Human pancreatic islets of Langerhans contain five distinct endocrine cell types, each producing a characteristic hormone. The dysfunction or loss of the insulin-producing β cells causes diabetes ...mellitus, a disease that harms millions. Until now, β cells were generally regarded as a single, homogenous cell population. Here we identify four antigenically distinct subtypes of human β cells, which we refer to as β1-4, and which are distinguished by differential expression of ST8SIA1 and CD9. These subpopulations are always present in normal adult islets and have diverse gene expression profiles and distinct basal and glucose-stimulated insulin secretion. Importantly, the β cell subtype distribution is profoundly altered in type 2 diabetes. These data suggest that this antigenically defined β cell heterogeneity is functionally and likely medically relevant.
Type 1 diabetes (T1D) occurs as a consequence of the autoimmune destruction of insulin-producing pancreatic beta (β) cells and commonly presents with insulin deficiency and unregulated glycemic ...control. Despite improvements in the medical management of T1D, life-threatening complications are still common. Beta-cell replication to replace lost cells may be achieved by using small-molecule mitogenic drugs, like harmine. However, the safe and effective delivery of such drugs to beta cells remains a challenge. This work aims to deploy an antibody conjugated nanocarrier platform to achieve cell-specific delivery of candidate therapeutic and imaging agents to pancreatic endocrine cells. We approached this goal by generating core-shell type micellar nanocarriers composed of the tri-block copolymer, Pluronic®F127 (PEO
-PPO
-PEO
). We decorated these nanocarriers with a pancreatic endocrine cell-selective monoclonal antibody (HPi1), with preference for beta cells, to achieve active targeting. The PPO-based hydrophobic core allows encapsulation of various hydrophobic cargoes, whereas the PEO-based hydrophilic shell curbs the protein adhesion, hence prolonging the nanocarriers' systemic circulation time. The nancarriers were loaded with quantum dots (QDots) that allowed nanocarrier detection both in-vitro and in-vivo. In-vitro studies revealed that HPi1 conjugated nanocarriers could target endocrine cells in dispersed islet cell preparations with a high degree of specificity, with beta cells exhibiting a fluorescent quantum dot signal that was approximately five orders of magnitude greater than the signal associated with alpha cells. In vivo endocrine cell targeting studies demonstrated that the HPi1 conjugated nanocarriers could significantly accumulate at the islet xenograft site. For drug delivery studies, the nanocarriers were loaded with harmine. We demonstrated that HPi1 conjugated nanocarriers successfully targeted and delivered harmine to human endocrine cells in a human islet xenograft model. In this model, targeted harmine delivery yielded an ~ 41-fold increase in the number of BrdU positive cells in the human islet xenograft than that observed in untreated control mice. By contrast, non-targeted harmine yielded an ~ 9-fold increase in BrdU positive cells. We conclude that the nanocarrier platform enabled cell-selective targeting of xenografted human pancreatic endocrine cells and the selective delivery of the hydrophobic drug harmine to those cells. Further, the dramatic increase in proliferation with targeted harmine, a likely consequence of achieving higher local drug concentrations, supports the concept that targeted drug delivery may promote more potent biological responses when using harmine and/or other drugs than non-targeting approaches. These results suggest that this targeted drug delivery platform may apply in drug screening, beta cell regenerative therapies, and/or diagnostic imaging in patients with type 1 diabetes.
PDX1
/NKX6-1
pancreatic progenitors (PPs) give rise to endocrine cells both in vitro and in vivo. This cell population can be successfully differentiated from human pluripotent stem cells (hPSCs) and ...hold the potential to generate an unlimited supply of β cells for diabetes treatment. However, the efficiency of PP generation in vitro is highly variable, negatively impacting reproducibility and validation of in vitro and in vivo studies, and consequently, translation to the clinic. Here, we report the use of a proteomics approach to phenotypically characterize hPSC-derived PPs and distinguish these cells from non-PP populations during differentiation. Our analysis identifies the pancreatic secretory granule membrane major glycoprotein 2 (GP2) as a PP-specific cell surface marker. Remarkably, GP2 is co-expressed with NKX6-1 and PTF1A in human developing pancreata, indicating that it marks the multipotent pancreatic progenitors in vivo. Finally, we show that isolated hPSC-derived GP2
cells generate β-like cells (C-PEPTIDE
/NKX6-1
) more efficiently compared to GP2
and unsorted populations, underlining the potential therapeutic applications of GP2.Pancreatic progenitors (PPs) can be derived from human pluripotent stem cells in vitro but efficiency of differentiation varies, making it hard to sort for insulin-producing cells. Here, the authors use a proteomic approach to identify the secretory granule membrane glycoprotein 2 as a marker for PDX1+/NKX6-1+ PPs.
