Production of fully functional mature beta cells from human pancreatic progenitors
11299711 · 2022-04-12
Assignee
Inventors
- Matthias Hebrok (Belmont, CA, US)
- Holger Andreas Russ (San Francisco, CA, US)
- Gopika G. Nair (San Francisco, CA, US)
Cpc classification
C12N2535/00
CHEMISTRY; METALLURGY
C12N2501/117
CHEMISTRY; METALLURGY
C12N2501/999
CHEMISTRY; METALLURGY
C12N2501/16
CHEMISTRY; METALLURGY
C12N5/0678
CHEMISTRY; METALLURGY
A61K35/545
HUMAN NECESSITIES
International classification
A61K35/545
HUMAN NECESSITIES
Abstract
Methods are provided for the simple, fast, effective and safe directed differentiation of embryonic stem cells into the mature beta cells of enriched beta clusters, wherein the beta cells rapidly and reliably secrete insulin in response to glucose levels. The cells are useful transplant therapeutics for diabetic individuals. These cells can also be used for drug screening purposes to identify factors/chemicals capable of increasing beta cell functions, proliferation, survival, and resistance to immune assault.
Claims
1. A method of producing human endocrine progenitor cells from pluripotent stem cells comprising: (a) incubating pluripotent stem cells (PSCs) in culture medium comprising Wnt3a, Activin A, TGβi, and keratinocyte growth factor (KGF) for 3-7 days; (b) exposing the cells resulting from step (a) to medium comprising retinoic acid (RA) for 2-3 days; and (c) culturing the cells resulting from step (b) in medium comprising RA, epidermal growth factor (EGF), and KGF, thereby producing a culture comprising greater than 70% PDX1.sup.+ NKX6.1.sup.+ human endocrine progenitor cells.
2. The method of claim 1 wherein incubating step (a) is five days, exposing step (b) is two days, or culturing step (c) is 3-5 days.
3. The method of claim 1 further comprising incubating the culture comprising PDX1.sup.+ NKX6.1.sup.+ endocrine progenitor cells in medium comprising Alki, T3, γ-secretase inhibitor XXi, LDN-193189, NEAA, N-acetyl Cysteine, zinc sulfate, glutamine supplement, heparin, and vitamin C for at least 8 days, thereby producing INS.sup.+ NKX6.1.sup.+ GCG.sup.− immature β-like cells.
4. The method of claim 3 further comprising sorting the PDX1.sup.+ NKX6.1.sup.+ cells in the culture to enrich for INS.sup.+ NKX6.1.sup.+ GCG.sup.− immature β-like cells.
5. The method of claim 4 further comprising re-aggregating the immature beta-like cells into clusters of about 100-150 μm.
6. The method of claim 5 further comprising culturing the re-aggregated clusters in medium comprising Alki, T3 and low glucose for at least 6 days, thereby producing mature functional beta cells in enhanced beta-clusters (eBCs).
7. The method of claim 6 wherein the eBCs respond to in vivo glucose challenges within three days of transplantation of the cells into a subject.
8. The method of claim 6 wherein culturing of the re-aggregated clusters produces no detectable cell types of exocrine pancreatic lineages.
9. The method of claim 6 wherein at least 95% of the eBC-cells express chromogranin A, or at least 95% of the eBC-cells eBCs stain positive for synaptophysin, or up to 80% of the eBC-cells are monohormonal C-peptide.sup.+ cells.
10. The method of claim 6 wherein 80% of the eBC-cells are double positive for C-peptide and NKX6.1.
11. The method of claim 6 wherein the basal oxygen consumption rate of eBCs is more than about 10.799 picomoles of oxygen consumed per minute per nanogram of C-peptide.
12. The method of claim 6 wherein the extracellular acidification rate of eBCs is at most 0.6922 mpH/minute per nanogram of C-peptide.
13. The method of claim 3 further comprising producing beta cells from INS.sup.+ NKX6.1.sup.+ GCG.sup.− immature β-like cells comprising (a) sorting the INS.sup.+ NKX6.1.sup.+ GCG.sup.− immature β-like cells in a culture to enrich for INS.sup.+ NKX6.1.sup.+ GCG.sup.− immature β-like cells; and (b) re-aggregating the cells into islet-sized clusters by maintaining the self-organized clusters in CMRL containing B27 or FBS, glutamamide supplement, NEAA, Rock inhibitor, ALKi II, vitamin C, T3, N-acetyl cysteine, zinc sulfate and heparin for at least 6 days, thereby producing mature beta cells.
14. The method of claim 6 wherein maturation of beta cells is achieved through activation of mitochondrial oxidative respiration.
Description
BRIEF DESCRIPTION OF THE DRAWING
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DETAILED DESCRIPTION
(32) The disclosure provides methods of generating fully functional mature β-cells in enriched 3 clusters from human pluripotent stem cells comprising the steps of exposing progenitor cells to temporally ordered inducing agents, cell sorting and re-aggregation of immature β-like cells generated from hPSCs to form enriched Beta-clusters (eBCs). In general terms, the methods disclosed herein provide for isolating hPSC-derived β-like cells using any technique known in the art, such as fluorescence-activated cell sorting (FACS) cytometry with the coding region for Green Fluorescence Protein (GFP) operably linked to the insulin promoter. In principle, other techniques are available, including Magnetic-activated cell sorting (MACS) with antibodies that specifically enrich for endocrine cells and/or deplete progenitor cells, antibodies raised against synthetic epitopes ectopically driven by the insulin promoter or selective destruction of progenitor cells using antibody-drug conjugates. In addition, mechanical isolation, taking advantage of specific properties of immature β-cells and pancreatic progenitors, is also contemplated. The isolated, or sorted, hPSC-derived β-like cells are re-aggregated into about 100-150 μm-sized clusters with about 500-2,000 cells/cluster in Aggrewell™ micro-wells, the GravityPLUS Hanging Drop System, or Perfecta3D Hanging Drop Plates. The clusters generated by this technique are called ‘enhanced β-clusters’ (eBCs). The eBCs are further cultured in optimized media conditions constituting ‘maturation media’ for 6-14 days. Medium ingredients for this culturing step include reduced glucose concentration (about 5.5 mM) as well as high concentrations of Alk5 inhibitor II and T3. An exemplary medium is CMRL (5.5 mM glucose) containing B27 (or FBS), Glutamax, NEAA, Rock inhibitor, ALKi II, vitamin C, T3, N-acetyl cysteine, zinc sulfate and heparin. These clusters exclusively constitute cells committed to the endocrine lineage, particularly enriched in the β-cell lineage.
(33) The disclosure provides eBCs, and methods of producing and using eBCs, that display superior functional properties in vitro and in vivo, analogous to human islets. Without wishing to be bound by theory, it is believed that, mechanistically, coalescence of immature β-like cells induces metabolic maturation and drives mitochondrial oxidative respiration, central elements of stimulus-secretion coupling in β-cells. The resulting mature β-cells are highly similar to primary adult β-cells by transcriptome, immunohistochemical, and functional analyses. The findings disclosed herein point to endocrine cell clustering as a significant step in the maturation of hPSC-derived-β-like-cells under cell culture conditions.
