Engineering functional human gastrointestinal organoid tissues using the three primary germ layers separately derived from pluripotent stem cells

2021 ◽  
Author(s):  
Niveda Udaykumar
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Germ Layers

scholarly journals A Src inhibitor regulates the cell cycle of human pluripotent stem cells and improves directed differentiation

2015 ◽  
Vol 210 (7) ◽  
pp. 1257-1268 ◽  
Author(s):  
Sundari Chetty ◽  
Elise N. Engquist ◽  
Elie Mehanna ◽  
Kathy O. Lui ◽  
Alexander M. Tsankov ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Directed Differentiation ◽  
Differentiation Potential ◽  
Germ Layers ◽  
Positive Effects ◽  
Src Inhibitor ◽  
Expression Of Genes

Driving human pluripotent stem cells (hPSCs) into specific lineages is an inefficient and challenging process. We show that a potent Src inhibitor, PP1, regulates expression of genes involved in the G1 to S phase transition of the cell cycle, activates proteins in the retinoblastoma family, and subsequently increases the differentiation propensities of hPSCs into all three germ layers. We further demonstrate that genetic suppression of Src regulates the activity of the retinoblastoma protein and enhances the differentiation potential of hPSCs across all germ layers. These positive effects extend beyond the initial germ layer specification and enable efficient differentiation at subsequent stages of differentiation.

185 Directed Differentiation of Porcine Induced Pluripotent Stem Cells into all Three Germ Layers via Embryoid Body Formation

2018 ◽  
Vol 30 (1) ◽  
pp. 232
Author(s):  
W. Chakritbudsabong ◽  
S. Pamonsupornvichit ◽  
L. Sariya ◽  
R. Pronarkngver ◽  
S. Chaiwattanarungruengpaisan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Induced Pluripotent Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Caspase 3 ◽  
Embryoid Body ◽  
Germ Layers ◽  
Significant Difference ◽  
Induced Pluripotent

Human induced pluripotent stem cells (iPSC) have been generated by reprogramming somatic cells using a cocktail of stem cell transcription factors but the application has been limited in transplantation therapies. The pig represents an ideal model for human clinical research, in part because of its similarity to human physiology and immunology but also because of its use in assessing side effects in long-term preclinical studies. Porcine induced pluripotent stem cells (piPSC) have been established in many studies but their differentiation pattern has not been reported. The aim of this study was to estimate the efficiency and pattern of differentiated piPSC into all 3 germ layers using embryoid body (EB) formation. Two piPSC lines (VSMUi001-A and VSMUi001-D) were induced from porcine embryonic fibroblasts by retroviral overexpression of 5 human reprogramming transcription factors (OCT4, SOX2, KLF4, c-MYC, and LIN28). For EB formation, the piPSC were harvested by treating with TrypLE™ Select (Thermo Fisher Scientific, Waltham, MA, USA) and the cells were cultured in nonadherent 96-well plates in piPSC media without growth factors. Data are expressed as mean ± SEM of at least 3 independent experiments. Statistical analyses were evaluated with Student t-tests for comparison between the 2 cell lines. Statistical significance was set at a P-value of < 0.05. The percentages of EB formation, which were calculated as the number of wells containing EB on Day 3 of differentiation, were 95.3 ± 3.42 and 89.1 ± 5.34 (VSMUi001-A and VSMUi001-D, respectively). However, there was no significant difference between the percentages of EB formation derived from the 2 cell lines. For EB size measurement, 20 EB per experiment were taken after incubation for 3, 7, 14, and 21 days. Both EB sizes increased over time (average diameter of 238.1 ± 6.18, 297.9 ± 4.10, 438.6 ± 13.33, and 728.8 ± 24.92 mm from VSMUi001-A, and 255.8 ± 5.12, 357.9 ± 3.94, 459.6 ± 11.88, and 439.4 ± 20.31 mm from VSMUi001-D). Moreover, both EB displayed homogeneity in size and shape (Day 3, 7), exhibited a cystic structure (Day 14), and a vesicular cavity was present (Day 21). For immunohistochemical analysis, both EB had lower levels of cleaved caspase 3, a marker of apoptotic cells, on Day 3 but higher levels of cleaved caspase 3 from Day 7 through 21. On the contrary, EB showed higher levels of Ki67, a marker of proliferating cells, on Day 3 but lower levels of Ki67 on Days 7, 14, and 21, respectively. In gene expression assessment, EB exhibited ectoderm gene (NeuroD1), mesoderm genes (TNNT2 and TNNI1), and endoderm genes (SOX17 and Endolase) at Day 7 and 21 by using RT-PCR. In conclusion, we report the successful in vitro formation of cystic EB from 2 piPSC lines, indicating that the piPSC could differentiate into 3 germ layers. This will allow researchers to unveil the roadmap of molecular cues needed for piPSC differentiation. This research project is supported by grants from the Mahidol University, Thailand.