Insulin-secreting β cells and glucagon-secreting α cells maintain physiological blood glucose levels, and their malfunction drives diabetes development. Using ChIP sequencing and RNA sequencing ...analysis, we determined the epigenetic and transcriptional landscape of human pancreatic α, β, and exocrine cells. We found that, compared with exocrine and β cells, differentiated α cells exhibited many more genes bivalently marked by the activating H3K4me3 and repressing H3K27me3 histone modifications. This was particularly true for β cell signature genes involved in transcriptional regulation. Remarkably, thousands of these genes were in a monovalent state in β cells, carrying only the activating or repressing mark. Our epigenomic findings suggested that α to β cell reprogramming could be promoted by manipulating the histone methylation signature of human pancreatic islets. Indeed, we show that treatment of cultured pancreatic islets with a histone methyltransferase inhibitor leads to colocalization of both glucagon and insulin and glucagon and insulin promoter factor 1 (PDX1) in human islets and colocalization of both glucagon and insulin in mouse islets. Thus, mammalian pancreatic islet cells display cell-type-specific epigenomic plasticity, suggesting that epigenomic manipulation could provide a path to cell reprogramming and novel cell replacement-based therapies for diabetes.
Background: Donor-site morbidity, limited numbers of cells, loss of phenotype during ex vivo expansion, and age-related decline in chondrogenic activity present critical obstacles to the use of ...autologous chondrocyte implantation for cartilage repair. Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells.
Hypothesis/Purpose: The authors hypothesized that juvenile chondrocyte would show stronger and more stable chondrogenic activity than adult cells in vitro and that juvenile cells pose little risk of immunologic incompatibility in adult hosts.
Study Design: Controlled laboratory study.
Methods: Cartilage samples were from juvenile (<13 years old) and adult (>13 years old) donors. The chondrogenic activity of freshly isolated human articular chondrocytes and of expanded cells after monolayer culture was measured by proteoglycan assay, gene expression analysis, and histology. Lymphocyte proliferation assays were used to assess immunogenic activity.
Results: Proteoglycan content in neocartilage produced by juvenile chondrocytes was 100-fold higher than in neocartilage produced by adult cells. Collagen type II and type IX mRNA in fresh juvenile chondrocytes were 100- and 700-fold higher, respectively, than in adult chondrocytes. The distributions of collagens II and IX were similar in native juvenile cartilage and in neocartilage made by juvenile cells. Juvenile cells grew significantly faster in monolayer cultures than adult cells (P = .002) and proteoglycan levels produced in agarose culture was significantly higher in juvenile cells than in adult cells after multiple passages (P < .001). Juvenile chondrocytes did not stimulate lymphocyte proliferation.
Conclusion: These results document a dramatic age-related decline in human chondrocyte chondrogenic potential and show that allogeneic juvenile chondrocytes do not stimulate an immunologic response in vivo.
Clinical Relevance: Juvenile human chondrocytes have greater potential to restore articular cartilage than adult cells, and may be transplanted without the fear of rejection, suggesting a new allogeneic approach to restoring articular cartilage in older individuals.
Innovations in the field of amphiphilic block copolymers have led to the development of a series of attractive polymer-based drug and gene delivery micellar formulations. The amphiphilic block ...copolymers' low critical micelle concentration (CMC) results in highly stable nanoscale micelles possessing favorable in vivo safety profiles and biocompatibility, making them an excellent carrier choice for the systemic administration of various poorly soluble drugs. These micelles can also be used as an actively targeted drug delivery system. The targeting is achieved by conjugating specific targeting ligand molecules to the micelle surface. The conjugation takes place at the hydrophilic termini of the copolymers, which forms the shell or surface of the nanomicelles. In our lab, we have developed a targeted Pluronic
F127-based nanoformulation to achieve targeting of specific cell types in the pancreas. To achieve active targeting based on the desired end application, we have conjugated several monoclonal antibodies (~150 kDa IgG) reactive to specific cell types in the pancreas by coupling lysine amino groups of the antibody to the p-nitrophenyl carbonate groups generated on the hydrophilic PEO segments of the Pluronic
F127. The resultant targeted nanomicelles demonstrated high binding specificity and targeting efficiency. These nanomicelles can be used to encapsulate and deliver hydrophobic imaging agents and/or water-insoluble therapeutic small molecules to specific pancreatic cell types, enabling the development of diverse tools for use in the diagnosis and/or treatment of pancreas pathologies.