(34) Given that hESC-derived polyhormonal cells have been shown to give rise to alpha cells (Rezania et al, 2011), we expected the in vitro generation of polyhormonal endocrine cells to result from premature assignment to the endocrine fate. To address this issue, a detailed step-wise analysis of pancreatic progenitor generation and endocrine induction was performed. Most current protocols efficiently establish PDX1.sup.+ progenitors by using Retinoic Acid in combination with molecules to inhibit bone morphogenic protein (BMP) and sonic hedgehog (SHH) signaling pathways, while simultaneously adding either fibroblast growth factor 10 or keratinocyte growth factor (KGF, also known as FGF7) (Rezania et al, 2012; Hua et al, 2013; Guo et al, 2013b; Nostro & Keller, 2012; Mfopou et al, 2010). Disclosed herein is the need to temporally control the introduction of inducing agents in the pathway of directed differentiation of embryonic stem cells to functional beta-like pancreatic cells. For example, the early or indiscriminate use of BMP inhibitors to specify pancreatic cells promotes the precocious induction of endocrine differentiation in PDX1.sup.+ pancreatic progenitors, which results in the formation of polyhormonal cells. BMP inhibitors do have a role in directed differentiation of ES cells to beta-like cells, but only if the inhibitors are introduced later in the process, i.e., after the cells have begun to express NKX6.1. Simplified culture conditions have been identified that replicate fetal endocrine development and allow for the precise and efficient generation of PDX1.sup.+ and PDX1.sup.+/NKX6.1.sup.+ progenitor populations without precocious activation of the endocrine marker NEUROG3. Subsequent induction of endocrine differentiation in correctly specified PDX1.sup.+/NKX6.1.sup.+ progenitor cells results in the formation of glucose-responsive insulin-expressing beta-like cells in vitro within, or less than, three weeks. Our study, therefore, details a simplified directed differentiation protocol that closely recapitulates key aspects of human endocrine development and results in the formation of large numbers of glucose-responsive beta-like cells under cell culture conditions.
(35) A simplified differentiation protocol is disclosed herein that replicates key steps of embryonic pancreas organogenesis for the defined generation of human pancreatic progenitor and endocrine cell types from human embryonic stem cells (hESCs) that results in the formation of glucose-responsive beta-like cells in vitro. A straightforward schematic comparing the protocol disclosed herein to conventional protocols is provided in
(36) Recently, two other groups have reported the derivation of glucose responsive beta-like cells from hESC cells that share many characteristics of the beta-like cells described herein (Rezania et al, 2014; Pagliuca et al, 2014). Both of these studies, however, focused on optimizing the later stages of direct differentiation, while employing parts of published protocols, namely the addition of RCN, to establish pancreatic progenitor populations. Data disclosed herein demonstrate that generation of pancreatic progenitors using this method also results in the undesirable generation of immature polyhormonal endocrine cells that lack expression of the critical beta cell transcription factor NKX6.1. Indeed, both published studies do note appreciable populations of C-peptide/insulin positive cells that lack NKX6.1 expression. We demonstrate that polyhormonal cells result from precocious endocrine induction in PDX1.sup.+ pancreatic progenitors (lacking NKX6.1 expression), which can be avoided by omitting BMP inhibitors during the pancreas specification stage. Further, our detailed analysis of the effects of individual RCN factors on expression of key pancreatic markers revealed that retinoic acid alone is sufficient to induce proficient generation of more than 98% PDX1.sup.+ pancreatic progenitors. Subsequent exposure to EGF and KGF results in the rapid and effective activation of NKX6.1 in these cells, generating PDX1.sup.+/NKX6.1.sup.+ progenitor cells with the ability to give rise to beta-like cells in vitro. These simplified differentiation conditions enable the efficient generation of human pancreatic and more restricted endocrine progenitor populations from pluripotent stem cells without unwanted formation of polyhormonal cells. This simplified differentiation protocol more closely resembles key aspects of early human pancreas development and, as such, represents an improvement over published protocols.
(37) Studies in rodents have shown an important role for Notch signaling in the endocrine differentiation of progenitor cells in vivo. While initially required for the generation of competent progenitor cells, a subsequent reduction of Notch signaling is necessary for the induction of NEUROG3 expression that initiates endocrine differentiation (Shih et al, 2012). In the context of in vitro differentiation, previous studies have shown that direct inhibition of Notch signaling by gamma secretase inhibitors or the use of BMP and TGFβ/ALK inhibitors results in increased insulin expression at later stages (Mfopou et al, 2010; Nostro et al, 2011; Pagliuca et al, 2014; Rezania et al, 2014). We employed BMP and Activin receptor-Like Kinase (ALK) inhibition over a 5-day window to induce NEUROG3 expression specifically in PDX1.sup.+/NKX6.1.sup.+ progenitors, which resulted in the efficient generation of INS.sup.+/NKX6.1.sup.+ beta-like cells, while only few polyhormonal cells were observed (about 3%, which is less than 5%). Likely, these unwanted cells originated from the small percentage of PDX1 pancreatic progenitors present at the time of endocrine induction. In contrast to the formation of PDX1.sup.+ and PDX1.sup.+/NKX6.1.sup.+ progenitors that occurs rapidly (36-48 hours after addition of inducing factor(s)) and uniformly in the majority of cells, endocrine differentiation occurs over a prolonged period and is confined to a small subset of total cells. This might be a reflection of the situation observed during normal human pancreas development, where only few progenitor cells initiate the endocrine differentiation program at any given time (Jennings et al, 2013). While simultaneous widespread induction of endocrine differentiation in a majority of PDX1.sup.+/NKX6.1.sup.+ progenitor population would greatly reduce differentiation time and increase beta-like cell yield, our results point to a regulation of NEUROG3 expression that requires subtle, yet temporally precise, adjustment that appears more complex than just Notch inhibition. As our differentiation protocol allows for a tight control of NEUROG3 expression, it could be used in future studies to identify novel regulators of NEUROG3 gene expression, and ideally to achieve uniform NEUROG3 activation during direct differentiation in vitro.
(38) While cadaveric islet preparations are widely accepted as the gold standard for studying human beta cells, several problems associated with their use remain. For example, their performance and utility depend on a number of confounding factors: genetic variance, age and life style of the donor, isolation time, islet purity and shipping conditions. By eliminating the constraints of availability and reproducibility, we expect hESC-derived beta cells to provide an important therapeutic and a tool to accelerate understanding of the biology of human beta cells.
(39) Without wishing to be bound by theory, the methodology disclosed herein resulted from approaching the problem of engineering fully functional mature β-cells by adopting a developmental perspective that recapitulated an important process in β-cell development unheeded in hPSC differentiations until now: the clustering or coalescence of newly born β-cells observed during islet formation in vivo. Islet formation is initiated towards the end of gestation in humans and rodents, wherein delaminating β-cells, coalesce into small islet-like aggregates that progressively form larger clusters during postnatal development[21]. This period of neonatal growth is coincident with gradual functional maturation of β-cells, which broadly encompasses acquisition of glucose sensing, dense core granule biogenesis, stimulus-secretion coupling, and ultimate metamorphosis into adult β-cells. Consistent with this, incorporating a re-aggregation step permits maturation of in vitro-generated hPSC-derived-β-like cells into eBCs with superior functional properties, including the stereotypical rapid and robust release of C-peptide highly synchronous with glucose concentrations, elevated calcium signaling on glucose stimulation that was ablated on removal of stimulus, highly sensitive K-ATP channels that can be reversibly closed and opened, and mitochondrial energization analogous to adult human islets. Disclosed herein are detailed functional analyses of in vitro hPSC-derived-β-cells, illuminating the remarkable similarity of in vitro-generated islet-like eBCs to human islets. The formation of these alpha and delta cells provides further evidence that the eBCs are capable of inducing the formation of beta islet structures that more closely resemble native human islets, and by structure and functional assay appear to be beta cells capable of providing, or restoring, glucose responsiveness in an organism. Notably, the size (100 μm) of eBCs falls within the range of smaller human islets that have better function in vitro and improved outcome post-transplantation[33]. eBCs function rapidly within a few days (3-days) of transplantation, like human islets, and prevent STZ-induced diabetes in mice. In addition to C-peptide-expressing cells, single-hormone-positive cells expressing glucagon and somatostatin organized in islet-like structures are observed in eBC grafts 48 days following transplantation, further supporting the robust function of eBCs in vivo.