scholarly journals Engineering functional human gastrointestinal organoid tissues using the three primary germ layers separately derived from pluripotent stem cells

2021 ◽  
Author(s):  
Alexandra K Eicher ◽  
Daniel O Kechele ◽  
Nambirajan Sundaram ◽  
H Matthew Berns ◽  
Holly M Poling ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Clinical Care ◽  
Regional Identity ◽  
Model Systems ◽  
Gastric Epithelium ◽  
Enteric Neurons ◽  
Patient Specific ◽  
Germ Layers

The development of human organoid model systems has provided new avenues for patient-specific clinical care and disease modeling. However, all organoid systems are missing important cell types that, in the embryo, get incorporated into organ tissues during development. Based on the concept of how embryonic organs are assembled, we developed an organoid assembly approach starting with cells from the three primary germ layers; enteric neuroglial, mesenchymal, and epithelial precursors, all separately derived from human pluripotent stem cells. From these we generated human gastric tissue containing differentiated glands, surrounded by layers of smooth muscle containing functional enteric neurons that controlled contractions of the engineered tissue. We used this highly tractable system to identify essential roles for the enteric nervous system in the growth and regional identity of the gastric epithelium and mesenchyme and for glandular morphogenesis of the antral stomach. This approach of starting with separately-derived germ layer components was applied to building more complex fundic and esophageal tissue, suggesting this as a new paradigm for tissue engineering.

285 GENERATION OF CANINE INDUCED PLURIPOTENT STEM CELLS FROM CANINE FETAL FIBROBLAST AND ADULT FIBROBLAST OF CLONED DOG

2013 ◽  
Vol 25 (1) ◽  
pp. 290 ◽  
Author(s):  
H. S. Kwon ◽  
H. J. Oh ◽  
D. H. Lee ◽  
D. E. Kim ◽  
S. K. Kang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
In Vitro Differentiation ◽  
Germ Layers ◽  
Fetal Fibroblast ◽  
Reprogramming Factors ◽  
Induced Pluripotent ◽  
Cloned Dog

Induced pluripotent stem cells (iPSC) derived from a patient’s fibroblasts have been used as fine resources for studying disease mechanisms and therapeutic strategies. The dog is considered invaluable in human disease research because its genetic diseases are strikingly similar to those of human. Therefore, we generated cloned dogs and transgenic cloned dogs via somatic cell nuclear transfer. In this study, we tried to derive canine iPSCs from canine fibroblasts to establish a way to make iPSC from skin fibroblasts of transgenic cloned dogs. We isolated canine fetal fibroblast (FF) from normal beagles and adult skin fibroblast (ASF) from cloned beagles. Both ASF and FF were infected with all-in-one retroviral vector that delivers human reprogramming factors (Oct4, Sox2, Klf4, c-Myc). Ten to twenty-one days after infection, the colony-shaped structure was picked and plated on a mouse embryonic fibroblast (MEF) feeder layer, pretreated with mitomycin C. Then, all cells were cultured with DMEM/F12 supplemented with 20% fetal bovine serum, 5 ng mL–1 basic fibroblast growth factor (bFGF), 5 ng mL–1 LIF, 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin. Alkaline phosphatase (AP) activity and expression of Oct4, Sox2, SSEA1, and SSEA4, were observed in the cells to characterise the iPS cell colonies. In vitro differentiation of 10th-passage canine iPSC was performed through embryonic body formation. About 50 canine iPS-like colonies were formed on a 100-mm dish. As a result, the canine iPSC from FF (iPSC-FF) and canine iPSC from ASF (iPSC-ASF) showed typical colony morphology, and both stained positively for AP. The expression of pluripotency-associated transcription factors Oct4 and Sox2 was positively displayed in iPSC-FF colonies. The stem cell markers SSEA1 and SSEA4 were negative in canine iPSC-FF. The canine iPS-FF spontaneously differentiated into all 3 germ layers in vitro, showing positive expressions of βIII-tubulin (ectoderm), α-SMA (mesoderm), and GATA6 (endoderm). As for iPS-ASF, characterisation and in vitro differentiation experiment are in progress. These results show that canine iPS-FF are similar to embryonic stem cells in terms of morphology and the ability to differentiate into 3 germ layers. Although we did not demonstrate complete verification of canine iPS-ASF of the cloned dog, their morphology, AP expression, and iPS-FF generation should indicate the possibility of iPSC production in the cloned dog. In conclusion, retroviral transduction of 4 human reprogramming factors can reprogram canine fetal fibroblasts into canine iPSC. The technique of producing canine iPSC will stimulate the utilisation of transgenic cloned dogs and expand the range of human diseases or therapeutic application. This study was supported by RDA (#PJ0089752012), RNL Bio (#550-20120006), IPET (#311011-05-1-SB010), Research Institute for Veterinary Science, and Nestlé Purina Korea.

