Dr. Holger Russ is interested in elucidating the underlying mechanisms that lead to the development of diabetes in humans. One specific focus of the lab is to investigate and elucidate molecular mechanisms that govern human beta cell development, maturation, replication, and function under steady state conditions and response’s to stress(es). Dr. Russ’ lab was among the first three groups demonstrating the generation of functional beta cells from human pluripotent stem cells under cell culture conditions. It is now possible to generate patient specific cell lines with the ability to differentiate into any cell type thus enabling the study of beta cell function in a specific genomic context. The Russ lab is taking advantage of recent breakthroughs in genome editing technology to establish different inducible CRISPR/Cas9 systems to facilitate rapid and precise gene modification of pluripotent stem- and its differentiated cell derivates. While the pancreatic beta cell is key to glucose homeostasis, the thymus gland also plays a critical role in the development of T1D. The Russ lab was the first to demonstrate the successful generation of human embryonic stem cell-derived thymic epithelial cells (TECs) by directed differentiation. Moving forward, the lab is determined to combine in vitro derived thymic epithelium with human T-cell progenitors either in vitro or in vivo to study diverse aspects of autoimmunity in a strictly human context.
Education: PhD: Tel Aviv University; Doktorvater: Shimon Efrat, 2011
Postdoctoral training: UCSF; Advisor: Matthias Hebrok, 2016
The emphasis of Dr.Russ`s research is on understanding the underlying molecular and cellular mechanisms resulting in autoimmune type 1 diabetes (T1D) in humans, with a focus on the insulin-producing beta cells. During his career, Dr. Russ successfully worked on different aspects of T1D, which led to several original and important contributions to the fields of beta-, thymus- and stem cell- biology. After completing his Diploma thesis in Heidelberg/Germany, Dr. Russ obtained his Ph.D. from Sackler School of Medicine at Tel Aviv University/Israel followed by postdoctoral work at the Diabetes Center at the University of California- San Francisco. Thereafter he established his independent research program at the Barbara Davis Center for Diabetes at UC- Anschutz Medical Campus. His lab employs state of the art human stem cell technology and primary human cell culture with genome engineering approaches to model and eventually treat patients suffering from diabetes. Dr.Russ`s long term goal is to find a cure for patients suffering from diabetes, maintain a productive and extramural funded research program, train and enable the next generation of scientists focused on T1D research; and take responsibility for developing and/or maintaining a cutting-edge diabetes research program.
Doctor of Medicine: University of Colorado School of Medicine, 2013
Resident in Pediatrics: University of Colorado School of Medicine/Children's Hospital Colorado, 2016
Fellowship in Pediatric Endocrinology: University of Colorado School of Medicine/Children's Hospital Colorado/Barbara Davis Center for Childhood Diabetes, 2019
Taylor is a K12 scholar and Assistant Professor of Pediatrics at the Barbara Davis Center for Diabetes. Her research project in the Russ Lab is “Elucidating the role of the type 1 diabetes risk gene PTPN2 on human beta cell function in health and disease.” Through this work, she is investigating new and exciting potential therapies for type 1 diabetes using stem cell biology and state of the art gene editing technologies. She aims to understand the role of high-risk genes associated with type 1 diabetes in the stem cell derived beta cell context. Using her knowledge from her clinical practice in the care of children with type 1 diabetes she hopes to utilize the expertise of the Russ Lab to investigate translational applications of stem cell derived beta cells.
Ph.D. Molecular Endocrinology, Orphan Nuclear Receptor Lab, School of Biological Sciences & Technology,Chonnam National University, South Korea.
M.Phil. Human Genetics, Bharathiayar University, India
M.S. Zoology, Jamal Mohamed College, Bharathidasan University, India
Bala is interested in exploring the molecular mechanisms underlying human beta cell maturation and investigating the translatome profiling in differentiated beta cells from normal and T1D patient-specific iPSCs. He always enjoys working as a team rather than squeezing his brain alone. He loves hiking, ice-skating, walking in trails and visiting new places with family. He also loves playing cricket, volleyball and skiing with friends. He enjoys trying all kinds of junk foods in between healthy food habits.
B.S. from Northern Arizona University
Doctoral Candidate
Molecular Biology Program
University of Colorado Anschutz Medical Campus
Funding: T32 Molecular Biology training grant (2018-2019)
Single Cell RNA-Seq Grant from the RNA Bioscience Initiative
Stephan’s work focuses on the generation of an in vitro functional human thymus organoid system to study the mechanisms of human thymic development and T cell selection. In his free time Stephan enjoys spending time with his wife and standard poodle, hiking, and playing disc-golf.
B.S. Cellular and Molecular Biology, The University of Texas at El Paso
PhD candidate
Molecular Biology Program
University of Colorado Anschutz Medical Campus
Roberto is interesting in uncovering the mechanisms of beta cell immune responses during an autoimmune attack as well as how to protect them
B.S in Biochemistry at University of Massachusetts Boston.
Masters of Science in Nutrition and Biomedicine at the Technical University of Munich.
Funding: T-32 training grant
Ali is interested in determining the underlying drivers of beta cell interconversion and heterogeneity in healthy and diseased states. His project focuses on determining the Fate of Stem Cell Derived Pancreatic β-like Cells (sBCs) Upon Transplantation Using Genetic Lineage Tracing.