(40) Neonatal β-cells change their transcriptional profile during maturation; expression of ‘allowed’ genes required for regulated insulin secretion is increased while ‘disallowed’ housekeeping genes are repressed to circumvent inappropriate function. ‘Allowed’ genes include those promoting mature β-cell identity such as PDX1, NKX6.1, NEUROD1, ISL1 and PAX6[12, 13, 27, 34]. Greater than 80-90% of cells in eBCs are co-positive for these transcription factors and C-peptide. Moreover, these markers are more strongly expressed in eBCs than immature d20 β-like-cells. Importantly, C-peptide is expressed at higher intensity in eBCs compared with immature d20 β-like cells[16], a finding in line with previous reports of enhanced insulin content in mature β-cells[7, 8]. Of note, eBCs contain high levels of MAFB, but are low in MAFA expression. This is reminiscent of ‘juvenile β-cells’, which are fully functional, expressing high levels of MAFB for several years before MAFA levels increase[10]. Maturation also increases expression of genes regulating glucose sensing and secretion; these genes are similarly expressed in eBCs and human islets. In particular, SLC30A8, vital for insulin granule maturation and exocytosis[35], is highly enriched in eBCs. Concurrent with increase in genes coding for critical aspects of mature β-cell function, ‘disallowed’ genes capable of decoupling stimulus-secretion need to fade during maturation. Two such genes, HK1 and LDHA, were examined for repressive DNA methylation marks and found them to be hypermethylated in eBCs in a pattern akin to human islets[9]. Thus, eBCs are functionally mature and equipped to appropriately respond to glucose excursions during meals while remaining inactive during fasting.
(41) Although the relevance of β-cell-cell contacts in human and mouse islet function has been underscored by several reports[36-38], the surprising outcome of clustering immature β-like-cells as disclosed herein was induction of mitochondrial metabolic maturation. β-cells in eBCs were enriched in oxidative metabolic pathways, including OXPHOS, electron transport chain, TCA cycle and ATP biosynthesis when compared with β-cells residing in progenitor-rich environments. We observed increased mitochondrial respiration, mitochondrial mass and differential membrane depolarization on glucose stimulation in β-cells of eBCs. Additionally, cristae in the mitochondrion of these mature β-cells were highly folded and stacked, a noteworthy observation given the recent discoveries linking dynamics of cristae morphology to function of OXPHOS system[31]. The findings disclosed herein are in concert with the transition from glycolysis-centric to OXPHOS-centric metabolism posited to occur during postnatal functional maturation[8, 17, 30, 39], a process that might in part be driven by coalescence of β-cells. Furthermore, clustering resulted in enrichment of ERRγ, a mitochondrial regulator of the metabolic transition, suggesting that re-aggregation activates endogenous processes driving increased mitochondrial activity.
(42) Correlation analysis from RNA-seq experiments unveiled that the hPSC-derived-β-cells are highly similar to primary β-cells (R=0.9253). Nonetheless, differences in expression of certain adult β-cell markers, including MAFA, UCN3 and G6PC2, were found between the two groups. These data are in line with an extended postnatal maturation period in humans lasting through childhood[11] wherein juvenile yet functionally mature β-cells have high expression of MAFB[10] but not MAFA as observed in the disclosed eBCs. Furthermore, emerging data sheds light on heterogeneity within β-cells of human islets, especially indicating the existence of distinct populations of β-cells with distinguishable functional capabilities. On closely comparing the ex vivo-generated β-cells with four β-cell subtypes described by Dorrell et al.[40], the cells were found to be CD9low and ST8SIA1low, and hence probably represent β1-subtype, the most abundant and glucose-responsive β-cell subtype. On the contrary, there was no enrichment of Fltp/Cfap126, a marker separating mature β-cells from replication competent β-cells in mice[41], in the mature fully functional β-cells compared with immature β-cells. Interestingly, Fltp/Cfap126 was also reported to increase upon endocrine cell clustering and compaction in that study. Although not Fltp/Cfap126, the RNA-seq results provided herein revealed enrichment of other cilia- and flagella-associated proteins, namely Cfap20, Cfap36 and Cfap97, in the mature versus immature hPSC-derived-β-cells (p<0.05).
(43) In summary, clustering/re-aggregation of immature β-like-cells has been demonstrated herein to be an important step in the maturation and generation of fully functional β-cells from hPSCs, in vitro. Our data strongly indicate that coalescence of immature β-like cells induces metabolic maturation of mitochondria capable of generating the necessary ATP currency for mature β-cell function such as insulin synthesis, packaging and exocytosis. This improved protocol provides the methodology for hPSC-derived-β-cell therapeutics for diabetes and delivers a cell type equivalent, if not identical, to human β-cells for such therapy as well as drug discovery and disease modeling.
(44) Taken together, the fast and simplified protocol disclosed herein provides precise temporal control over the generation of subsequent pancreatic progenitor and endocrine cell types and results in the establishment of human beta-like cells that exhibit glucose responsiveness in vitro and in vivo. The suspension-based direct differentiation approach is scalable, and the ability to produce large numbers of beta-like cells provides a safe and effective cell therapy to patients suffering from diabetes. Furthermore, through the production and maintenance of different developmental cell populations, the approach can be used for more detailed investigations into human pancreas development and human beta cell function that were previously impossible due to limited donor material, such as large scale drug screens and genome-wide gene function studies.
(45) The following examples illustrate embodiments of the disclosure.
Example 1
(46) Materials and Methods
(47) Cell Culture
(48) Undifferentiated MEL1 INS.sup.GFP/w reporter cells (Micallef et al, 2012) were maintained on mouse embryo fibroblast feeder layers (Millipore) in hESC media as described (Guo et al, 2013b). Suspension-based differentiations were carried out as follows. Briefly, confluent cultures were dissociated into single cell suspension by incubation with TrypLE (Gibco). Cells were counted and each well of 6-well low-adherence plates were seeded with 5.5×10.sup.6 cells in 5.5 ml hES media supplemented with 10 ng/ml Activin A (R&D systems) and 10 ng/ml HeregulinB1 (Peprotech). Plates were placed on an orbital shaker at 100 rpm to induce sphere formation, as described (Schulz et al, 2012). To induce definitive endoderm differentiation, aggregates were collected 24 hours later in a 50 ml falcon tube, allowed to settle by gravity, washed once with PBS and re-suspended in d1 media (RPMI (Gibco) containing 0.2% FBS, 1:5000 ITS (Gibco), 100 ng/ml Activin A, and 50 ng/ml WNT3a (R&D systems)). Clusters from 3 wells were combined into 2 wells at this point and distributed into fresh low-attachment plates in 5.5 ml d1 media. Media thereafter was changed daily, by removing either 4.5 ml media (at the end of d1) or 5.5 ml media the following days and adding back 5.5 ml fresh media until day 9. After day 9, only 5 ml of media was removed and added daily.
(49) Differentiation employing published protocols has been described (Schulz et al, 2012; Rezania et al, 2012). Media in our simplified differentiation protocol consists of, d2: RPMI containing 0.2% FBS, 1:2000 ITS, and 100 ng/ml Activin A; d3: RPMI containing 0.2% FBS, 1:1000 ITS, 2.5 μM TGFbi IV (CalBioChem), and 25 ng/ml KGF (R&D systems); d4-5: RPMI containing 0.4% FBS, 1:1000 ITS, and 25 ng/ml KGF. d6-7: DMEM (Gibco) with 25 mM Glucose containing 1:100 B27 (Gibco), 3 nM TTNBP (Sigma); d8: DMEM with 25 mM Glucose containing 1:100 B27, 3 nM TTNBP, and 50 ng/ml EGF (R&D systems); d9: DMEM with 25 mM Glucose containing 1:100 B27, 50 ng/ml EGF, and 50 ng/ml KGF. d10-14: DMEM with 25 mM Glucose containing 1:100 B27, 500 nM LDN-193189 (Stemgent), 30 nM TATA-Binding Protein (TBP; Millipore), 1000 nM Alki II (Axxora), and 25 ng/ml KGF; d15-21: DMEM with 2.8 mM Glucose containing 1:100 Glutamax (Gibco) and 1:100 NEAA (Gibco). Human islets were from Prodo Laboratories or the UCSF Islets and Cellular Production Facility.
