogenous electrical fields as directional signals in development, repair and invasion. Bioessays 2003;25:759 –766. Nuccitelli R. Ionic currents in morphogenesis. Experientia 1988;44: 657– 666. Diez H, Fischer A, Winkler A et al. Hypoxia-mediated activation of Dll4-Notch-Hey2 signaling in endothelial progenitor cells and adoption of arterial cell fate. Exp Cell Res 2007;313:1–9. D’Ippolito G, Diabira S, Howard GA et al. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells. Bone 2006;39:513–522. Grayson WL, Zhao F, Izadpanah R et al. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol 2006;207:331–339. Gustafsson MV, Zheng X, Pereira T et al. Hypoxia requires notch signaling to maintain the undifferentiated cell state. Dev Cell 2005;9: 617– 628. Czyz J, Wobus A. Embryonic stem cell differentiation: The role of extracellular factors. Differentiation 2001;68:167–174. Li WJ, Jiang YJ, Tuan RS. Chondrocyte phenotype in engineered fibrous matrix is regulated by fiber size. Tissue Eng 2006;12:1775–1785. Liu H, Collins SF, Suggs LJ. Three-dimensional culture for expansion and differentiation of mouse embryonic stem cells. Biomaterials 2006; 27:6004 – 6014. Liu H, Lin J, Roy K. Effect of 3D scaffold and dynamic culture condition on the global gene expression profile of mouse embryonic stem cells. Biomaterials 2006;27:5978 –5989. Luther MJ, Davies E, Muller D et al. Cell-to-cell contact influences proliferative marker expression and apoptosis in MIN6 cells grown in islet-like structures. Am J Physiol Endocrinol Metab 2005;288: E502–E509. Pipeleers DG, Schuit FC, in’t Veld PA et al. Interplay of nutrients and hormones in the regulation of insulin release. Endocrinology 1985;117: 824 – 833.


3164

46 Bosco D, Orci L, Meda P. Homologous but not heterologous contact increases the insulin secretion of individual pancreatic B-cells. Exp Cell Res 1989;184:72– 80. 47 Hopcroft DW, Mason DR, Scott RS. Structure-function relationships in pancreatic islets: Support for intraislet modulation of insulin secretion. Endocrinology 1985;117:2073–2080. 48 Lifson N, Kramlinger KG, Mayrand RR et al. Blood flow to the rabbit pancreas with special reference to the islets of Langerhans. Gastroenterology 1980;79:466 – 473. 49 Jansson L. The regulation of pancreatic islet blood flow. Diabetes Metab Rev 1994;10:407– 416. 50 Giuliani M, Moritz W, Bodmer E et al. Central necrosis in isolated hypoxic human pancreatic islets: Evidence for postisolation ischemia. Cell Transplant 2005;14:67–76. 51 Papas KK, Avgoustiniatos ES, Tempelman LA et al. High-density culture of human islets on top of silicone rubber membranes. Transplant Proc 2005;37:3412–3414. 52 Biro GP, Blais P. Perfluorocarbon blood substitutes. Crit Rev Oncol Hematol 1987;6:311–374. 53 Mates vanLobensels E, Anderson JC, Hildebrandt J et al. Modeling diffusion limitation of gas exchange in lungs containing perfluorocarbon. J Appl Physiol 1999;86:273–284. 54 Radisic M, Deen W, Langer R et al. Mathematical model of oxygen distribution in engineered cardiac tissue with parallel channel array perfused with culture medium containing oxygen carriers. Am J Physiol Heart Circ Physiol 2005;288:H1278 –H1289. 55 Domıńguez-Bendala J, Klein D, Ribeiro M et al. TAT-mediated neurogenin 3 protein transduction stimulates pancreatic endocrine differentiation in vitro. Diabetes 2005;54:720 –726. 56 Fornoni A, Cobianchi L, Sanabria NY et al. The l-isoform but not d-isoforms of a JNK inhibitory peptide protects pancreatic beta-cells. Biochem Biophys Res Commun 2007;354:227–233. 57 Li H, Edlund H. Persistent expression of Hlxb9 in the pancreatic epithelium impairs pancreatic development. Dev Biol 2001;240:247–253. 58 Avgoustiniatos ES, Colton CK. Effect of external oxygen mass transfer resistances on viability of immunoisolated tissue. Ann N Y Acad Sci 1997;831:145–167. 59 Raleigh JA, Miller GG, Franko AJ et al. Fluorescence immunohistochemical detection of hypoxic cells in spheroids and tumours. Br J Cancer 1987;56:395– 400. 60 Selander L, Edlund H. Nestin is expressed in mesenchymal and not epithelial cells of the developing mouse pancreas. Mech Dev 2002;113: 189 –192. 61 Pictet R, Rutter, W. J. Development of the embryonic endocrine pancreas. In: Steiner DF, Frankel, N., eds. Handbook of Physiology, Vol 1. Baltimore: Williams & Wilkins, 1972:25– 66. 62 Collombat P, Hecksher-Sorensen J, Broccoli V et al. The simultaneous loss of Arx and Pax4 genes promotes a somatostatin-producing cell fate specification at the expense of the alpha- and beta-cell lineages in the mouse endocrine pancreas. Development 2005;132:2969 –2980.

63 Collombat P, Mansouri A, Hecksher-Sorensen J et al. Opposing actions of Arx and Pax4 in endocrine pancreas development. Genes Dev 2003; 17:2591–2603. 64 Ritz-Laser B, Estreicher A, Gauthier BR et al. The pancreatic beta-cellspecific transcription factor Pax-4 inhibits glucagon gene expression through Pax-6. Diabetologia 2002;45:97–107. 65 Kadesch T. Notch signaling: The demise of elegant simplicity. Curr Opin Genet Dev 2004;14:506 –512. 66 Apelqvist A, Li H, Sommer L et al. Notch signalling controls pancreatic cell differentiation. Nature 1999;400:877– 881. 67 Edlund H. Developmental biology of the pancreas. Diabetes 2001; 50(suppl 1):S5–S9. 68 Hart A, Papadopoulou S, Edlund H. Fgf10 maintains notch activation, stimulates proliferation, and blocks differentiation of pancreatic epithelial cells. Dev Dyn 2003;228:185–193. 69 Jensen J, Pedersen EE, Galante P et al. Control of endodermal endocrine development by Hes-1. Nat Genet 2000;24:36 – 44. 70 Lammert E, Brown J, Melton DA. Notch gene expression during pancreatic organogenesis. Mech Dev 2000;94:199 –203. 71 Murtaugh LC, Stanger BZ, Kwan KM et al. Notch signaling controls multiple steps of pancreatic differentiation. Proc Natl Acad Sci U S A 2003;100:14920 –14925. 72 Cejudo-Martin P, Johnson RS. A new notch in the HIF belt: How hypoxia impacts differentiation. Dev Cell 2005;9:575–576. 73 Pear WS, Simon MC. Lasting longer without oxygen: The influence of hypoxia on Notch signaling. Cancer Cell 2005;8:435– 437. 74 Sainson RC, Harris AL. Hypoxia-regulated differentiation: Let’s step it up a Notch. Trends Mol Med 2006;12:141–143. 75 Colen KL, Crisera CA, Rose MI et al. Vascular development in the mouse embryonic pancreas and lung. J Pediatr Surg 1999;34:781–785. 76 Emerling BM, Platanias LC, Black E et al. Mitochondrial reactive oxygen species activation of p38 mitogen-activated protein kinase is required for hypoxia signaling. Mol Cell Biol 2005;25:4853– 4862. 77 Arnold RS, Shi J, Murad E et al. Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1. Proc Natl Acad Sci U S A 2001;98:5550 –5555. 78 Sauer H, Neukirchen W, Rahimi G et al. Involvement of reactive oxygen species in cardiotrophin-1-induced proliferation of cardiomyocytes differentiated from murine embryonic stem cells. Exp Cell Res 2004;294: 313–324. 79 Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 2001;11:173–186. 80 Funato Y, Michiue T, Asashima M et al. The thioredoxin-related redoxregulating protein nucleoredoxin inhibits Wnt-beta-catenin signalling through dishevelled. Nat Cell Biol 2006;8:501–508. 81 Wells JM, Esni F, Boivin GP et al. Wnt/beta-catenin signaling is required for development of the exocrine pancreas. BMC Dev Biol 2007;7:4.

See www.StemCells.com for supplemental material available online.