PhD from Ludwig Maximilian University of Munich, Germany
MSc from University of Canterbury, New Zealand
BSc from Wittenberg University, Ohio
Patent: Systems and Methods for Query and Index Optimization for Retrieving Data in Instances of a Formulation Data Structure from a Database. 2018. Publication Number: WO/2018/187306. American Chemical Society.
Amanda is focused on elucidating the role of the thymus in the development of type I diabetes and autoimmune disease. She implements the techniques developed by the lab of directed differentiation to generate human embryonic stem-cell derived thymic epithelial cells, with the aim of gaining deeper insight into the biology and function of the thymus. In her free time she enjoys transforming traditional recipes into healthy, guilt-free versions and sharing these on her website to promote healthy dietary changes. When not optimizing lab processes or cooking, you will find her out in nature with her family exploring all the various hiking and outdoor opportunities Colorado has to offer.
Fiona joined us in 2017 from bonny Scotland, she has a BSc hon. in Biochemistry from the University of Edinburgh and studied for a Masters and PhD in Stem Cell Biology at the University of Cambridge – funded by a competitive 4-year PhD Studentship from the British Heart Foundation. After completing her PhD Fiona undertook a short secondment working as Policy Advisor at the Department for Business Innovation and Skills (BIS) in Her Majesty’s Government, Westminster. From 2015 to 2017 she worked as post-doctoral scientist in a startup company focusing on the transdifferentiation of exocrine pancreatic tissue to functional beta cells at the University of Aberdeen. Fiona’s research interests focus on developing regenerative medicine based therapies for diabetes specifically through the directed differentiation of human pluripotent cells. Endocrine differentiation, beta cell maturation and beta cell functionality have become her specialist subjects!
B.S. in Biology from Saint Michael's College, Vermont
Shane is interested in understanding the role of a type 1 diabetes risk gene in human beta cell development and function through genome editing stem cell derived beta-like cells, and using inhibitors in primary human islets. Shane spends his free time in the Rocky mountains scoping out the perfect camp site or skiing fresh powder at one of the ski resorts.
BS in Biology from University of Copenhagen 2013-2016
MS in Human Biology from University of Copenhagen 2016-2018
Master's thesis project under supervision of Dr. Palle Serup at Novo Nordisk Foundation Center for Stem Cell Biology 2017-2018 (Title: Characterization of hESC-derived Pancreatic Progenitors)
Maria is interested in generating pancreatic beta-cells from human pluripotent stem cells, and in better understanding the molecular mechanisms underlying this development, particularly mitochondrial metabolism. Being a type 1 diabetic herself, she is also extremely interested in the translational aspect of our research, and in how we can make our stem cell-derived beta-cells suitable for transplantation and protect them from an autoimmune attack. In her free time she likes to spend as much time as possible in the mountains. She enjoys hiking, camping, horseback riding, fishing, skiing, and climbing 14’ers. She enjoys traveling to places she has never been before.
MBA in Computer Science from Utkal UniversityB.S from R.D Women's College, Utkal University, India
Prabha is interested in bioinformatics and MATLAB. Her goal is to study the epigenetic and transcriptional characteristics of human beta cells and find the underlying mechanisms that lead to Type 1 Diabetes. She enjoys exploring new places and loves spending time with her kid and family
Ramos, S.A., Morton, J.J., Yadav, P., Reed, B., Alizadeh, S.I., Shilleh, A.H., Perrenoud, L., Jaggers, J., Kappler, J., Jimeno, A. and Russ, H.A., 2021. Hudish, L.I., Bubak, A., Triolo, T.M., Niemeyer, C.S., Sussel, L., Nagel, M., Taliaferro, J.M. and Russ, H.A., 2020. Russell, R., Carnese, P.P., Hennings, T.G., Walker, E.M., Russ, H.A., Liu, J.S., Giacometti, S., Stein, R. and Hebrok, M., 2020. Goering, R., Hudish, L.I., Guzman, B.B., Raj, N., Bassell, G.J., Russ, H.A., Dominguez, D. and Taliaferro, J.M., 2020. Zhou, X., Nair, G.G., Russ, H.A., Belair, C.D., Li, M.L., Shveygert, M., Hebrok, M. and Blelloch, R., 2019. Liebau, S., Russ, H.A. and Kleger, A., 2019. Bubak, A.N., Como, C.N., Coughlan, C.M., Johnson, N.R., Hassell Jr, J.E., Mescher, T., Niemeyer, C.S., Mahalingam, R., Cohrs, R.J., Boyd, T.D., Potter, H., Russ, H.A., Nagel, M.A., 2019. Nair, G.G., Liu, J.S., Russ, H.A., Tran, S., Saxton, M.S., Chen, R., Juang, C., Li, M.L., Nguyen, V.Q., Giacometti, S. and Puri, S., 2019. Castro-Gutierrez, R., Michels, A.W. and Russ, H.A., 2018. Puri, S., Roy, N., Russ, H.A., Leonhardt, L., French, E.K., Roy, R., Bengtsson, H., Scott, D.K., Stewart, A.F. and Hebrok, M., 2018. Docherty, F.M. and Russ, H.A., 2018. Faleo, G., Russ, H.A., Wisel, S., Parent, A.V., Nguyen, V., Nair, G.G., Freise, J.E., Villanueva, K.E., Szot, G.L., Hebrok, M. and Tang, Q., 