(50) Mice
(51) NOD.Cg-Prkdcscid Il2rgtm1 Wjl/SzJ mice (NSG) were obtained from Jackson Laboratories. Mice used in this study were maintained according to protocols approved by the University of California, San Francisco Committee on Laboratory Animal Resource Center. For kidney capsule grafts, approximately 5.0×10.sup.6 hESC differentiated cells in spheres and 4000 human islet equivalents were transplanted as described (Russ & Efrat, 2011; Szot et al, 2007). For glucose-induced insulin secretion, mice were fasted overnight and serum was collected before and after intraperitoneal administration of 3 g/kg D-glucose solution. For induction of diabetes, mice were administered 35 mg/kg streptozotocin via intraperitoneal injection for 5 days. Graft bearing kidneys were removed for immunofluorescence analysis. No statistical method was employed to determine sample size, mice were not randomized and analysis was not blinded.
(52) Cell Sorting and Flow Cytometric Analysis
(53) Briefly, spheres were collected and allowed to settle by gravity. Clusters were washed once in PBS and dissociated by gentle pipetting after 12-15 minutes incubation in Accumax (innovative cell technologies). For sorting, cell suspension were filtered and re-suspended in FACS buffer consisting of phosphate-buffered saline (PBS) (UCSF cell culture facility) containing 2 mM EDTA (Ambion) and 1% BSA (Sigma). Dead cells were excluded by DAPI (Sigma) staining. Cell sorting was performed on a FACS Aria II (BD Bioscience). For flow-based analysis, dissociated cells were fixed with 4% paraformaldehyde (Electron Microscopy Science) for 15 minutes at room temperature, followed by two washes in PBS. Samples were either stored at 4 C or immediately stained with directly conjugated antibodies. Data analysis was performed with FlowJo software. Mouse Glucagon and mouse human C-peptide antibodies were conjugated in-house by the UCSF Antibody Core and/or with Antibody Labeling Kits (Molecular Probes) according to manufacturer's instructions. Commercially available directly conjugated antibodies, i.e., antibodies Human PAX6-Alexa647, Islet-1-PE, NKX6.1-Alexa647, NKX6.1-PE, ChromograninA-PE, NeuroD1-Alexa647, PDX1-PE, and Ki67-Alexa647, were from BD Bioscience.
(54) Electron Microscopic Analysis
(55) Spheres were fixed by adding 37° C. 0.1M sodium cacodylate solution (Sigma) containing 2% paraformaldehyde (Electron Microscopy Science) and 2.5% glutaraldehyde (Electron Microscopy Science), 3 mM CaCl.sub.2 (Sigma), final pH 7.4. Spheres were then transferred to 4° C. for approximately 18 hours, followed by standard processing and analysis by the Electron Microscope Lab/Diabetes Center Microscope Core.
(56) Immunofluorescence Analysis
(57) Spheres were fixed for 15-30 minutes at room temperature with 4% paraformaldehyde, followed by multiple washes in PBS. Whole mount staining was performed in suspension, by first blocking overnight at 4° C. in blocking buffer consisting of CAS-block (Invitrogen) with 0.2% TritonX (Fisher). Primary antibodies were incubated overnight at 4° C. in blocking buffer, followed by washes in PBS containing 0.1% Tween-20 (PBS-T, Sigma) and incubation in appropriate secondary antibodies diluted in PBS-T overnight at 4° C. The next day, clusters were washed in PBS-T followed by PBS and mounted with Vectashield (Vector) on glass slides. For sectioning of clusters, spheres were embedded in 2% Agar (Sigma), followed by dehydration, paraffin embedding, and sectioning. Cut sections were rehydrated and treated with an antigen retrieval solution (Biogenex) before incubation with primary antibodies overnight at 4° C. in blocking buffer. The next day, sections were washed 3 times in PBS-T and incubated with appropriate secondary antibodies for 30-40 minutes at room temperature in PBS-T. Appropriate Alexa-conjugated secondary antibodies were purchased from JAX or Molecular Probes and used at 1:500 dilutions. Slides were washed in PBS-T and PBS before mounting in Vectashield. Nuclei were visualized with DAPI. Images were acquired using a Leica SP5 microscope or a Zeiss ApoTome. Primary antibodies were employed as indicated in Table 1.
(58) TABLE-US-00001 TABLE 1 Antigen Species Dilution Manufacturer Human C-peptide Mouse 1:200 Chemicon Human C-peptide Rat 1:1000 DSHB Insulin Mouse 1:1000 Sigma Insulin Guinea pig 1:500 DAKO Glucagon Mouse 1:1000 Sigma NKX6.1 Mouse 1:100 DSHB NKX2.2 Mouse 1:20 DSHB PDX1 Goat 1:200 R&D systems Human NEUROG3 Sheep 1:300 R&D systems Ki67 Rabbit 1:100 NovoCastra
(59) qPCR Analysis
(60) Total RNA was isolated with TRIZOL (Sigma) or micro/mini RNAeasy kit (Qiagen) and reverse transcribed using the iScript cDNA Kit (Bio-Rad) according to manufacturer's instructions. qPCR analysis was performed on an ABI 7900 HT Fast Real-Time PCR System (Applied Biosystems) and CFX Connect Real Time System (Biorad) using standard protocols. Primers were Taqman Probes (Applied Biosystems) and/or as published previously (Liu et al, 2014). P-values were calculated using a two-tailed student's t-test.
(61) Content Analysis
(62) Insulin, human C-peptide and proinsulin analyses were performed by measuring an aliquot of acidic ethanol lysed clusters with commercially available ELISA kits (Insulin Cat. 80-INSMR-CH10, human C-peptide cat. 80-CPTHU-CH01, and proinsulin Cat. 80-PINHUT-CH01; all from Alpco). Total DNA was quantified by PicoGreen (Invitrogen) assay and normalized to the percentage of C-peptide-positive cells in each sample.
(63) Western Blotting for Proinsulin/Insulin:
(64) Cell lysates were resolved on 4-12% acrylamide gradient SDS-PAGE gels (NuPAGE, Invitrogen) normalized to cellular DNA (Quant-iT dsDNA, Molecular Probes). The samples were then electrotransferred to nitrocellulose membranes and immunoblotted with guinea pig anti-insulin, which recognizes both proinsulin and insulin, as previously described (Haataja et al, 2013). Immunoblotting with anti-tubulin was used as a confirmatory loading control. HRP-conjugated secondary antibodies (Jackson ImmunoResearch) were used for enhanced chemiluminescence detection (Millipore). The analysis was performed four times with isolated human islets used as a positive control.
(65) Glucose Stimulated Insulin Secretion
(66) Human islets or hES-derived spheres were transferred into tubes and washed twice with Krebs-Ringer Bicarbonate buffer (KRB) containing 2.8 mM Glucose. Samples were incubated for one hour in 2.8 mM glucose containing KRB to allow equilibration of cells. The 2.8 mM buffer was removed and replaced with fresh KRB containing 2.8 mM glucose for one hour followed by incubation for another hour in KRB containing 16.7 mM glucose. After the incubation period, buffers were collected for human C-peptide-specific ELISA analysis using a commercially available kit (Alpco).
