An Efficient Method for the Differentiation of Human iPSC-Derived Endoderm toward Enterocytes and Hepatocytes

The endoderm, differentiated from human induced pluripotent stem cells (iPSCs), can differentiate into the small intestine and liver, which are vital for drug absorption and metabolism. The development of human iPSC-derived enterocytes (HiEnts) and hepatocytes (HiHeps) has been reported. However, pharmacokinetic function-deficiency of these cells remains to be elucidated. Here, we aimed to develop an efficient differentiation method to induce endoderm formation from human iPSCs. Cells treated with activin A for 168 h expressed higher levels of endodermal genes than those treated for 72 h. Using activin A (days 0–7), CHIR99021 and PI−103 (days 0–2), and FGF2 (days 3–7), the hiPSC-derived endoderm (HiEnd) showed 97.97% CD−117 and CD−184 double-positive cells. Moreover, HiEnts derived from the human iPSC line Windy had similar or higher expression of small intestine-specific genes than adult human small intestine. Activities of the drug transporter P-glycoprotein and drug-metabolizing enzyme cytochrome P450 (CYP) 3A4/5 were confirmed. Additionally, Windy-derived HiHeps expressed higher levels of hepatocyte- and pharmacokinetics-related genes and proteins and showed higher CYP3A4/5 activity than those derived through the conventional differentiation method. Thus, using this novel method, the differentiated HiEnts and HiHeps with pharmacokinetic functions could be used for drug development.

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Generation of tetracycline-controllable CYP3A4-expressing Caco-2 cells by the piggyBac transposon system

Scientific Reports ◽

10.1038/s41598-021-91160-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Moe Ichikawa ◽

Hiroki Akamine ◽

Michika Murata ◽

Sumito Ito ◽

Kazuo Takayama ◽

...

Keyword(s):

Small Intestine ◽

Drug Absorption ◽

Cell Model ◽

Negative Effects ◽

Piggybac Transposon ◽

Drug Metabolizing Enzyme ◽

Metabolizing Enzyme ◽

Cyp3a4 Expression

AbstractCaco-2 cells are widely used as an in vitro intestinal epithelial cell model because they can form a monolayer and predict drug absorption with high accuracy. However, Caco-2 cells hardly express cytochrome P450 (CYP), a drug-metabolizing enzyme. It is known that CYP3A4 is the dominant drug-metabolizing enzyme in human small intestine. In this study, we generated CYP3A4-expressing Caco-2 (CYP3A4-Caco-2) cells and attempted to establish a model that can simultaneously evaluate drug absorption and metabolism. CYP3A4-Caco-2 cells were generated by piggyBac transposon vectors. A tetracycline-controllable CYP3A4 expression cassette (tet-on system) was stably transduced into Caco-2 cells, thus regulating the levels of CYP3A4 expression depending on the doxycycline concentration. The CYP3A4 expression levels in CYP3A4-Caco-2 cells cultured in the presence of doxycycline were similar to or higher than those of adult small intestine. The CYP3A4-Caco-2 cells had enough ability to metabolize midazolam, a substrate of CYP3A4. CYP3A4 overexpression had no negative effects on cell proliferation, barrier function, and P-glycoprotein activity in Caco-2 cells. Thus, we succeeded in establishing Caco-2 cells with CYP3A4 metabolizing activity comparable to in vivo human intestinal tissue. This cell line would be useful in pharmaceutical studies as a model that can simultaneously evaluate drug absorption and metabolism.

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Differentiation of human induced pluripotent stem cells into erythroid cells

Stem Cell Research & Therapy ◽

10.1186/s13287-020-01998-9 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Mohsen Ebrahimi ◽

Mehdi Forouzesh ◽

Setareh Raoufi ◽

Mohammad Ramazii ◽

Farhoodeh Ghaedrahmati ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Promising Alternative ◽