Although the lymphatic system arises as an extension of venous vessels in the embryo, little is known about the role of circulating progenitors in the maintenance or development of lymphatic ...endothelium. Here, we investigated whether hematopoietic stem cells (HSCs) have the potential to give rise to lymphatic endothelial cells (LEC).
Following the transfer of marked HSCs into irradiated recipients, donor-derived LEC that co-express the lymphatic endothelial markers Lyve-1 and VEGFR-3 were identified in several tissues. HSC-derived LEC persisted for more than 12 months and contributed to approximately 3-4% of lymphatic vessels. Donor-derived LECs were not detected in mice transplanted with common myeloid progenitors and granulocyte/macrophage progenitors, suggesting that myeloid lineage commitment is not a requisite step in HSC contribution to lymphatic endothelium. Analysis of parabiotic mice revealed direct evidence for the existence of functional, circulating lymphatic progenitors in the absence of acute injury. Furthermore, the transplantation of HSCs into Apc(Min/+) mice resulted in the incorporation of donor-derived LEC into the lymphatic vessels of spontaneously arising intestinal tumors.
Our results indicate that HSCs can contribute to normal and tumor associated lymphatic endothelium. These findings suggest that the modification of HSCs may be a novel approach for targeting tumor metastasis and attenuating diseases of the lymphatic system.
The erythropoietin receptor (EpoR) is widely expressed but its renoprotective action is unexplored. To examine the role of EpoR in vivo in the kidney, we induced acute kidney injury (AKI) by ...ischemia-reperfusion in mice with different EpoR bioactivities in the kidney. EpoR bioactivity was reduced by knockin of wild-type human EpoR, which is hypofunctional relative to murine EpoR, and a renal tubule-specific EpoR knockout. These mice had lower EPO/EpoR activity and lower autophagy flux in renal tubules. Upon AKI induction, they exhibited worse renal function and structural damage, more apoptosis at the acute stage (<7 days), and slower recovery with more tubulointerstitial fibrosis at the subacute stage (14 days). In contrast, mice with hyperactive EpoR signaling from knockin of a constitutively active human EpoR had higher autophagic flux, milder kidney damage, and better renal function at the acute stage but, surprisingly, worse tubulointerstitial fibrosis and renal function at the subacute stage. Either excess or deficient EpoR activity in the kidney was associated with abnormal peritubular capillaries and tubular hypoxia, creating a "U-shaped" relationship. The direct effects of EpoR on tubular cells were confirmed in vitro by a hydrogen peroxide model using primary cultured proximal tubule cells with different EpoR activities. In summary, normal erythropoietin (EPO)/EpoR signaling in renal tubules provides defense against renal tubular injury maintains the autophagy-apoptosis balance and peritubular capillary integrity. High and low EPO/EpoR bioactivities both lead to vascular defect, and high EpoR activity overides the tubular protective effects in AKI recovery.
The efficient generation of hepatocytes from human pluripotent stem cells (hPSCs) requires the induction of a proper endoderm population, broadly characterized by the expression of the cell surface ...marker CXCR4. Strategies to identify and isolate endoderm subpopulations predisposed to the liver fate do not exist. In this study, we generated mouse monoclonal antibodies against human embryonic stem cell-derived definitive endoderm with the goal of identifying cell surface markers that can be used to track the development of this germ layer and its specification to a hepatic fate. Through this approach, we identified two endoderm-specific antibodies, HDE1 and HDE2, which stain different stages of endoderm development and distinct derivative cell types. HDE1 marks a definitive endoderm population with high hepatic potential, whereas staining of HDE2 tracks with developing hepatocyte progenitors and hepatocytes. When used in combination, the staining patterns of these antibodies enable one to optimize endoderm induction and hepatic specification from any hPSC line.
We describe here the humanisation of two mouse monoclonal antibodies that bind to surface markers on human pancreatic islet endocrine cells. Monoclonal antibodies produced by the HIC1-2B4 and ...HIC0-4F9 mouse hybridomas bind distinct surface molecules expressed on endocrine cells and have been validated for a number of experimental methods including immunohistochemistry and live cell sorting by flow cytometry. Variable region framework and first constant region domain sequences were replaced with that from compatible human antibody sequences, and the resulting recombinant antigen-binding fragments were cloned and expressed in mouse myeloma cells. ELISA, fluorescent immunohistochemistry, and flow cytometry were used to assess the specificity of the humanised antibody fragments. Purification and binding analyses indicated that human islet endocrine cell binding was retained in the humanised antibody fragments. These humanised, recombinant antibody fragments have a lower risk of eliciting adverse responses from a patient's immune system and, therefore, have highly improved clinical potential. Thus, they may be used to image, target or carry cargo specifically to islet cells in human patients.
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