(67) Additional Materials and Methods Used in Examples 8-13
(68) Cell Culture
(69) Mel1 Ins-GFP human embryonic stem cells were maintained and propagated on mouse embryonic fibroblasts (MEFs) in hESC media. The cells were passaged by enzymatic dissociation using TrypLE (Gibco). To initiate differentiation, confluent hESC cultures were dissociated into single-cell suspension using TrypLE, counted and seeded at 5.5×10.sup.6 cells/well in 6-well suspension plates in 5.5 ml hESC media supplemented with 10 ng/ml Activin A (R&D Systems) and 10 ng/ml heregulinB (Peprotech). The plates were incubated at 37° C. and 5% CO.sub.2 on an orbital shaker at 100 rpm to induce 3D sphere formation. After 24 hours, the spheres were collected in 50 ml falcon tubes, allowed to settle by gravity, washed once with PBS and resuspended in day 1 media. The resuspended spheres in day 1 media were distributed into fresh 6-well suspension plates with 5.5 ml of media/well. Thereafter, media was changed every day. Until day 3, spheres were fed by removing 5 ml of media and replenishing with 5.5 ml of fresh media. From day 4 until day 20, 4.5 ml media was removed and 5 ml of fresh media was added. Media compositions are as follows: Day 1: RPMI (Gibco) containing 0.2% FBS, 1:5,000 ITS (Gibco), 100 ng/ml activin A, and 50 ng/ml WNT3a (R&D Systems). Day 2: RPMI containing 0.2% FBS, 1:2,000 ITS, and 100 ng/ml activin A; Day 3: RPMI containing 0.2% FBS, 1:1,000 ITS, 2.5 μM TGFbi IV (CalBioChem), and 25 ng/ml KGF (R&D Systems); Day 4-5: RPMI containing 0.4% FBS, 1:1,000 ITS, and 25 ng/ml KGF; Day 6-7: DMEM (Gibco) with 25 mM glucose containing 1:100 B27 (Gibco), 3 nM TTNBP (Sigma); Day 8: DMEM with 25 mM glucose containing 1:100 B27, 3 nM TTNBP, and 50 ng/ml EGF (R&D Systems); Day 9-11: DMEM with 25 mM glucose containing 1:100 B27, 50 ng/ml EGF, and 50 ng/ml KGF; Day 12-20: DMEM with 25 mM glucose containing 1:100 B27, 1:100 Glutamax (Gibco), 1:100 NEAA (Gibco), 10 μM ALKi II (Axxora), 500 nM LDN-193189 (Stemgent), 1 μM Xxi (Millipore), 1 μM T3 (Sigma-Aldrich), 10-2000 μM (e.g., 0.5 mM) vitamin C, 1 mM N-acetyl cysteine (Sigma-Aldrich), 10 μM zinc sulfate (Sigma-Aldrich) and 10 μg/ml of heparin sulfate. Day 20: The spheres were collected, incubated with Accumax briefly and dissociated into single cells for flow cytometry. Live GFPhigh cells were sorted on Aria II at low flow rates and re-aggregated in Aggrewell™ 400 plates (StemCell Technologies) at 1,000 cells/cluster in CMRL containing 1:100 B27 (or 10% FBS), 1:100 Glutamax (Gibco), 1:100 NEAA (Gibco), 10 μM ALKi II (Axxora), 0.5 mM vitamin C, 1 μM T3 (Sigma-Aldrich), 1 mM N-acetyl cysteine (Sigma-Aldrich), 10 μM zinc sulfate (Sigma-Aldrich) and 10 μg/ml of heparin sulfate. Day 22-23: The re-aggregated enhanced Beta-clusters (eBCs) were transferred from Aggrewells into 6-well suspension plates and placed on orbital shakers at 100 rpm. From day 23 onwards, eBCs were maintained in 6-well suspension plates until the end of the experiment. Media was changed every third day following re-aggregation. Human islets used in the experiments were obtained from the UCSF Islet Core. They were used for functional and RNA-seq analysis within 24-48 hours of isolation.
(70) Immunofluorescent Staining
(71) Clusters were fixed with 4% PFA for 15 minutes at room temperature, washed with PBS, and stored at 4° C. until processing for paraffin sectioning. Clusters were first embedded in 2% agar, followed by dehydration, paraffin embedding, and sectioning at 5 μm thickness. Cut sections were then stained according to the protocol described previously (Russ et al., EMBO J 2015). Primary antibodies employed are detailed in Table 2. Secondary antibodies were conjugated AlexaFluors 488, 568 and 647 (Molecular probes) of appropriate species, and were used at 1:500 dilution. Cells were counterstained with DAPI to mark nuclei. Slides were mounted with coverslips using Prolong® Diamond Antifade reagent (Invitrogen). Images were generated using a Leica SP5 confocal microscope or a Zeiss apotome.
(72) TABLE-US-00002 TABLE 2 Antigen Species Dilution Manufacturer Human C-peptide Mouse 1:200 Chemicon Human C-peptide Rat 1:1000 DSHB Insulin Mouse 1:1000 Sigma Insulin Guinea pig 1:500 DAKO Glucagon Mouse 1:1000 Sigma NKX6.1 Mouse 1:100 DSHB NKX2.2 Mouse 1:20 DSHB PDX1 Goat 1:200 R&D systems SOX9 Amylase CK19
(73) Flow Cytometry
(74) Clusters at indicated stages were dissociated, fixed, permeabilized and stained for various intracellular markers for quantitative analysis on LSRFortessa X20 Dual, as described previously Russ et al., EMBO J 2015). Data were analyzed with FlowJo software. Anti-Glucagon and anti-human C-peptide antibodies were conjugated in-house using the Molecular Probes Antibody Labeling Kits according to manufacturer's instructions. Antibody details are listed in Table 3.
(75) TABLE-US-00003 TABLE 3 Antibody Manufacturer Human PAX6-Alexa647 BD Bioscience Islet-1-PE BD Bioscience NKX6.1-Alexa647 BD Bioscience NKX6.1-PE BD Bioscience ChromograninA-PE BD Bioscience NeuroD1-Alexa647 BD Bioscience PDX1-PE BD Bioscience
(76) Quantitative Real-Time PCR
(77) Human islets and hPSC-derived clusters were harvested at indicated stages of differentiation by homogenization in TRIzol (Invitrogen) or Buffer RLT (Qiagen), and RNA was isolated and purified using RNeasy Mini/Micro kits (Qiagen). qPCR was performed using gene expression assays from Applied Biosystems. Thermo Fisher Scientific Taqman Assay identifications are given in Table 5 in Example 10. The comparative threshold (ΔΔCT) method was used to quantify transcript abundance.
(78) Dynamic Insulin Secretion Assay
(79) An in-house built perifusion system was used for dynamic secretion assays. Twenty-thirty hPSC-derived clusters or human islets were placed on 10 μm TCTP filters (Isopore™ membrane) in plastic chambers that were immersed in a 37° C. water bath. Under temperature- and CO.sub.2-controlled conditions, the clusters were perifused at 1 ml/minute with Krebs-Ringer buffer (KRB) using a peristaltic pump (Isomatec IPC). After an initial hour-long preincubation in 2.8 mM KRB, alternating low (2.8 mM) and high (20 mM) glucose and 30 mM KCL were perfused through the system. Flow-through was collected over the course of the experiment, and C-peptide levels were measured using the STELLUX® Chemi Human C-peptide ELISA kit (Alpco). After the experiment, clusters/islets were recovered from the membrane and their insulin and DNA content were measured by acid-ethanol extraction and Quant-iT PicoGreen dsDNA Assay Kit, respectively.
(80) Islet Perifusion and Imaging for Calcium Flux and Mitochondrial Depolarization Analysis
(81) All imaging and perifusion experiments were conducted according to previously described methods (Adewala et al., Biomed. Microdevices 12:409-417, 2010; Shaikh et al., Lab on a Chip 9:97-106, 2009). In brief, hPSC-derived clusters and islets were incubated in 2 mL of Krebs buffer containing both 5 μM Fura-2/AM and 2.5 μM Rhodamine 123 fluorescent dyes (Molecular Probes, Calif.) for 30 minutes prior to loading the device. The islets were then loaded into the temperature-equilibrated microfluidic device mounted on an inverted epifluorescence microscope (Leica DMI 4000B, location). Dual-wavelength Fura-2/AM was excited at 340 and 380 nm and fluorescent emission was detected at 510 nm. Intracellular Ca.sup.2+ was expressed as a ratio of fluorescent emission intensity F340/F380(%). Rh123 was excited at 490±10 nm, and emission was measured at 530±10 nm. Fura-2 and Rh123 fluorescence emission spectra were filtered using a Fura-2/FITC polychroic beamsplitter and double band emission filter (Chroma Technology. Part number: 73100bs). These images were collected with a CCD (Retiga-SRV, Fast 1394, QImaging). SimplePCI software (Hamamatsu Corp. location) was used for image acquisition and analysis. Both fluorescence signals were expressed as “change-in-percentage” after being normalized against basal intensity levels established before stimulation.