Human Ipscs ◽

Induced Pluripotent ◽

Human Ipsc

AbstractDuring the last years, several strategies have been made to obtain mature erythrocytes or red blood cells (RBC) from the bone marrow or umbilical cord blood (UCB). However, UCB-derived hematopoietic stem cells (HSC) are a limited source and in vitro large-scale expansion of RBC from HSC remains problematic. One promising alternative can be human pluripotent stem cells (PSCs) that provide an unlimited source of cells. Human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are self-renewing progenitors that can be differentiated to lineages of ectoderm, mesoderm, and endoderm. Several previous studies have revealed that human ESCs can differentiate into functional oxygen-carrying erythrocytes; however, the ex vivo expansion of human ESC-derived RBC is subjected to ethical concerns. Human iPSCs can be a suitable therapeutic choice for the in vitro/ex vivo manufacture of RBCs. Reprogramming of human somatic cells through the ectopic expression of the transcription factors (OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG) has provided a new avenue for disease modeling and regenerative medicine. Various techniques have been developed to generate enucleated RBCs from human iPSCs. The in vitro production of human iPSC-derived RBCs can be an alternative treatment option for patients with blood disorders. In this review, we focused on the generation of human iPSC-derived erythrocytes to present an overview of the current status and applications of this field.

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Human Induced Pluripotent Stem-Cell-Derived Cardiomyocytes as Models for Genetic Cardiomyopathies

International Journal of Molecular Sciences ◽

10.3390/ijms20184381 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4381 ◽

Cited By ~ 10

Author(s):

Andreas Brodehl ◽

Hans Ebbinghaus ◽

Marcus-André Deutsch ◽

Jan Gummert ◽

Anna Gärtner ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Promising Alternative ◽

Human Ipscs ◽

Genetic Landscape ◽

Inherited Cardiomyopathies ◽

Induced Pluripotent ◽

Human Ipsc

In the last few decades, many pathogenic or likely pathogenic genetic mutations in over hundred different genes have been described for non-ischemic, genetic cardiomyopathies. However, the functional knowledge about most of these mutations is still limited because the generation of adequate animal models is time-consuming and challenging. Therefore, human induced pluripotent stem cells (iPSCs) carrying specific cardiomyopathy-associated mutations are a promising alternative. Since the original discovery that pluripotency can be artificially induced by the expression of different transcription factors, various patient-specific-induced pluripotent stem cell lines have been generated to model non-ischemic, genetic cardiomyopathies in vitro. In this review, we describe the genetic landscape of non-ischemic, genetic cardiomyopathies and give an overview about different human iPSC lines, which have been developed for the disease modeling of inherited cardiomyopathies. We summarize different methods and protocols for the general differentiation of human iPSCs into cardiomyocytes. In addition, we describe methods and technologies to investigate functionally human iPSC-derived cardiomyocytes. Furthermore, we summarize novel genome editing approaches for the genetic manipulation of human iPSCs. This review provides an overview about the genetic landscape of inherited cardiomyopathies with a focus on iPSC technology, which might be of interest for clinicians and basic scientists interested in genetic cardiomyopathies.

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EXPRESSION PROFILES OF DRUG-METABOLIZING ENZYME CYP3A AND DRUG EFFLUX TRANSPORTER MULTIDRUG RESISTANCE 1 SUBFAMILY mRNAS IN RAT SMALL INTESTINE

Drug Metabolism and Disposition ◽

10.1124/dmd.31.10.1235 ◽

2003 ◽

Vol 31 (10) ◽

pp. 1235-1239 ◽

Cited By ~ 59

Author(s):

Kohji Takara ◽

Noriaki Ohnishi ◽

Sayo Horibe ◽

Teruyoshi Yokoyama

Keyword(s):

Small Intestine ◽

Multidrug Resistance ◽

Expression Profiles ◽

Drug Efflux ◽

Rat Small Intestine ◽

Efflux Transporter ◽

Drug Metabolizing Enzyme ◽

Metabolizing Enzyme

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Drug metabolizing enzyme and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells

Biochemical Pharmacology ◽

10.1016/j.bcp.2013.06.029 ◽

2013 ◽

Vol 86 (5) ◽

pp. 691-702 ◽

Cited By ~ 68

Author(s):

Maria Ulvestad ◽

Pär Nordell ◽

Annika Asplund ◽

Marie Rehnström ◽

Susanna Jacobsson ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Protein Profiles ◽

Transporter Protein ◽

Drug Metabolizing Enzyme ◽

Induced Pluripotent ◽

Metabolizing Enzyme

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Human iPSC banking: barriers and opportunities

Journal of Biomedical Science ◽

10.1186/s12929-019-0578-x ◽

2019 ◽

Vol 26 (1) ◽

Cited By ~ 26

Author(s):

Ching-Ying Huang ◽

Chun-Lin Liu ◽

Chien-Yu Ting ◽

Yueh-Ting Chiu ◽

Yu-Che Cheng ◽

...

