Development of a Personalized Intestinal Fibrosis Model Using Human Intestinal Organoids Derived From Induced Pluripotent Stem Cells

Abstract Background Intestinal fibrosis is a serious complication of Crohn’s disease. Numerous cell types including intestinal epithelial and mesenchymal cells are implicated in this process, yet studies are hampered by the lack of personalized in vitro models. Human intestinal organoids (HIOs) derived from induced pluripotent stem cells (iPSCs) contain these cell types, and our goal was to determine the feasibility of utilizing these to develop a personalized intestinal fibrosis model. Methods iPSCs from 2 control individuals and 2 very early onset inflammatory bowel disease patients with stricturing complications were obtained and directed to form HIOs. Purified populations of epithelial and mesenchymal cells were derived from HIOs, and both types were treated with the profibrogenic cytokine transforming growth factor β (TGFβ). Quantitative polymerase chain reaction and RNA sequencing analysis were used to assay their responses. Results In iPSC-derived mesenchymal cells, there was a significant increase in the expression of profibrotic genes (Col1a1, Col5a1, and TIMP1) in response to TGFβ. RNA sequencing analysis identified further profibrotic genes and demonstrated differential responses to this cytokine in each of the 4 lines. Increases in profibrotic gene expression (Col1a1, FN, TIMP1) along with genes associated with epithelial-mesenchymal transition (vimentin and N-cadherin) were observed in TGFβ -treated epithelial cells. Conclusions We demonstrate the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. This now permits the generation of near unlimited quantities of patient-specific cells that could be used to reveal cell- and environmental-specific mechanisms underpinning intestinal fibrosis.

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DEVELOPMENT OF A PERSONALIZED MODEL OF INTESTINAL FIBROSIS USING HUMAN INTESTINAL ORGANOIDS DERIVED FROM INDUCED PLURIPOTENT STEM CELLS

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa347.085 ◽

2021 ◽

Vol 27 (Supplement_1) ◽

pp. S35-S35

Author(s):

Hannah Estrada ◽

Shachi Patel ◽

Shervin Rabizadeh ◽

Stephan Targan ◽

Robert Barrett

Keyword(s):

Stem Cells ◽

Epithelial Cells ◽

Pluripotent Stem Cells ◽

Cell Types ◽

Mesenchymal Cells ◽

Intestinal Epithelial ◽

Intestinal Fibrosis ◽

Intestinal Organoids ◽

Induced Pluripotent

Abstract Background Intestinal fibrosis is a serious complication of inflammatory bowel disease (IBD) with > 20% of Crohn’s disease patients developing this complication within 10 years of diagnosis. Despite improvements in anti-inflammatory medication, its incidence remains stubbornly high and thus far surgical intervention remains the only treatment option. Numerous cell types including intestinal epithelial and mesenchymal cells are implicated in this process, yet studies are hampered by the lack of personalized in vitro models. One potential avenue that would permit a personalized approach is to utilize human intestinal organoids (HIOs) derived from induced pluripotent stem cells (iPSCs). iPSCs can be generated from any individual, faithfully recapitulate the genetics of the host and can be directed to form HIOs that contain both epithelial and mesenchymal cells. Our goal was to determine the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. Methods iPSCs from two control individuals and two very early onset-IBD (VEOIBD) patients with stricturing complications were obtained and directed to form HIOs. Given HIOs are heterogeneous in terms of size, shape and ratio of mesenchymal to epithelial cells, they were firstly dissociated to a single cell suspension and EpCAM was used to positively select for epithelial cells using magnetic activated cellular sorting. These EpCAM+ cells were then seeded onto transwells and EpCAM- cells were seeded as monolayers in 10% serum containing media. Both cell types were treated with the profibrotic cytokine TGFβ, and changes in the expression of selected genes were analyzed. Results iPSCs from all 4 individuals could be directed to form HIOs containing both epithelial (E-cadherin+) and mesenchymal (vimentin+) cells (see Fig. 1). In the TGFβ-treated mesenchymal cell population, expression of N-cadherin and Col1a1 was significantly increased in all four lines after 8 and 48hrs respectively, with the highest increase occurring in cells derived from VEOIBD patient 2 (see Table 1). In the TGFβ-treated epithelial cell population, Col1a1 and fibronectin expression was increased in all lines after 96hrs with the highest fold change occurring in cells derived from VEOIBD patient 1 (fibronectin) and 2 (Col1a1). Conclusion We demonstrate the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. We show that iPSCs generated from all selected individuals could be directed to form HIOs and that responses to the profibrotic cytokine TGFβ can be examined in both intestinal epithelial and mesenchymal cells. This now permits the generation of near unlimited quantities of patient specific cells that could be used to reveal cell and environmental specific mechanisms underpinning intestinal fibrosis which may ultimately lead to personalized treatments. Fluorescent images of human intestinal organoids generated from A) Control 1, B) Control 2, C) VEOIBD patient 1 and D) VEOIBD patient 2 that were immunostained with vimentin (green), E-cadherin (red) and counterstainied with DAPI (blue). All images X20

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Utilising Induced Pluripotent Stem Cells in Neurodegenerative Disease Research: Focus on Glia

International Journal of Molecular Sciences ◽

10.3390/ijms22094334 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4334

Author(s):

Katrina Albert ◽

Jonna Niskanen ◽

Sara Kälvälä ◽

Šárka Lehtonen

Keyword(s):

Stem Cells ◽

Neurodegenerative Diseases ◽

Neurodegenerative Disease ◽

Induced Pluripotent Stem Cells ◽

Glial Cells ◽

Pluripotent Stem Cells ◽

Cell Types ◽

Human Ipscs ◽

Induced Pluripotent

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism’s somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer’s disease and Parkinson’s disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.

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Generation of Sheep Induced Pluripotent Stem Cells With Defined DOX-Inducible Transcription Factors via piggyBac Transposition

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.785055 ◽

2021 ◽

Vol 9 ◽

Author(s):

Moning Liu ◽

Lixia Zhao ◽

Zixin Wang ◽

Hong Su ◽

Tong Wang ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Cell Types ◽

T Antigen ◽

Sv40 Large T Antigen ◽

Adult Individual ◽

Induced Pluripotent

Pluripotent stem cells (PSCs) have the potential to differentiate to all cell types of an adult individual and are useful for studying mammalian development. Establishing induced pluripotent stem cells (iPSCs) capable of expressing pluripotent genes and differentiating to three germ layers will not only help to explain the mechanisms underlying somatic reprogramming but also lay the foundation for the establishment of sheep embryonic stem cells (ESCs) in vitro. In this study, sheep somatic cells were reprogrammed in vitro into sheep iPSCs with stable morphology, pluripotent marker expression, and differentiation ability, delivered by piggyBac transposon system with eight doxycycline (DOX)-inducible exogenous reprogramming factors: bovine OCT4, SOX2, KLF4, cMYC, porcine NANOG, human LIN28, SV40 large T antigen, and human TERT. Sheep iPSCs exhibited a chimeric contribution to the early blastocysts of sheep and mice and E6.5 mouse embryos in vitro. A transcriptome analysis revealed the pluripotent characteristics of somatic reprogramming and insights into sheep iPSCs. This study provides an ideal experimental material for further study of the construction of totipotent ESCs in sheep.

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Using induced pluripotent stem cells to understand retinal ciliopathy disease mechanisms and develop therapies

Biochemical Society Transactions ◽

10.1042/bst20160156 ◽

2016 ◽

Vol 44 (5) ◽

pp. 1245-1251 ◽

Cited By ~ 14

Author(s):

David A. Parfitt ◽

Amelia Lane ◽

Conor Ramsden ◽

Katarina Jovanovic ◽

Peter J. Coffey ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Retinal Degeneration ◽

Pluripotent Stem Cells ◽

Photoreceptor Cells ◽

Cell Types ◽

Disease Mechanisms ◽

Wide Range ◽

Induced Pluripotent

The photoreceptor cells in the retina have a highly specialised sensory cilium, the outer segment (OS), which is important for detecting light. Mutations in cilia-related genes often result in retinal degeneration. The ability to reprogramme human cells into induced pluripotent stem cells and then differentiate them into a wide range of different cell types has revolutionised our ability to study human disease. To date, however, the challenge of producing fully differentiated photoreceptors in vitro has limited the application of this technology in studying retinal degeneration. In this review, we will discuss recent advances in stem cell technology and photoreceptor differentiation. In particular, the development of photoreceptors with rudimentary OS that can be used to understand disease mechanisms and as an important model to test potential new therapies for inherited retinal ciliopathies.