(82) RNA-Sequencing Library Preparation and Data Analysis
(83) Total RNA was isolated from d27 eBCs and Unsorted-Reagg clusters; GFP-high cells FACS sorted from d27 eBCs and Unsorted-Reagg clusters and d20 immature clusters; and β-cells FACS sorted from adult human islets using RNeasy Micro kits (Qiagen). RNA isolated from sorted cells was further concentrated employing RNA Clean & Concentrator (Zymo research). Only RNA samples with an RNA Integrity Number (RIN) >7 were used to generate libraries for deep sequencing. Ribosomal RNA (rRNA) was depleted by RiboGone (Clontech), and 325-350 bp-sized strand-specific cDNA libraries were prepared using the SMARTer Stranded Total RNA Sample Prep Kit—Low Input Mammalian Kit (Clontech). The samples were further sequenced on an Illumina HiSeq 4000 instrument generating paired-end 100-base-pair reads. Read qualities were assessed by the FASTQC tool on Galaxy. Reads were then mapped to the human genome (hg38) using TopHat version 2.1.0 (Trapnell, et al., Bioinformatics 25:1105-1111, 2009) using the corresponding sample's mean inner distance between mate pairs, library-type fr-firststrand for strand determination leaving other parameters at default settings. The tuxedo suite tools were used for further analysis: rRNA and tRNA reads were masked during transcript assembly using Cufflinks and differential expression analysis was performed using Cuffdiff. Seeking an unbiased approach to pathway analysis, we used the Gene set enrichment analysis (GSEA) tool developed by Broad Institute (Mootha et al., Nat. Genet. 34:267-273, 2003; Subramanian et al., PNAS 102:15545-15550, 2005) that identifies groups of coordinately regulated genes present in gene sets annotated in the Molecular Signatures Database (MSigDB). The ranking metric used was ‘tTest’, number of permutations employed was 1000 and MSigDB collections used were hallmark gene sets, curated gene sets such as KEGG, and GO gene sets such as biological process, molecular function and cellular component. The program R was used to plot scatter plots and calculate correlation coefficients. The online tool Morpheus, from the Broad Institute, was used to generate heatmaps and perform hierarchical clustering.
(84) Bisulfite Sequencing
(85) DNA samples were bisulfite-converted and purified using the Epitect Plus kit (Qiagen). Bisulfite-treated DNA was used as template to amplify the regions of interest using the bisulfite-converted DNA primers described previously (Dhawan et al., J. Clin. Invest. 125:2851-2860, 2015). Annealing temperatures were 64° C. for HK1 and 61° C. for LDHA using KAPA HiFi Uracil+ DNA polymerase and ReadyMix. PCR products were gel purified and used for TOPO-TA cloning (Invitrogen), followed by Sanger sequencing. Bisulfite sequencing data were aligned and analyzed using the Sequencher software. In the resulting data, each line of the diagram represents one clone and 8-10 clones were analyzed per sample. Filled circles represent methylated CpGs and open (white) circles represent unmethylated ones.
(86) Electron Microscopy
(87) Clusters were spun down and excess media was removed, followed by addition of ice-cold fixative (0.1 M sodium cacodylate solution containing 2% paraformaldehyde and 2.5% gluteraldehyde, 3 mM CaCl.sub.2, final pH 7.4) and incubation on ice for 20 minutes. The clusters were further processed using standard transmission electron microscopy procedures and analyzed by the Gladstone Electron Microscopy Core.
(88) In Vitro Metabolic Flux Analysis
(89) Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in real time using an XFe24 extracellular flux analyzer (Seahorse Bioscience). hPSC-derived clusters and human islets were first rinsed with pH-adjusted (7.4) XF base media (sodium bicarbonate-free) supplemented with 3 mM glucose. Twenty-thirty clusters/islets were placed per well of an islet plate and each experiment had two replicates per group. This was followed by insertion of a mesh to prevent movement of clusters during the assay and incubation in a non-CO.sub.2 incubator at 37° C. for at least one hour. Three baseline measurements were taken, following which glucose (2.8 or 20 mM), Oligomycin (5 μM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 μM), and rotenone and antimycin A (5 μM) were injected sequentially. OCR and ECAR were measured at 37° C. in real time throughout the assay period. OCR and ECAR were normalized to average baseline measurement and expressed as percent change during the course of the experiment. The insulin content of each well was also determined by STELLUX® Chemi Human C-peptide ELISA kit (Alpco).
(90) Mitochondria Experiments
(91) For estimation of mitochondrial mass, dissociated cell populations were incubated in warm media containing MitoID at a dilution of 1:10,000 for one hour at 37° C. with gentle shaking. Following washes with PBS, dissociated cells were fixed with 4% PFA, stained with anti-C peptide and anti-NKX6.1 antibodies and analyzed on LSRFortessa X20 Dual (BD Biosciences). For estimation of mitochondrial membrane potential, MitoTracker Red CM-H2XRos (Thermo Fisher Scientific), a mitotracker dye that fluoresces only on oxidation in live cells, was used. Dissociated cell populations were incubated with 500 nm MitoTracker Red CM-H2XRos in KRB containing either 2.8 or 20 mM glucose for one hour at 37° C. with gentle shaking. Cells were fixed and further stained with antibodies against C-peptide and NKX6.1 before FACS analysis. For measuring mitochondrial DNA copy number, DNA was isolated from indicated cell populations using QIAamp DNA Micro Kit (Qiagen). qPCR was used to determine the ratio of mitochondrial mtDNA 16S rRNA gene to nuclear ß-2-microglobulin (B2M) gene using SYBR Green (Roche) based detection. Human specific primers used for mtDNA 16S rRNA were: 5′-GCCTTCCCCCGTAAATGATA-3′ (SEQ ID NO:1) and 5′-TTATGCGATTACCGGGCTCT-3′ (SEQ ID NO:2), and for B2M were: 5′-TGCTGTCTCCATGTTTGATGTATCT-3′ (SEQ ID NO:3) and 5′-TCTCTGCTCCCCACCTCTAAGT-3′ (SEQ ID NO:4).
(92) Mouse Studies
(93) NOD.Cg-Prkdcscid I12rgtm1Wjl/SzJ mice (NSG) were obtained from Jackson Laboratories and bred in the facility at the University of California, San Francisco. Male mice between the age group of 10-16 weeks were used in this study and were maintained according to protocols approved by the University of California, San Francisco, Institutional Animal Care and Use Committee. Mice were anaesthetized with isoflurane and transplanted with eBCs under the kidney capsule. Two cohorts of mice were transplanted with 700 eBCs (about 700,000 cells) and one cohort received 4000 eBCs (about 4×10.sup.6 cells). For in vivo glucose challenge experiments, mice were fasted overnight, and serum was collected by sub-mandibular bleeding before, and 30 minutes following, an intraperitoneal injection of 2 g/kg D-glucose solution. Kidneys bearing grafts were removed at indicated time points for immunofluorescent and H&E staining. For determining the ability of the grafts to protect against diabetes, age-matched control and eBC-transplanted mice were injected with multiple low-doses of STZ (35 mg/kg/d for 5 days), and monitored for hyperglycemia. Control mice that were overtly diabetic either died or had to be euthanized. A survival nephrotectomy was performed to remove the grafts 50 days after transplant, following which blood glucose levels of the mice were monitored.
(94) Statistical Analyses
(95) Statistical tests performed for specific data are described in the brief descriptions of the corresponding figures. In brief, under the assumption of normal distribution, two-tailed unpaired t-tests (Student's t-test) were used if standard deviation (SD) was equal or two-tailed unpaired t-tests with Welch's correction were used if SD was unequal to compare various groups in
Example 2
(96) Pancreatic Differentiation of hESCs Using a Large-Scale Culture System Results in Two Distinct Subsets of Insulin-Producing Cells.
(97) To generate pancreatic beta-like cells from human PSC, we established a scalable three-dimensional suspension culture system based on previously reported methods (Schulz et al, 2012; Rezania et al, 2012) (
Example 3
(98) Defining the Temporal Activities of Individual Signaling Factors to Efficiently Generate PDX1.sup.+ and PDX1.sup.+/NKX6.1.sup.+ Pancreas Progenitor Populations while Preventing Precocious Induction of Endocrine Differentiation.
(99) To characterize the type of progenitors present in differentiating cultures at the point of endocrine induction, we performed a detailed time course analysis for the expression of pancreatic markers PDX1, NKX6.1, NEUROG3, GCG and INS (
Example 4
(100) Recapitulating Human Pancreas Organogenesis to Generate Endocrine Progenitors
(101) This improved and simplified differentiation protocol provides the basis for subsequent efficient formation of insulin-producing cells in suspension (
Example 5
(102) Efficient Generation of PDX1.sup.+/NKX6.1.sup.+ Pancreatic Progenitor Cells Prior to Endocrine Induction Results in Glucose Responsive Beta-Like Cells.
(103) To test the expectation that precocious activation of NEUROG3 expression results in immature polyhormonal cells and not INS/NKX6.1 double positive beta-like cells, we initiated endocrine differentiation at day 7 in PDX1.sup.+ pancreatic progenitors by exposing the cells to NEUROG3 inducers ALKi and Noggin (
(104) To further characterize gene expression in beta-like cells at days 19-20, we took advantage of the GFP live marker to compare sorted GFP.sup.+ beta-like cells and GFP− populations to purified human islets. hESC-derived beta-like cells showed high levels of insulin gene transcripts, comparable to cadaveric islet preparations, while GFP-negative populations exhibit only insignificant levels of the hormone (
Example 6
(105) hESC-Derived Beta-Like Cells Remain Glucose Responsive after Short Term Transplantation.
(106) To determine whether hESC-derived beta-like cells can maintain their glucose responsiveness in vivo, we transplanted approximately 5 million cells under the kidney capsule of immunodeficient mice (days 19-21 spheres consisting of progenitors and beta-like cells). Mice transplanted with 4000 human islets served as controls. Seven to 10 days post-surgery, human C-peptide levels were measured in overnight-fasted mice, before and after the administration of a glucose bolus. As expected, mice that received human islet grafts exhibited low levels of insulin secretion upon fasting, followed by a marked increase in circulating insulin after glucose challenge (average of 221±116 pM,
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Example 7
(108) Re-Aggregation of Induced Beta-Like Cells Improves Yield and Functionality
(109) Human pluripotent stem cells (hPSCs) have been successfully coaxed down the pancreatic lineage into functional insulin-producing cells in vitro, using 3D and planar cell culture systems. However, these protocols generate a mixed population of pancreatic progenitors that are yet uncommitted to the endocrine lineage and insulin-producing cells that are potentially in the process of maturation. It has become apparent that this heterogeneous population is significantly distinct from adult islets, which are mainly composed of fully matured endocrine cells. Developmental studies in mice have shown that early endocrine-committed precursors delaminate from the pancreatic epithelium, cluster together, and then undergo their final maturation secluded from the epithelial niche.
(110)
INTRODUCTION
(111) Recently, we and others have demonstrated the generation of insulin-producing cells in vitro from human pluripotent stem cells.sup.1,2,3; however, a heterogeneous population of uncommitted pancreatic progenitors (about 60-70%) and immature insulin-producing cells (about 30-40%) are produced in these studies.
(112) These insulin-producing cells do not exhibit robust response to glucose in dynamic perifusion assays, indicating that they are not mature or equivalent to islet beta cells.
(113) During development, endocrine commitment is followed by delamination from the pancreatic epithelium, migration and clustering of newly born endocrine cells. It is in these clusters that islet cells, including beta cells, mature into functional cells. Re-aggregation of newly generated beta cells into clusters resembling islets leads to maturation and improved functional properties.
(114) Generation of Insulin-Producing Beta-Like Cells from hESCs.
(115) In
(116) The strategy for re-aggregation of beta cells involved generating immature beta-like cells as described herein and subjecting such cells at day 19-20 of culture to a sorting procedure to enrich for beta-like cells, followed by at least two days of incubation to allow the beta-like cells to self-organize into enhanced beta clusters (eBCs).
(117) Beta-Clusters are Highly Enriched in Bona Fide Beta Cell Markers.
(118) In
(119) Beta-clusters demonstrate significantly greater functional properties than non-enriched hESC-derived beta cell population, in vitro. In
(120) Beta-Clusters Possess Highly Coupled Mitochondria.
(121) On exposure to high glucose, the mitochondrial membrane potential in Beta-clusters decreases in a similar fashion to human islets, as shown in
(122) Beta-Clusters Show Biphasic Calcium Response to Glucose.
(123) Single clusters were perifused with KRB containing varying amounts of glucose and KCL in microfluidic chambers, with the results shown in
(124) Beta-Clusters Function In Vivo.
(125) In
(126) Conclusions
(127) Current beta cell differentiation protocols from hPSCs do not generate homogeneous populations of mature beta cells. In the work disclosed herein, protocols were developed that recapitulated endocrine cell clustering that occurs during the maturation process in embryonic development by sorting and re-aggregating beta cells formed from an Ins-GFP hES cell line. Re-aggregated Beta-clusters are highly enriched for beta cell markers, including c-peptide, PDX1, and NKX6.1, compared to the heterogeneous populations generated during differentiation. The Beta-clusters maintain high levels of expression of these critical genes on prolonged cell culture. Moreover, these Beta-clusters demonstrate significantly better functional properties. In vitro, these clusters showed an improved stimulation in static glucose-stimulated insulin secretion assays and dynamically secrete human C-peptide in perifusion systems in a similar fashion to human islets. They show a decrease in their mitochondrial potential upon stimulation and exhibit a biphasic calcium response to glucose akin to human islets. In vivo, these clusters secrete human C-peptide in response to glucose challenge as early as three days following transplantation and are functional at least until 45 days post transplant. In summary, a new technique to isolate and generate homogeneous and highly pure islet-like endocrine clusters that have enhanced functional capacity has been developed, as disclosed herein.
REFERENCES FOR EXAMPLE 7 ONLY
(128) 1. Rezania, et al. Nature Biotech 32, 1121-1133, 2014. 2. Pagliuca, et al. Cell 159(2), 428-439, 2014. 3. Russ, et al. EMBO J 34(13), 1759-1772, 2015. 4. Nair, et al. Curr. Op in Genetics & Dev. 32, 171-180, 2015.