2017. Chang, R., Faleo, G., Russ, H.A., Parent, A.V., Elledge, S.K., Bernards, D.A., Allen, J.L., Villanueva, K., Hebrok, M., Tang, Q. and Desai, T.A., 2017. Abstract: Dorrell, C., Schug, J., Canaday, P.S., Russ, H.A., Tarlow, B.D., Grompe, M.T., Horton, T., Hebrok, M., Streeter, P.R., Kaestner, K.H. and Grompe, M., 2016. Zhu, S., Russ, H.A., Wang, X., Zhang, M., Ma, T., Xu, T., Tang, S., Hebrok, M. and Ding, S., 2016. Russ, H.A., Landsman, L., Moss, C.L., Higdon, R., Greer, R.L., Kaihara, K., Salamon, R., Kolker, E. and Hebrok, M., 2016. Sintov, E., Nathan, G., Knoller, S., Pasmanik-Chor, M., Russ, H.A. and Efrat, S., 2015. Russ, H.A., Parent, A.V., Ringler, J.J., Hennings, T.G., Nair, G.G., Shveygert, M., Guo, T., Puri, S., Haataja, L., Cirulli, V., Blelloch, R., Greg, L. Szot., Peter, Arvan. and Matthias, Hebrok., 2015. Russ, H.A., and Hebrok, M., 2014. Morris IV, J.P., Greer, R., Russ, H.A., von Figura, G., Kim, G.E., Busch, A., Lee, J., Hertel, K.J., Kim, S., Mcmanus, M. and Hebrok, M., 2014. Von Figura, G., Fukuda, A., Roy, N., Liku, M.E., Morris IV, J.P., Kim, G.E., Russ, H.A., Firpo, M.A., Mulvihill, S.J., Dawson, D.W. and Ferrer, J., 2014. Li, K., Zhu, S., Russ, H.A., Xu, S., Xu, T., Zhang, Y., Ma, T., Hebrok, M. and Ding, S., 2014. Parent, A.V., Russ, H.A., Khan, I.S., LaFlam, T.N., Metzger, T.C., Anderson, M.S. and Hebrok, M., 2013. Efrat, S. and Russ, H.A., 2012. Bar, Y., Russ, H.A., Sintov, E., Anker-Kitai, L., Knoller, S. and Efrat, S., 2012. Russ, H.A., and Efrat, S., 2011. Russ, H.A., Sintov, E., Anker-Kitai, L., Friedman, O., Lenz, A., Toren, G., Farhy, C., Pasmanik-Chor, M., Oron-Karni, V., Ravassard, P. and Efrat, S., 2011. Bar-Nur, O., Russ, H.A., Efrat, S. and Benvenisty, N., 2011. Russ, H.A. and Efrat, S., 2011. Russ, H.A., Ravassard, P., Kerr-Conte, J., Pattou, F. and Efrat, S., 2009. Bar, Y., Russ, H.A., Knoller, S., Ouziel-Yahalom, L. and Efrat, S., 2008. Russ, H.A., Bar, Y., Ravassard, P. and Efrat, S., 2008. Links .....
Generation of functional human thymic cells from induced pluripotent stem cells. Journal of Allergy and Clinical Immunology.
Abstract:
Abstract
Background: The thymus is a glandular organ that is essential for the formation of the adaptive immune system by educating developing T cells. The thymus is most active during childhood and involutes around the time of adolescence, resulting in a severe reduction or absence of naive T-cell output. The ability to generate a patient-derived human thymus would provide an attractive research platform and enable the development of novel cell therapies.
Objectives: This study sought to systematically evaluate signaling pathways to develop a refined direct differentiation protocol that generates patient-derived thymic epithelial progenitor cells from multiple induced pluripotent stem cells (iPSCs) that can further differentiate into functional patient-derived thymic epithelial cells on transplantation into athymic nude mice.
Methods: Directed differentiation of iPSC generated TEPs that were transplanted into nude mice. Between 14 and 19 weeks posttransplantation, grafts were removed and analyzed by flow cytometry, quantitative PCR, bulk RNA sequencing, and single-cell RNA sequencing for markers of thymic-cell and T-cell development.
Results: A direct differentiation protocol that allows the generation of patient-derived thymic epithelial progenitor cells from multiple iPSC lines is described. On transplantation into athymic nude mice, patient-derived thymic epithelial progenitor cells further differentiate into functional patient-derived thymic epithelial cells that can facilitate the development of T cells. Single-cell RNA sequencing analysis of iPSC-derived grafts shows characteristic thymic subpopulations and patient-derived thymic epithelial cell populations that are indistinguishable from TECs present in primary neonatal thymus tissue.
Conclusions: These findings provide important insights and resources for researchers focusing on human thymus biology.
2020
Modeling Hypoxia-Induced neuropathies using a fast and scalable human motor neuron differentiation system. Stem Cell Reports.
Abstract:
Human motor neuron (MN) diseases encompass a spectrum of disorders. A critical barrier to dissecting disease mechanisms is the lack of appropriate human MN models. Here, we describe a scalable, suspension-based differentiation system to generate functional human MN diseases in 3 weeks. Using this model, we translated recent findings that mRNA mis-localization plays a role in disease development to the human context by establishing a membrane-based system that allows efficient fractionation of MN cell soma and neurites. In response to hypoxia, used to mimic diabetic neuropathies, MNs upregulated mitochondrial transcripts in neurites; however, mitochondria were decreased. These data suggest that hypoxia may disrupt translation of mitochondrial mRNA, potentially leading to neurite damage and development of neuropathies. We report the development of a novel human MN model system to investigate mechanisms of disease affecting soma and/or neurites that facilitates the rapid generation and testing of patient-specific MN diseases.
