11 research outputs found
Differentiating Embryonic Stem Cells Pass through âTemporal Windowsâ That Mark Responsiveness to Exogenous and Paracrine Mesendoderm Inducing Signals
BACKGROUND: Mesendoderm induction during embryonic stem cell (ESC) differentiation in vitro is stimulated by the Transforming Growth Factor and Wingless (Wnt) families of growth factors. PRINCIPAL FINDINGS: We identified the periods during which Bone Morphogenetic Protein (BMP) 4, Wnt3a or Activin A were able to induce expression of the mesendoderm marker, Mixl1, in differentiating mouse ESCs. BMP4 and Wnt3a were required between differentiation day (d) 1.5 and 3 to most effectively induce Mixl1, whilst Activin A induced Mixl1 expression in ESC when added between d2 and d4, indicating a subtle difference in the requirement for Activin receptor signalling in this process. Stimulation of ESCs with these factors at earlier or later times resulted in little Mixl1 induction, suggesting that the differentiating ESCs passed through 'temporal windows' in which they sequentially gained and lost competence to respond to each growth factor. Inhibition of either Activin or Wnt signalling blocked Mixl1 induction by any of the three mesendoderm-inducing factors. Mixing experiments in which chimeric EBs were formed between growth factor-treated and untreated ESCs revealed that BMP, Activin and Wnt signalling all contributed to the propagation of paracrine mesendoderm inducing signals between adjacent cells. Finally, we demonstrated that the differentiating cells passed through 'exit gates' after which point they were no longer dependent on signalling from inducing molecules for Mixl1 expression. CONCLUSIONS: These studies suggest that differentiating ESCs are directed by an interconnected network of growth factors similar to those present in early embryos and that the timing of growth factor activity is critical for mesendoderm induction
Generation of monohormonal INSULIN+ cells from human embryonic stem cells
Human Embryonic Stem Cells (hESCs) are immortal, pluripotent cells derived from the
inner cell mass of the pre implantation embryo. These cells have the potential to
differentiate into all cell types including insulin producing beta cells, which could provide
an alternative to cadaverâderived islets for the treatment of type 1 diabetes.
We have explored the formation of pancreatic progenitor cells and mature ÎČâcells using
hESC reporter lines which express GFP under the control of regulatory sequences from the
PDX1 and INSULIN genes respectively. Using serumâfree media, we have found that specific
combinations of factors applied in a sequential fashion result in the formation of a
pancreatic precursor population, which can be further differentiated to hormone
expressing cells. These endocrine cells have been shown to be a heterogeneous population
that contains a substantial population of INSULIN expressing cells that do not express
either glucagon or somatostatin, of which a subpopulation expresses NKX6.1.
An INSULINGFP/w hESC reporter line was further modified by the addition of a luciferase
transgene under the control of an EF1α promoter, which allowed for in vivo imaging of
transplanted INSULIN expressing cells. Differentiated TgâEF1αLuc.INSULINGFP/w cells were
sorted based on INSULINâGFP expression, and transplanted under the kidney capsule of immunocompromised mice and assessed for the retention of INSULINâGFP+ cells. It was
found that the transplanted INSULIN+ cells had the ability to form monohormonal
endocrine cells of three different lineages. Additionally it was seen that persisting
INSULIN+ cells coâexpressed transcription factors associated with betaâcell maturity, such
as NKX6.1 and MAFA
Generation of iPSC lines from peripheral blood mononuclear cells from 5 healthy adults
Abstract: We describe the generation and characterization of 5 human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of healthy adult individuals. The PBMCs were reprogrammed using non-integrating Sendai viruses containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. The iPSC lines exhibited a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. These iPSC lines can be used as controls in studying disease mechanisms
Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system
The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract
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Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system
The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract
GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives
The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that ÎČ-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives