RESUMO
Scalable processes are requisite for the robust biomanufacturing of human pluripotent stem cell (hPSC)-derived therapeutics. Toward this end, we demonstrate the xeno-free expansion and directed differentiation of human embryonic and induced pluripotent stem cells to definitive endoderm (DE) in a controlled stirred suspension bioreactor (SSB). Based on previous work on converting hPSCs to insulin-producing progeny, differentiation of two hPSC lines was optimized in planar cultures yielding up to 87% FOXA2+ /SOX17+ cells. Next, hPSCs were propagated in an SSB with controlled pH and dissolved oxygen. Cultures displayed a 10- to 12-fold increase in cell number over 5-6 days with the maintenance of pluripotency (>85% OCT4+ ) and viability (>85%). For differentiation, SSB cultures yielded up to 89% FOXA2+ /SOX17+ cells or ~ 8 DE cells per seeded hPSC. Specification to DE cell fate was consistently more efficient in the bioreactor compared to planar cultures. Hence, a tunable strategy is established that is suitable for the xeno-free manufacturing of DE cells from different hPSC lines in scalable SSBs. This study advances bioprocess development for producing a wide gamut of human DE cell-derived therapeutics.
Assuntos
Reatores Biológicos , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Linhagem Celular , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
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.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Células Secretoras de Insulina/fisiologia , Pâncreas/citologia , Animais , Glicemia/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/terapia , Células-Tronco Embrionárias/citologia , Glucose/farmacologia , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/transplante , Camundongos , Camundongos SCID , Camundongos Transgênicos , EstreptozocinaRESUMO
Besides its role in exocrine differentiation, pancreas-specific transcription factor 1a (PTF1a) is required for pancreas specification from the foregut endoderm and ultimately for endocrine cell formation. Examining the early role of PTF1a in pancreas development has been challenging due to limiting amounts of embryonic tissue material for study. Embryonic stem cells (ESCs) which can be differentiated in vitro, and without limit to the amount of experimental material, can serve as a model system to study these early developmental events. To this end, we derived and characterized a mouse ESC line with tetracycline-inducible expression of PTF1a (tet-Ptf1a mESCs). We found that transient ectopic expression of PTF1a initiated the pancreatic program in differentiating ESCs causing cells to activate PDX1 expression in bud-like structures resembling pancreatic primordia in vivo. These bud-like structures also expressed progenitor markers characteristic of a developing pancreatic epithelium. The epithelium differentiated to generate a wave of NGN3+ endocrine progenitors, and further formed cells of all three pancreatic lineages. Notably, the insulin+ cells in the cultures were monohormonal, and expressed PDX1 and NKX6.1. PTF1a-induced cultures differentiated into significantly more endocrine and exocrine cells and the ratio of endocrine-to-exocrine cell differentiation could be regulated by retinoic acid (RA) and nicotinamide (Nic) signaling. Moreover, induced cultures treated with RA and Nic exhibited a modest glucose response. Thus, this tet-Ptf1a ESC-based in vitro system is a valuable new tool for interrogating the role of PTF1a in pancreas development and in directing differentiation of ESCs to endocrine cells.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Células Endócrinas/metabolismo , Pâncreas/metabolismo , Fatores de Transcrição/genética , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/ultraestrutura , Células Endócrinas/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/genética , Insulina/metabolismo , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Genéticos , Niacinamida/farmacologia , Organogênese/genética , Pâncreas/citologia , Pâncreas/crescimento & desenvolvimento , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Tretinoína/farmacologiaRESUMO
Cell replacement therapy using ß cells derived from stem cells is a promising alternative to conventional diabetes treatment options. Although current differentiation methods produce glucose-responsive ß cells, they can also yield populations of undesired endocrine progenitors and other proliferating cell types that might interfere with long-term islet function and safety of transplanted cells. Here, we describe the generation of an array of monoclonal antibodies against cell surface markers that selectively label stem cell-derived islet cells. A high-throughput screen identified promising candidates, including three clones that mark a high proportion of endocrine cells in differentiated cultures. A scalable magnetic sorting method was developed to enrich for human pluripotent stem cell (hPSC)-derived islet cells using these three antibodies, leading to the formation of islet-like clusters with improved glucose-stimulated insulin secretion and reduced growth upon transplantation. This strategy should facilitate large-scale production of functional islet clusters from stem cells for disease modeling and cell replacement therapy.
Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Células-Tronco Pluripotentes , Diferenciação Celular , Glucose/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
Type 1 diabetic patients with severe hypoglycemia unawareness have benefitted from cellular therapies, such as pancreas or islet transplantation; however, donor shortage and the need for immunosuppression limits widespread clinical application. We previously developed an intravascular bioartificial pancreas (iBAP) using silicon nanopore membranes (SNM) for immunoprotection. To ensure ample nutrient delivery, the iBAP will need a cell scaffold with high hydraulic permeability to provide mechanical support and maintain islet viability and function. Here, we examine the feasibility of superporous agarose (SPA) as a potential cell scaffold in the iBAP. SPA exhibits 66-fold greater hydraulic permeability than the SNM along with a short (<10 µm) diffusion distance to the nearest islet. SPA also supports short-term functionality of both encapsulated human islets and stem-cell-derived enriched ß-clusters in a convection-based system, demonstrated by high viability (>95%) and biphasic insulin responses to dynamic glucose stimulus. These findings suggest that the SPA scaffold will not limit nutrient delivery in a convection-based bioartificial pancreas and merits continued investigation.
Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Pâncreas Artificial , Sefarose/química , Transplante de Células-Tronco/métodos , Alicerces Teciduais , Adulto , Diabetes Mellitus Tipo 1/terapia , Glucose/farmacologia , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas , Membranas Artificiais , Nanoporos , SilícioRESUMO
Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease.
Assuntos
Fator 6 Nuclear de Hepatócito/genética , Pâncreas/fisiologia , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Fator 6 Nuclear de Hepatócito/metabolismo , Células-Tronco Embrionárias Humanas , Humanos , Transcrição GênicaRESUMO
Diabetes mellitus, which affects more than 463 million people globally, is caused by the autoimmune ablation or functional loss of insulin-producing ß-cells, and prevalence is projected to continue rising over the next decades. Generating ß-cells to mitigate the aberrant glucose homeostasis manifested in the disease has remained elusive. Substantial advances have been made in producing mature ß-cells from human pluripotent stem cells that respond appropriately to dynamic changes in glucose concentrations in vitro and rapidly function in vivo following transplantation in mice. Other potential avenues to produce functional ß-cells include: transdifferentiation of closely related cell types (for example, other pancreatic islet cells such as α-cells, or other cells derived from endoderm); the engineering of non-ß-cells that are capable of modulating blood sugar; and the construction of synthetic 'cells' or particles mimicking functional aspects of ß-cells. This Review focuses on the current status of generating ß-cells via these diverse routes, highlighting the unique advantages and challenges of each approach. Given the remarkable progress in this field, scalable bioengineering processes are also discussed for the realization of the therapeutic potential of derived ß-cells.
Assuntos
Diferenciação Celular , Diabetes Mellitus/terapia , Células Secretoras de Insulina/fisiologia , Células-Tronco Pluripotentes/fisiologia , Células-Tronco/fisiologia , Animais , Reatores Biológicos , Blastocisto/citologia , Células-Tronco Embrionárias/fisiologia , Humanos , Imunossupressores , Lactente , Recém-Nascido , Ilhotas Pancreáticas/fisiologia , Camundongos , Transplante de Células-TroncoRESUMO
Differentiation of human embryonic stem cells into pancreatic ß 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 ß cell counterparts. The barrier(s) to in vitro ß 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 ß 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 ß 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 ß cell maturation in vitro.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Humanos , Camundongos , MicroRNAs/genética , Interferência de RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
Human but not mouse islets transplanted into immunodeficient NSG mice effectively accumulate lipid droplets (LDs). Because chronic lipid exposure is associated with islet ß-cell dysfunction, we investigated LD accumulation in the intact human and mouse pancreas over a range of ages and states of diabetes. Very few LDs were found in normal human juvenile pancreatic acinar and islet cells, with numbers subsequently increasing throughout adulthood. While accumulation appeared evenly distributed in postjuvenile acinar and islet cells in donors without diabetes, LDs were enriched in islet α- and ß-cells from donors with type 2 diabetes (T2D). LDs were also found in the islet ß-like cells produced from human embryonic cell-derived ß-cell clusters. In contrast, LD accumulation was nearly undetectable in the adult rodent pancreas, even in hyperglycemic and hyperlipidemic models or 1.5-year-old mice. Taken together, there appear to be significant differences in pancreas islet cell lipid handling between species, and the human juvenile and adult cell populations. Moreover, our results suggest that LD enrichment could be impactful to T2D islet cell function.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Células Secretoras de Glucagon/patologia , Células Secretoras de Insulina/patologia , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/patologia , Gotículas Lipídicas/patologia , Células Acinares/patologia , Células Acinares/ultraestrutura , Adolescente , Adulto , Fatores Etários , Idoso , Animais , Criança , Pré-Escolar , Diabetes Mellitus Experimental/patologia , Células-Tronco Embrionárias , Feminino , Células Secretoras de Glucagon/ultraestrutura , Humanos , Lactente , Células Secretoras de Insulina/ultraestrutura , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/ultraestrutura , Gotículas Lipídicas/ultraestrutura , Masculino , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Pessoa de Meia-Idade , Ratos , Doadores de Tecidos , Adulto JovemRESUMO
In the version of this article originally published, the Gene Expression Omnibus (GEO) accession number listed in the data availability section was incorrectly given as GSE10979 instead of GSE109795. The sentence should read "RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE109795," and the code should link to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109795. The error has been corrected in the HTML and PDF versions of the paper.