Loss of the transcription factor MAFB limits β-cell derivation from human PSCs. Nature Communications, 11(1), pp.1-15.
Abstract:
Next generation sequencing studies have highlighted discrepancies in β-cells which exist between mice and men. Numerous reports have identified MAF BZIP Transcription Factor B (MAFB) to be present in human β-cells postnatally, while its expression is restricted to embryonic and neo-natal β-cells in mice. Using CRISPR/Cas9-mediated gene editing, coupled with endocrine cell differentiation strategies, we dissect the contribution of MAFB to β-cell development and function specifically in humans. Here we report that MAFB knockout hPSCs have normal pancreatic differentiation capacity up to the progenitor stage, but favor somatostatin- and pancreatic polypeptide–positive cells at the expense of insulin- and glucagon-producing cells during endocrine cell development. Our results describe a requirement for MAFB late in the human pancreatic developmental program and identify it as a distinguishing transcription factor within islet cell subtype specification. We propose that hPSCs represent a powerful tool to model human pancreatic endocrine development and associated disease pathophysiology.
FMRP promotes RNA localization to neuronal projections through interactions between its RGG domain and G-quadruplex RNA sequences. Elife, 9, p.e52621.
Abstract:
The sorting of RNA molecules to subcellular locations facilitates the activity of spatially restricted processes. We have analyzed subcellular transcriptomes of FMRP-null mouse neuronal cells to identify transcripts that depend on FMRP for efficient transport to neurites. We found that these transcripts contain an enrichment of G-quadruplex sequences in their 3′ UTRs, suggesting that FMRP recognizes them to promote RNA localization. We observed similar results in neurons derived from Fragile X Syndrome patients. We identified the RGG domain of FMRP as important for binding G-quadruplexes and the transport of G-quadruplex-containing transcripts. Finally, we found that the translation and localization targets of FMRP were distinct and that an FMRP mutant that is unable to bind ribosomes still promoted localization of G-quadruplex-containing messages. This suggests that these two regulatory modes of FMRP may be functionally separated. These results provide a framework for the elucidation of similar mechanisms governed by other RNA-binding proteins. 2019
LIN28B Impairs the Transition of hESC-Derived β Cells from the Juvenile to Adult State. Stem Cell Reports.
Abstract:
Differentiation of human embryonic stem cells into pancreatic β cells holds great promise for the treatment of diabetes. Recent advances have led to the production of glucose-responsive insulin-secreting cells in vitro, but resulting cells remain less mature than their adult primary β cell counterparts. The barrier(s) to in vitro β cell maturation are unclear. Here, we evaluated a potential role for microRNAs. MicroRNA profiling showed high expression of let-7 family microRNAs in vivo, but not in in vitro differentiated β cells. Reduced levels of let-7 in vitro were associated with increased levels of the RNA binding protein LIN28B, a negative regulator of let-7 biogenesis. Ablation of LIN28B during human embryonic stem cell (hESC) differentiation toward β cells led to a more mature glucose-stimulated insulin secretion profile and the suppression of juvenile-specific genes. However, let-7 overexpression had little effect. These results uncover LIN28B as a modulator of β cell maturation in vitro.
Stem Cell Derived Organoids in Human Disease and Development. Stem Cells International.
Varicella Zoster Virus Infection of Primary Human Spinal Astrocytes Produces Intracellular Amylin, Amyloid-beta, and an Amyloidogenic Extracellular Environment.
The Journal of infectious diseases.
Abstract:
Background: Herpes zoster is linked to amyloid-associated diseases, including dementia, macular degeneration, and diabetes mellitus, in epidemiological studies. Thus, we examined whether varicella-zoster virus (VZV)-infected cells produce amyloid.
Methods: Production of intracellular amyloidogenic proteins (amylin, amyloid precursor protein [APP], and amyloid-β [Aβ]) and amyloid, as well as extracellular amylin, Aβ, and amyloid, was compared between mock- and VZV-infected quiescent primary human spinal astrocytes (qHA-sps). The ability of supernatant from infected cells to induce amylin or Aβ42 aggregation was quantitated. Finally, the amyloidogenic activity of viral peptides was examined.
Results: VZV-infected qHA-sps, but not mock-infected qHA-sps, contained intracellular amylin, APP, and/or Aβ, and amyloid. No differences in extracellular amylin, Aβ40, or Aβ42 were detected, yet only supernatant from VZV-infected cells induced amylin aggregation and, to a lesser extent, Aβ42 aggregation into amyloid fibrils. VZV glycoprotein B (gB) peptides assembled into fibrils and catalyzed amylin and Aβ42 aggregation.
Conclusions: VZV-infected qHA-sps produced intracellular amyloid and their extracellular environment promoted aggregation of cellular peptides into amyloid fibrils that may be due, in part, to VZV gB peptides. These findings suggest that together with host and other environmental factors, VZV infection may increase the toxic amyloid burden and contribute to amyloid-associated disease progression.
Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived β cells.
Nature cell biology, 21(2), p.263.