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Comparison analysis of Bacillus Calmette-Guerin induced Autophagy mechanisms in Macrophages Derived from Human Induced Pluripotent Stem Cells and THP-1

10.21203/rs.3.rs-26931/v1 ◽

2020 ◽

Author(s):

Mengyi Zhu ◽

Danping Hong ◽

Ouyang Li ◽

Ahmed Salah Hassan ◽

Yanqin Li ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Public Health Problem ◽

Sequencing Analysis ◽

Bacillus Calmette Guerin ◽

Submicroscopic Structure ◽

Induced Pluripotent

Abstract Background Tuberculosis (TB) remains a major global public health problem and the leading cause of mortality by a single infectious agent. TB is a chronic infectious disease that is primarily caused by Mycobacterium tuberculosis (Mtb). Macrophage (Mφ) are the main hosts of Mtb, the interaction between Mtb and Mφ plays an important role in the pathogenesis of TB.Summary The macrophages used in the current study are mostly derived from tumor cell lines or peripheral blood mononuclear cells (PBMC), but the application of such cells still have many problems needed to be sloved, such as the loss of function due to changes in genetic structure and the difficulty in cell acquisition. Human induced pluripotent stem cells (hiPS) represent an innovative source for the standardized in vitro generation of Mφ, and show novel promise in exploring disease pathogenesis, particularly TB. Current studies have revealed that autophagy plays a central role in the interaction between Mtb and Mφ, but the molecular mechanism involoved remains unclear and the exact role of hiPS-derived macrophages (hiPS-Mφ) in regulating autophagy induced by Mtb also remains unclear. To investigate the similarities and differences in hiPS-Mφ and THP-1-Mφ in anti-tuberculosis immunity, this study successfully obtained macrophages derived from hiPS and THP-1, then explored the mechanism behind Bacillus Calmette-Guerin (BCG)-induced autophagy through transcriptome sequencing analysis, qPCR, Western Blot Analysis and cell submicroscopic structure observation etc.. Our findings revealed that BCG infection of hiPS-Mφ and THP-1-Mφ would promote autophagy by regulating the expression of autophagy-related genes, which also indicated that the BCG-induced autophagy in hiPS-Mφ and THP-1-Mφ may be associated with PI3K/AKT/mTOR signaling pathway. However, there are some differences in the mechanism by which BCG infects macrophages from different sources and induces autophagy. Considering the above findings, we have provided novel insights into the role of macrophages along with autophagy in the anti-tuberculosis immune mechanism and the possibility of establishing an in vitro hiPS-Mφ-TB disease model.

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In vitro pathological modelling using patient-specific induced pluripotent stem cells: the case of progeria

Biochemical Society Transactions ◽

10.1042/bst20110659 ◽

2011 ◽

Vol 39 (6) ◽

pp. 1775-1779 ◽

Cited By ~ 11

Author(s):

Xavier Nissan ◽

Sophie Blondel ◽

Marc Peschanski

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Systemic Disease ◽

Accelerated Aging ◽

Cell Types ◽

Patient Specific ◽

Cellular Mechanisms ◽

Induced Pluripotent

Progeria, also known as HGPS (Hutchinson–Gilford progeria syndrome), is a rare fatal genetic disease characterized by an appearance of accelerated aging in children. This syndrome is typically caused by mutations in codon 608 (C1804T) of the gene encoding lamins A and C, LMNA, leading to the production of a truncated form of the protein called progerin. Owing to their unique potential to self-renew and to differentiate into any cell types of the organism, pluripotent stem cells offer a unique tool to study molecular and cellular mechanisms related to this global and systemic disease. Recent studies have exploited this potential by generating human induced pluripotent stem cells from HGPS patients' fibroblasts displaying several phenotypic defects characteristic of HGPS such as nuclear abnormalities, progerin expression, altered DNA-repair mechanisms and premature senescence. Altogether, these findings provide new insights on the use of pluripotent stem cells for pathological modelling and may open original therapeutic perspectives for diseases that lack pre-clinical in vitro human models, such as HGPS.

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Induced pluripotent Stem Cells: Where we are currently?

Naucni casopis urgentne medicine - Halo 194 ◽

10.5937/halo26-27861 ◽

2020 ◽

Vol 26 (3) ◽

pp. 153-161

Author(s):

Nemanja Rančić ◽

Sanja Raščanin ◽

Milijana Miljković ◽

Mirjana Jovanović

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Drug Testing ◽

Embryonic Stem ◽

Cell Types ◽

Cell Genome ◽

Induced Pluripotent ◽

Tissue Rejection

Induced Pluripotent Stem Cells (iPSCs) are a type of pluripotent stem cells generated by reprogramming an adult somatic cell genome to the stage of a pluripotent stem cell in vitro by inducing a forced expression of specific transcription factors that are important for the maintenance of pluripotency. The iPSCs seem to be very similar to Embryonic Stem Cells (ESCs) in terms of morphology, cell surface markers and gene expression levels, but recent studies have demonstrated some differences between the two cell types. However, iPSCs might have potential application in regenerative medicine, transplantation, drug testing, disease modelling, and avoidance of tissue rejection and with less ethical concern than ESCs. This paper aims to present the most important characteristics of iPSCs which have therapeutic significance.

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