RESUMO
Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional ß 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 ß cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature ß-like cells to form islet-sized enriched ß-clusters (eBCs). eBCs display physiological properties analogous to primary human ß 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 ß 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 ß cells that resemble their endogenous counterparts.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células Endócrinas/citologia , Fibroblastos/citologia , Células-Tronco Embrionárias Humanas/citologia , Células Secretoras de Insulina/citologia , Animais , Células Cultivadas , Células-Tronco Embrionárias/fisiologia , Células Endócrinas/fisiologia , Fibroblastos/fisiologia , Glucose/farmacologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/citologia , Camundongos , Mitocôndrias/metabolismoRESUMO
Islet ß cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that ß cells of newborns secrete more insulin than adults in response to similar intracellular Ca2+ concentrations, suggesting differences in the Ca2+ sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca2+ binding paralog of the ß cell Ca2+ sensor Syt7, increased by â¼8-fold during ß cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 affects insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-ßH1 cells, a human ß cell line. These findings reveal the role that altered Ca2+ sensing plays in regulating ß cell maturation.
Assuntos
Cálcio/farmacologia , Glucose/farmacologia , Células Secretoras de Insulina/citologia , Insulina/metabolismo , Sinaptotagminas/metabolismo , Animais , Transporte Biológico , Diferenciação Celular/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipoglicemiantes/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Edulcorantes/farmacologia , Sinaptotagminas/genéticaRESUMO
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.
Assuntos
Células Secretoras de Insulina/metabolismo , Isquemia/terapia , Transplante das Ilhotas Pancreáticas , Transplante de Células-Tronco , Células-Tronco/metabolismo , Aminoácidos/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Isquemia/patologia , Camundongos Endogâmicos C57BL , Oxigênio/farmacologia , Ácido Pirúvico/farmacologia , Células-Tronco/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Sobrevivência de Tecidos/efeitos dos fármacosRESUMO
Transcription factors are tools repetitively used by the embryo to generate a variety of lineages. Hence, their context of activation is an important determinant of their ability to specifically trigger certain cell fates, but not others. The context is also consequential when considering directing differentiation of embryonic stem cells (ESCs). In this study, we sought to assess the context of pancreatic transcription factor 1a (PTF1a) activation in reference to its propancreatic effects in mouse ESCs (mESCs). We hypothesized that an enriched endodermal population would respond to PTF1a and trigger the pancreatic program more effectively than a spontaneously differentiated population. Using an in vitro model of pancreas development that we recently established, we found that inducing PTF1a in highly enriched definitive endoderm did not promote pancreatic differentiation but induction in more differentiated endoderm, specifically posterior foregut endoderm, did form pancreatic progenitors. These progenitors never underwent terminal differentiation to endocrine or acinar phenotype. However, a short 3D culture period, prior to PTF1a induction, led to the generation of monohormonal insulin(+) cells and amylase-expressing cells. Our findings suggest that enriched posterior foregut endoderm is competent to respond to PTF1a's propancreatic activity; but a 3D culture environment is essential for terminal differentiation of pancreatic progenitors.