Abstract: Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional β cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and β cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature β-like cells to form islet-sized enriched β-clusters (eBCs). eBCs display physiological properties analogous to primary human β cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell clustering induces metabolic maturation by driving mitochondrial oxidative respiration, a process central to stimulus–secretion coupling in mature β cells. eBCs display glucose-stimulated insulin secretion as early as three days after transplantation in mice. In summary, replicating aspects of endocrine cell clustering permits the generation of stem-cell-derived β cells that resemble their endogenous counterparts. 2018
β Cell replacement: improving on the design.
Current Opinion in Endocrinology, Diabetes and Obesity, 25(4), pp.251-257.
Abstract:
Purpose of review: Here we summarize recent advancements in β cell replacement as a therapy for type 1 diabetes.
Recent findings: β cell replacement therapy has been proposed as a cure for type 1 diabetes with the introduction of the Edmonton protocol for cadaveric islet transplantation. To allow widespread use of this approach, efforts have focused on establishing an abundant source of insulin-producing β cells, protecting transplanted cells from ischemia-mediated death, immune rejection, and re-occurring autoimmunity. Recent developments addressing these issues include generation of insulin-producing cells from human pluripotent stem cells, different encapsulation strategies and prevention of ischemia upon transplant.
Summary: Despite significant advances in generating functional β cells from human pluripotent stem cells, several key challenges remain in regard to the survival of β cell grafts, protection from (auto-) immune destruction and implementation of additional safety mechanisms before a stem cell-based cell replacement therapy approach can be widely applied. Taking current findings into consideration, we outline a multilayered approach to design immune-privileged β cells from stem cells using state of the art genome editing technologies that if successfully incorporated could result in great benefit for diabetic patients and improve clinical results for cell replacement therapy.
Replication confers β cell immaturity.
Nature communications, 9(1), p.485.
Abstract:
Pancreatic β cells are highly specialized to regulate systemic glucose levels by secreting insulin. In adults, increase in β-cell mass is limited due to brakes on cell replication. In contrast, proliferation is robust in neonatal β cells that are functionally immature as defined by a lower set point for glucose-stimulated insulin secretion. Here we show that β-cell proliferation and immaturity are linked by tuning expression of physiologically relevant, non-oncogenic levels of c-Myc. Adult β cells induced to replicate adopt gene expression and metabolic profiles resembling those of immature neonatal β that proliferate readily. We directly demonstrate that priming insulin-producing cells to enter the cell cycle promotes a functionally immature phenotype. We suggest that there exists a balance between mature functionality and the ability to expand, as the phenotypic state of the β cell reverts to a less functional one in response to proliferative cues.
Cell–Cell Interactions Driving Differentiation of Adult Pancreatic Stem Cells. 2017
Mitigating ischemic injury of stem cell-derived insulin-producing cells after transplant.
Stem cell reports, 9(3), pp.807-819.
Abstract:
The advent of large-scale in vitro differentiation of human stem cell-derived insulin-producing cells (SCIPC) has brought us closer to treating diabetes using stem cell technology. However, decades of experiences from islet transplantation show that ischemia-induced islet cell death after transplant severely limits the efficacy of the therapy. It is unclear to what extent human SCIPC are susceptible to ischemia. In this study, we show that more than half of SCIPC die shortly after transplantation. Nutrient deprivation and hypoxia acted synergistically to kill SCIPC in vitro. Amino acid supplementation rescued SCIPC from nutrient deprivation, likely by providing cellular energy. Generating SCIPC under physiological oxygen tension of 5% conferred hypoxia resistance without affecting their differentiation or function. A two-pronged strategy of physiological oxygen acclimatization during differentiation and amino acid supplementation during transplantation significantly improved SCIPC survival after transplant.
Nanoporous immunoprotective device for stem-cell-derived β-cell replacement therapy.
ACS nano, 11(8), pp.7747-7757.
Encapsulation of human embryonic stem-cell-differentiated beta cell clusters (hES-βC) holds great promise for cell replacement therapy for the treatment of diabetics without the need for chronic systemic immune suppression. Here, we demonstrate a nanoporous immunoprotective polymer thin film cell encapsulation device that can exclude immune molecules while allowing exchange of oxygen and nutrients necessary for in vitro and in vivo stem cell viability and function. Biocompatibility studies show the device promotes neovascular formation with limited foreign body response in vivo. The device also successfully prevented teratoma escape into the peritoneal cavity of mice. Long-term animal studies demonstrate evidence of engraftment, viability, and function of cells encapsulated in the device after 6 months. Finally, in vivo study confirms that the device was able to effectively immuno-isolate cells from the host immune system. 2016
Human islets contain four distinct subtypes of β cells.
Nature communications, 7, p.11756.
Abstract:
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.
Human pancreatic beta-like cells converted from fibroblasts.
Nature communications, 7, p.10080.
Abstract:
Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Here we show the conversion of human fibroblasts towards an endodermal cell fate by employing non-integrative episomal reprogramming factors in combination with specific growth factors and chemical compounds. On initial culture, converted definitive endodermal progenitor cells (cDE cells) are specified into posterior foregut-like progenitor cells (cPF cells). The cPF cells and their derivatives, pancreatic endodermal progenitor cells (cPE cells), can be greatly expanded. A screening approach identified chemical compounds that promote the differentiation and maturation of cPE cells into functional pancreatic beta-like cells (cPB cells) in vitro. Transplanted cPB cells exhibit glucose-stimulated insulin secretion in vivo and protect mice from chemically induced diabetes. In summary, our study has important implications for future strategies aimed at generating high numbers of functional beta cells, which may help restoring normoglycemia in patients suffering from diabetes.
Dynamic proteomic analysis of pancreatic mesenchyme reveals novel factors that enhance human embryonic stem cell to pancreatic cell differentiation.
Stem cells international, 2016.
Abstract:
Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.
2015
Inhibition of ZEB1 expression induces redifferentiation of adult human β cells expanded in vitro.
Scientific reports, 5, p.13024.
Abstract:
In-vitro expansion of functional adult human β-cells is an attractive approach for generating insulin-producing cells for transplantation. However, human islet cell expansion in culture results in loss of β-cell phenotype and epithelial-mesenchymal transition (EMT). This process activates expression of ZEB1 and ZEB2, two members of the zinc-finger homeobox family of E-cadherin repressors, which play key roles in EMT. Downregulation of ZEB1 using shRNA in expanded β-cell-derived (BCD) cells induced mesenchymal-epithelial transition (MET), β-cell gene expression, and proliferation attenuation. In addition, inhibition of ZEB1 expression potentiated redifferentiation induced by a combination of soluble factors, as judged by an improved response to glucose stimulation and a 3-fold increase in the fraction of C-peptide-positive cells to 60% of BCD cells. Furthermore, ZEB1 shRNA led to increased insulin secretion in cells transplanted in vivo. Our findings suggest that the effects of ZEB1 inhibition are mediated by attenuation of the miR-200c target genes SOX6 and SOX2. These findings, which were reproducible in cells derived from multiple human donors, emphasize the key role of ZEB1 in EMT in cultured BCD cells and support the value of ZEB1 inhibition for BCD cell redifferentiation and generation of functional human β-like cells for cell therapy of diabetes.
Controlled induction of human pancreatic progenitors produces functional beta‐like cells in vitro.
The EMBO journal, 34(13), pp.1759-1772.
Abstract:
Directed differentiation of human pluripotent stem cells into functional insulin‐producing beta‐like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non‐functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1+ and subsequent PDX1+/NKX6.1+ pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1+/NKX6.1+ progenitors produces glucose‐responsive beta‐like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short‐term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta‐like cells. 2014
Taming the young and restless—epigenetic gene regulation in pancreas and beta‐cell precursors.
The EMBO journal, 33(19), pp.2135-2136.
Abstract:
The in vivo assessment of epigenetic changes during mouse pancreatic beta‐cell differentiation reveals surprising differences to directed, in vitro differentiation of human embryonic stem cells. New findings reported in this issue of The EMBO Journal further identify Ezh2 as a critical determinant of endocrine progenitor number and could instruct improved protocols for stem cell‐based therapies.
Dicer regulates differentiation and viability during mouse pancreatic cancer initiation.
PloS one, 9(5), p.e95486.
Abstract:
miRNA levels are altered in pancreatic ductal adenocarcinoma (PDA), the most common and lethal pancreatic malignancy, and intact miRNA processing is essential for lineage specification during pancreatic development. However, the role of miRNA processing in PDA has not been explored. Here we study the role of miRNA biogenesis in PDA development by deleting the miRNA processing enzyme Dicer in a PDA mouse model driven by oncogenic Kras. We find that loss of Dicer accelerates Kras driven acinar dedifferentiation and acinar to ductal metaplasia (ADM), a process that has been shown to precede and promote the specification of PDA precursors. However, unconstrained ADM also displays high levels of apoptosis. Dicer loss does not accelerate development of Kras driven PDA precursors or PDA, but surprisingly, we observe that mouse PDA can develop without Dicer, although at the expense of proliferative capacity. Our data suggest that intact miRNA processing is involved in both constraining pro-tumorigenic changes in pancreatic differentiation as well as maintaining viability during PDA initiation.
The chromatin regulator Brg1 suppresses formation of intraductal papillary mucinous neoplasm and pancreatic ductal adenocarcinoma.
Nature cell biology, 16(3), p.255.
Abstract:
Pancreatic ductal adenocarcinoma (PDA) develops through distinct precursor lesions, including pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). However, genetic features resulting in IPMN-associated PDA (IPMN–PDA) versus PanIN-associated PDA (PanIN-PDA) are largely unknown. Here we find that loss of Brg1, a core subunit of SWI/SNF chromatin remodelling complexes, cooperates with oncogenic Kras to form cystic neoplastic lesions that resemble human IPMN and progress to PDA. Although Brg1-null IPMN–PDA develops rapidly, it possesses a distinct transcriptional profile compared with PanIN-PDA driven by mutant Kras and hemizygous p53 deletion. IPMN–PDA also is less lethal, mirroring prognostic trends in PDA patients. In addition, Brg1 deletion inhibits Kras-dependent PanIN development from adult acinar cells, but promotes Kras-driven preneoplastic transformation in adult duct cells. Therefore, this study implicates Brg1 as a determinant of context-dependent Kras-driven pancreatic tumorigenesis and suggests that chromatin remodelling may underlie the development of distinct PDA subsets.
Small molecules facilitate the reprogramming of mouse fibroblasts into pancreatic lineages.
Cell stem cell, 14(2), pp.228-236.