GENERATION AND CHARACTERISATION OF INDUCED PLURIPOTENT STEM CELLS (iPSCs) FROM ADULT CANINE FIBROBLASTS

2012 ◽  
Vol 24 (1) ◽  
pp. 285
Author(s):  
Jorge A. Piedrahita ◽  
Sehwon Koh ◽  
Natasha Olby
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Es Cells ◽  
Ips Cells ◽  
Embryoid Bodies ◽  
Germ Layers ◽  
Induced Pluripotent ◽  
Derivatives Of

Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can give rise to derivatives of all three germ layers and thus have great potential in regenerative medicine. In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing four transcription factors, Oct3/4, Klf4, Sox2 and c-Myc (OKSM). In his presentation we will describe the derivation of iPS cells from adult canine fibroblast by retroviral OSKM transduction. The isolated canine iPS cells were expanded in three different iPS culture media (FGF2, LIF and FGF2 plus LIF) and only the cells cultured in FGF2 plus LIF showed strong AP activity expressed pluripotency markers, POU5F1 (OCT4), SOX2, NANOG and LIN28 as well as ES cells-specific genes (PODXL, DPPA5, FGF5, REX1 and LAMP1). In vitro differentiation by formation of embryoid bodies (EBs) and directed differentiation showed cell derivatives of all three germ layers as confirmed by expression for AFP, CXCR4 and SOX17 (endoderm), desmin (DES), vimentin (VIM), MSX1 and BMP2 (mesoderm) and glial fibrillary acidic protein (GFAP), TUJ1, NCAM and bIII-tubulin (TUBB, ectoderm). In vivo, the putative canine iPS cells formed simple teratomas that expressed markers for all three germ layers. In summary, we were able to derive induced pluripotent cells from adult somatic cells by using four transcription factors. The isolated canine iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF and LIF is still unknown. This work was funded by a grant from the America Kennel Club to JAP.

scholarly journals Generating Human Gastruloids from Human Embryonic Stem Cells

2020 ◽  
Author(s):  
Naomi Moris ◽  
Kerim Anlas ◽  
Julia Schroeder ◽  
Sabitri Ghimire ◽  
Tina Balayo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Convergent Extension ◽  
Germ Layers ◽  
Trilineage Differentiation ◽  
Shape Changes

Abstract Gastruloids are aggregates of Pluripotent Stem Cells (PSCs) which, when exposed to differentiation medium and plated within defined conditions, undergo trilineage differentiation to all three germ layers (mesoderm, ectoderm and endoderm) with constitutive cell types organised spatiotemporally along 3 axes (Becarri et. al. 2018). They also undergo morphological shape changes including axial elongation through convergent extension cell movements. The gastruloid method has been well-established using mouse Embryonic Stem Cells (mESCs) and mouse induced PSCs (iPSCs) (van den Brink et. al., 2014; Turner et. al. 2016a; Turner et. al. 2016b; Bailie-Johnson et. al. 2015; Turner et. al. 2017a; Turner et. al. 2017b, Beccari et. al., 2018, See method in Girgin et. al., 2018). Here, we describe a new method to generate equivalent gastruloids from human pluripotent stem cells (hPSCs). hPSCs are able to generate axially elongated human gastruloids with evidence of spatially organised germ layers and comparable features to their mouse counterparts (Moris et. al., 2020).

scholarly journals Nox4-IGF2 Axis Promotes Differentiation of Embryoid Body Cells Into Derivatives of the Three Embryonic Germ Layers

2021 ◽  
Author(s):  
Jusong Kim ◽  
Jaewon Kim ◽  
Hee Jung Lim ◽  
Sanghyuk Lee ◽  
Yun Soo Bae ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Second Messengers ◽  
Differentiation Markers ◽  
Germ Layers ◽  
Cellular Processes ◽  
Signaling Axis ◽  
Induced Pluripotent

AbstractReactive oxygen species (ROS) play important roles as second messengers in a wide array of cellular processes including differentiation of stem cells. We identified Nox4 as the major ROS-generating enzyme whose expression is induced during differentiation of embryoid body (EB) into cells of all three germ layers. The role of Nox4 was examined using induced pluripotent stem cells (iPSCs) generated from Nox4 knockout (Nox4−/−) mouse. Differentiation markers showed significantly reduced expression levels consistent with the importance of Nox4-generated ROS during this process. From transcriptomic analyses, we found insulin-like growth factor 2 (IGF2), a member of a gene family extensively involved in embryonic development, as one of the most down-regulated genes in Nox4−/− cells. Indeed, addition of IGF2 to culture partly restored the differentiation competence of Nox4−/− iPSCs. Our results reveal an important signaling axis mediated by ROS in control of crucial events during differentiation of pluripotent stem cells. Graphical Abstract

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