Example 8
(129) Recapitulating Endocrine Cell Clustering Promotes Coalescence into Islet-Like Structures In Vitro
(130) The end product of current β-cell differentiation protocols consists of a mixed population of PDX1.sup.+ NKX6.1.sup.+ pancreatic progenitors and differentiated endocrine cells that have not yet reached full maturity, in addition to less defined lineages and potentially rare undifferentiated stem cells. Importantly, these protocols do not recapitulate the critical process of endocrine cell clustering that occurs during islet formation in vivo, which is a prerequisite for functional maturation[21, 22, 24]. To address this problem and mimic endocrine cell coalescence into islets, we sought to remove progenitor cells and re-aggregate hPSC-derived-β-like-cells into clusters. Using the INS.sup.GFP/W reporter cell line, we first optimized the differentiation protocol to efficiently generate up to 60% C-peptide.sup.+ Glucagon.sup.− monohormonal cells in 19-20 days (mean±SEM=54.8±1.5%)(
(131) At day 26-27 (d26-27), eBCs are exclusively endocrine with about 95% of cells expressing chromogranin A (
(132) TABLE-US-00004 TABLE 4 Percentage of total cells Population d19-20 d26-27 eBCs C-peptide+Glucagon− 54.8 ± 1.5 83.5 ± 1.7 C-peptide+PDX1+ 44.2 ± 2.8 86.1 ± 1.2 C-peptide+NKX6.1+ 48.7 ± 1.8 78.1 ± 1.8 C-peptide+NKX2.2+ 59.7 ± 1.5 79.9 ± 2.4 C-peptide+PAX6+ 40.7 ± 5.2 79.3 ± 2.2 C-peptide+ISL1+ 39.7 ± 2.9 80.8 ± 1.7 C-peptide+NEUROD1+ 55.7 ± 0.5 86.5 ± 0.9 C-peptide+Chga+ 55.8.7 ± 1.4 90.4 ± 1.8 C-peptide−PDX1+ 24.5 ± 2.4 3.2 ± 0.7 C-peptide−NKX6.1+ 36.9 ± 1.7 5.5 ± 0.3 PDX1+NKX6.1+C- 33.5 ± 2.5 4.0 ± 0.7 peptide−
Example 9
(133) eBCs Display Physiological Properties Similar to Human Islets In Vitro
(134) Current hPSC-derived-β-cells[2, 3, 25] are characterized by limited insulin secretion in response to glucose; especially they do not dynamically respond to glucose challenges in vitro, a trait indicative of an immature phenotype. To determine whether the observed increases in β-cell markers upon re-aggregation convey mature functional β-cell properties, we tested the dynamic response of d27 eBCs to alternating low and high glucose and KCL in a perifusion system. A rapid and marked first phase response was observed similar to human islets, albeit at slightly lower levels (
(135) To further probe the functional properties of eBCs, we sought to investigate the cascade of events preceding insulin-secretion in a mature β-cell. Summarily, oxidation of glucose drives mitochondrial charging and ATP production, thus increasing the cellular phosphate potential (ATP/ADP ratio), which in turn causes closure of ATP-sensitive potassium (K-ATP) channels leading to depolarization of plasma membrane, and finally culminating in calcium influx and insulin secretion. First, we examined cytosolic calcium flux upon stimulation with glucose and KCL using Fura-2/AM in microfluidic chambers. d20 clusters, which contain progenitors and endocrine cells, displayed a slow rise in calcium flux upon stimulation with high glucose that did not terminate on reduction of glucose concentration to baseline (
Example 10
(136) Transcriptome-Wide Analysis of eBCs Reveals Hallmarks of Function and Maturation
(137) The in vitro physiological analyses implied that clustering of β-cells confers exceptional functional properties. To further understand why eBCs display significantly better functional properties than d20 clusters that consist of a mix of progenitors and endocrine cells, a genome-wide transcriptome analysis was completed. INS.sup.GFP-high+ cells were sorted from eBCs and d20 clusters by FACS, and total RNA sequencing (RNA-seq) was performed (
(138) TABLE-US-00005 TABLE 5 Genes Taqman assay ids PDX1 Hs00426216_ml LDHA Hs00855332_gl SLC30A8 Hs00545183_ml MAFB Hs00271378_sl KCNK1 Hs00158428_ml KCNK3 Hs00605529_ml PCSK1 Hs01026107_ml KCNJ11 Hs00265026_s1
(139) TABLE-US-00006 TABLE 6 FPKM values D20 immature- D27 NEC- D27 eBC- Human Adult Gene name Beta beta Beta Islet-Beta ATP6V0D2 0 0.0124323 0.0513565 0.536991 Genes involved ATP5J 32.3957 15.3792 75.0129 177.73 in OXPHOS ATP5L 89.6481 60.5105 199.082 366.38 ATP6V1D 34.1963 24.4457 83.0309 139.125 ATP5O 66.5281 51.8313 152.828 221.862 ATP5C1 103.5 90.7585 267.492 277.976 NDUFA5 16.6862 17.115 66.9206 84.4922 COX6C 153.492 106.578 402.644 322.381 SNAP23 3.80459 3.23178 10.4768 14.4522 Genes involved VAMP4 5.12791 5.91566 23.3995 21.8029 in vesicular SEC22B 34.8315 43.5547 84.9389 163.532 transport STX7 7.54882 9.17629 20.4759 19.4536 NQO1 1.93235 6.05353 11.0637 21.3156 Regulation of cellular amino acid metabolic process CALM2 279.292 219.769 664.21 1085.96 Positive TXN 55.9843 37.6364 168.024 258.546 regulation of HMGB1 80.8789 67.5733 181.76 220.723 DNA binding SLC8A2 2.51685 11.3662 7.75431 9.03834 Calcium:Sodium SLC8A3 0.316007 0.793365 1.58513 3.22293 antiporter activity MRPL13 21.2227 11.3279 48.6998 80.2054 Mitochondrial translational elongation GOLT1B 13.1734 8.82642 39.4514 40.6625 Golgi mediated transport
(140) Emerging evidence indicates that β-cell maturation not only requires the cellular machinery enabling insulin synthesis and stimulus-secretion coupling, but also the reduction of disallowed genes that interfere with glucose sensing. DNA methylation has been recently implicated in repression[9], and hence we analyzed CpG-rich regions within loci of critical glucose-secretion decoupling genes: HK1 and LDHA. Bisulfite sequencing of differentially methylated regions of HK1 and LDHA in eBCs showed hypermethylation akin to the pattern observed in human islets[9] (
Example 11
(141) β-Cells Residing in a Highly Enriched Endocrine-Niche are Distinct from β-Cells in a Progenitor-Rich Niche
(142) The data indicate that eBCs, as a population, are more mature than immature (d20) clusters. To dissect the changes in the state of INS.sup.− GFP.sup.+ cells caused by coalescence, RNA-seq was conducted on GFP-high cells isolated from cell aggregates at these various stages (
Example 12
(143) β-Cell Clustering Induces Metabolic Maturation of Mitochondria
(144) RNA-seq results implicate activation of OXPHOS, electron transport chain and ATP production as potentially essential steps for maturation of eBCs. In order to assess mitochondrial respiratory function at a phenotypic level, we performed the Cell Mito stress test using a Seahorse XFe24 analyzer. eBCs (
(145) As expected, GSEA also indicated enrichment of the components of the ‘inner mitochondrial membrane complexes’, the location of OXPHOS, in β-cells upon re-aggregation/clustering (
Example 13
(146) eBCs Rapidly Function In Vivo
(147) A distinctive feature of human islets and mature β-cells is their ability to function in vivo within days after transplantation. Yet, published protocols have reported positive responses to in vivo glucose challenges only 2-6 weeks after transplantation of their in vitro hPSC-derived-β-cells[2, 3, 25]. To test the functionality of our cells in vivo, we transplanted 700 eBCs (about 700,000 cells) of which about 80% are C-peptide.sup.+ NKX6.1.sup.+ (about 560,000 β-cells) into non-diabetic NSG mice. eBCs secreted more C-peptide after an acute glucose challenge as early as 3 days post-transplant (7/8 animals) and maintained their function even 30-days after transplant (
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(149) Each of the references listed below and cited throughout the disclosure is incorporated by reference herein in its entirety, or in relevant part, as would be apparent from context. The disclosed subject matter has been described with reference to various specific embodiments and techniques. It should be understood, however, that many variations and modifications may be made while remaining within the spirit and scope of the disclosed subject matter.