Abstract:
Pancreatic β cells are of great interest for the treatment of type 1 diabetes. A number of strategies already exist for the generation of β cells, but a general approach for reprogramming nonendodermal cells into β cells could provide an attractive alternative in a variety of contexts. Here, we describe a stepwise method in which pluripotency reprogramming factors were transiently expressed in fibroblasts in conjunction with a unique combination of soluble molecules to generate definitive endoderm-like cells that did not pass through a pluripotent state. These endoderm-like cells were then directed toward pancreatic lineages using further combinations of small molecules in vitro. The resulting pancreatic progenitor-like cells could mature into cells of all three pancreatic lineages in vivo, including functional, insulin-secreting β-like cells that help to ameliorate hyperglycemia. Our findings may therefore provide a useful approach for generating large numbers of functional β cells for disease modeling and, ultimately, cell-based therapy. 2013
Generation of functional thymic epithelium from human embryonic stem cells that supports host T cell development.
Cell stem cell, 13(2), pp.219-229.
Abstract:
Inducing immune tolerance to prevent rejection is a key step toward successful engraftment of stem-cell-derived tissue in a clinical setting. Using human pluripotent stem cells to generate thymic epithelial cells (TECs) capable of supporting T cell development represents a promising approach to reach this goal; however, progress toward generating functional TECs has been limited. Here, we describe a robust in vitro method to direct differentiation of human embryonic stem cells (hESCs) into thymic epithelial progenitors (TEPs) by precise regulation of TGFβ, BMP4, RA, Wnt, Shh, and FGF signaling. The hESC-derived TEPs further mature into functional TECs that support T cell development upon transplantation into thymus-deficient mice. Importantly, the engrafted TEPs produce T cells capable of in vitro proliferation as well as in vivo immune responses. Thus, hESC-derived TEP grafts may have broad applications for enhancing engraftment in cell-based therapies as well as restoring age- and stress-related thymic decline. 2012
Making β cells from adult tissues.
Trends in Endocrinology & Metabolism, 23(6), pp.278-285.
Abstract:
β-Cell replacement represents an attractive prospect for diabetes therapy. Although much hope has been placed on derivation of insulin-producing cells from human pluripotent stem cells, this approach continues to face considerable challenges. Cells from adult human tissues, with both stem/progenitor and mature phenotypes, offer a possible alternative. This review summarizes recent progress in two major strategies based on this cell source, ex vivo expansion of human islet β cells and conversion of non-β cells into insulin-producing cells by nuclear reprogramming, and examines the obstacles that remain to be overcome for bringing these strategies closer to clinical application in diabetes therapy.
Redifferentiation of expanded human pancreatic β-cell-derived cells by inhibition of the NOTCH pathway.
Journal of Biological Chemistry, 287(21), pp.17269-17280.
Abstract:
In vitro expansion of β-cells from adult human pancreatic islets would overcome donor β-cell shortage for cell replacement therapy for diabetes. Using a β-cell-specific labeling system we have shown that β-cell expansion is accompanied by dedifferentiation resembling epithelial-mesenchymal transition and loss of insulin expression. Epigenetic analyses indicate that key β-cell genes maintain open chromatin structure in expanded β-cell-derived (BCD) cells, although they are not transcribed. In the developing pancreas important cell-fate decisions are regulated by NOTCH receptors, which signal through the Hairy and Enhancer of Split 1 (HES1) transcription regulator. We have reported that BCD cell dedifferentiation and proliferation in vitro correlate with reactivation of the NOTCH pathway. Inhibition of HES1 expression using shRNA during culture initiation results in reduced β-cell replication and dedifferentiation, suggesting that HES1 inhibition may also affect BCD cell redifferentiation following expansion. Here, we used HES1 shRNA to down-regulate HES1 expression in expanded human BCD cells, showing that HES1 inhibition is sufficient to induce BCD cell redifferentiation, as manifested by a significant increase in insulin expression. Combined treatment with HES1 shRNA, cell aggregation in serum-free medium, and a mixture of soluble factors further stimulated the redifferentiation of BCD cells. In vivo analyses demonstrated the ability of the redifferentiated cells to replace β-cell function in hyperglycemic immunodeficient mice. These findings demonstrate the redifferentiation potential of ex vivo expanded BCD cells and the reproducible differentiating effect of HES1 inhibition in these cells. 2011
Development of human insulin-producing cells for cell therapy of diabetes.
Pediatr Endocrinol Rev, 9(2), pp.590-597.
Abstract:
Diabetes mellitus is characterized by the loss of insulin-producing beta cells. While conventional treatment results in severe long-term complications, cell replacement therapy is a promising approach for the cure of this disease. However, its application is severally limited by the shortage of donor tissue. Hence, great research efforts concentrate on the development of an abundant cell source of functional beta-like cells, by pursuing three main strategies: Expansion of human donor beta cells in vitro, reprogramming of other cell types, and directed differentiation of pluripotent stem cells, both embryonic and patient-derived. The goal of all these approaches has been the generation of cells with properties that closely resemble the beta-cell phenotype, in particular production and storage of adequate amounts of mature insulin, and its regulated release in response to physiological signals. Here we review recent progress in all three approaches and discuss their advantages as well as remaining challenges.
Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.
PloS one, 6(9), p.e25566.
Abstract:
Background Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.
Methodology/Principal Finding Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.
Conclusions/Significance These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening.
Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells.
Cell stem cell, 9(1), pp.17-23.
Abstract:
Human induced pluripotent stem cells (HiPSCs) appear to be highly similar to human embryonic stem cells (HESCs). Using two genetic lineage-tracing systems, we demonstrate the generation of iPSC lines from human pancreatic islet beta cells. These reprogrammed cells acquired markers of pluripotent cells and differentiated into the three embryonic germ layers. However, the beta cell-derived iPSCs (BiPSCs) maintained open chromatin structure at key beta-cell genes, together with a unique DNA methylation signature that distinguishes them from other PSCs. BiPSCs also demonstrated an increased ability to differentiate into insulin-producing cells both in vitro and in vivo, compared with ESCs and isogenic non-beta iPSCs. Our results suggest that the epigenetic memory may predispose BiPSCs to differentiate more readily into insulin producing cells. These findings demonstrate that HiPSC phenotype may be influenced by their cells of origin, and suggest that their skewed differentiation potential may be advantageous for cell replacement therapy.
In-Vivo Functional Assessment of Engineered Human Insulin-Producing Cells (Artech House). 2009
Epithelial-mesenchymal transition in cells expanded in vitro from lineage-traced adult human pancreatic beta cells.
PloS one, 4(7), p.e6417.
Abstract:
Background In-vitro expansion of functional beta cells from adult human islets is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes. Using genetic cell-lineage tracing we have recently shown that beta cells cultured from adult human islets undergo rapid dedifferentiation and proliferate for up to 16 population doublings. These cells have raised interest as potential candidates for redifferentiation into functional insulin-producing cells. Previous work has associated dedifferentiation of cultured epithelial cells with epithelial-mesenchymal transition (EMT), and suggested that EMT generates cells with stem cell properties. Here we investigated the occurrence of EMT in these cultures and assessed their stem cell potential.
Methodology/Principal Findings Using cell-lineage tracing we provide direct evidence for occurrence of EMT in cells originating from beta cells in cultures of adult human islet cells. These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC). However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types.
Conclusions/Significance These findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells. 2008
HES-1 is involved in adaptation of adult human β-cells to proliferation in vitro.
Diabetes, 57(9), pp.2413-2420.
Abstract:
OBJECTIVE— In vitro expansion of β-cells from adult human islets could solve the tissue shortage for cell replacement therapy of diabetes. Culture of human islet cells typically results in <16 cell doublings and loss of insulin expression. Using cell lineage tracing, we demonstrated that the expanded cell population included cells derived from β-cells. Understanding the molecular mechanisms involved in β-cell fate in vitro is crucial for optimizing expansion and redifferentiation of these cells. In the developing pancreas, important cell-fate decisions are regulated by NOTCH receptors, which signal through the hairy and enhancer of split (HES)-1 transcriptional regulator. Here, we investigated the role of the NOTCH signaling pathway in β-cell dedifferentiation and proliferation in vitro.
RESEARCH DESIGN AND METHODS—Isolated human islets were dissociated into single cells. β-Cells were genetically labeled using a Cre-lox system delivered by lentiviruses. Cells were analyzed for changes in expression of components of the NOTCH pathway during the initial weeks in culture. HES-1 expression was inhibited by a small hairpin RNA (shRNA), and the effects on β-cell phenotype were analyzed.
RESULTS— Human β-cell dedifferentiation and entrance into the cell cycle in vitro correlated with activation of the NOTCH pathway and downregulation of the cell cycle inhibitor p57. Inhibition of HES-1 expression using shRNA resulted in significantly reduced β-cell replication and dedifferentiation.
CONCLUSIONS—These findings demonstrate that the NOTCH pathway is involved in determining β-cell fate in vitro and suggest possible molecular targets for induction of β-cell redifferentiation following in vitro expansion.
In vitro proliferation of cells derived from adult human β-cells revealed by cell-lineage tracing.
Diabetes, 57(6), pp.1575-1583.
Abstract:
OBJECTIVE— Expansion of insulin-producing β-cells from adult human islets could alleviate donor shortage for cell-replacement therapy of diabetes. A major obstacle to development of effective expansion protocols is the rapid loss of β-cell markers in the cultured cells. Here, we report a genetic cell-lineage tracing approach for following the fate of cultured β-cells.
RESEARCH DESIGN AND METHODS— Cells dissociated from isolated human islets were infected with two lentiviruses, one expressing Cre recombinase under control of the insulin promoter and the other, a reporter cassette with the structure cytomegalovirus promoter-loxP-DsRed2-loxP-eGFP.
RESULTS— β-Cells were efficiently and specifically labeled by the dual virus system. Label+, insulin− cells derived from β-cells were shown to proliferate for a maximum of 16 population doublings, with an approximate doubling time of 7 days. Isolated labeled cells could be expanded in the absence of other pancreas cell types if provided with medium conditioned by pancreatic non–β-cells. Analysis of mouse islet cells by the same method revealed a much lower proliferation of labeled cells under similar culture conditions.
CONCLUSIONS— Our findings provide direct evidence for survival and dedifferentiation of cultured adult human β-cells and demonstrate that the dedifferentiated cells significantly proliferate in vitro. The findings confirm the difference between mouse and human β-cell proliferation under our culture conditions. These findings demonstrate the feasibility of cell-specific labeling of cultured primary human cells using a genetic recombination approach that was previously restricted to transgenic animals.
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