Keyword(s):

Pluripotent Stem Cells ◽

Disease Modeling ◽

Toxicity Assessment ◽

Cell Therapies ◽

The Past ◽

Human Ipscs ◽

Ongoing Development ◽

Barriers And Opportunities ◽

Induced Pluripotent ◽

Human Ipsc

Abstract The introduction of induced pluripotent stem cells (iPSCs) has opened up the potential for personalized cell therapies and ushered in new opportunities for regenerative medicine, disease modeling, iPSC-based drug discovery and toxicity assessment. Over the past 10 years, several initiatives have been established that aim to collect and generate a large amount of human iPSCs for scientific research purposes. In this review, we compare the construction and operation strategy of some iPSC banks as well as their ongoing development. We also introduce the technical challenges and offer future perspectives pertaining to the establishment and management of iPSC banks.

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Human iPSCs-Derived Endothelial Cells with Mutation in HNF1A as a Model of Maturity-Onset Diabetes of the Young

Cells ◽

10.3390/cells8111440 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1440 ◽

Cited By ~ 2

Author(s):

Kachamakova-Trojanowska ◽

Stepniewski ◽

Dulak

Keyword(s):

Endothelial Cells ◽

Microvascular Complications ◽

Maturity Onset Diabetes ◽

Isogenic Lines ◽

Onset Diabetes ◽

Human Ipscs ◽

Induced Pluripotent ◽

Biallelic Mutations ◽

Human Ipsc ◽

The Impact

Patients with HNF1A-maturity-onset diabetes of the young (MODY) often develop endothelial dysfunction and related microvascular complications, like retinopathy. As the clinical phenotype of HNF1A-MODY diabetes varies considerably, we used human induced pluripotent stem cells (hiPSCs) from two healthy individuals (control) to generate isogenic lines with mutation in HNF1A gene. Subsequently, control hiPSCs and their respective HNF1A clones were differentiated toward endothelial cells (hiPSC-ECs) and different markers/functions were compared. Human iPSC-ECs from all cell lines showed similar expression of CD31 and Tie-2. VE-cadherin expression was lower in HNF1A-mutated isogenic lines, but only in clones derived from one control hiPSCs. In the other isogenic set and cells derived from HNF1A-MODY patients, no difference in VE-cadherin expression was observed, suggesting the impact of the genetic background on this endothelial marker. All tested hiPSC-ECs showed an expected angiogenic response regardless of the mutation introduced. Isogenic hiPSC-ECs responded similarly to stimulation with pro-inflammatory cytokine TNF- with the increase in ICAM-1 and permeability, however, HNF1A mutated hiPSC-ECs showed higher permeability in comparison to the control cells. Summarizing, both mono- and biallelic mutations of HNF1A in hiPSC-ECs lead to increased permeability in response to TNF- in normal glycemic conditions, which may have relevance to HNF1A-MODY microvascular complications.

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In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells

Blood ◽

10.1182/blood-2012-06-434407 ◽

2013 ◽

Vol 121 (8) ◽

pp. 1255-1264 ◽

Cited By ~ 136

Author(s):

Giovanni Amabile ◽

Robert S. Welner ◽

Cesar Nombela-Arrieta ◽

Anna Morena D'Alise ◽

Annalisa Di Ruscio ◽

...

Keyword(s):

Stem Cells ◽

Stromal Cells ◽

Pluripotent Stem Cells ◽

Hematopoietic Cells ◽

Immunodeficient Mice ◽

Human Ipscs ◽

Key Points ◽

Induced Pluripotent ◽

Human Ipsc

Key Points Human hematopoietic cells develop within human iPSC-derived teratomas in immunodeficient mice. Co-transplantation of OP9 stromal cells along with human iPSCs increases hematopoietic specification within teratomas.

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13. A Novel Method of Culturing Murine Induced Pluripotent Stem Cells (iPSCs)

Inquiry@Queen's Undergraduate Research Conference Proceedings ◽

10.24908/iqurcp.8805 ◽

2016 ◽

Author(s):

Kirsten Sjonnesen

Keyword(s):

Stem Cells ◽

Large Scale ◽

Embryonic Stem ◽

Cell Lineages ◽

Human Ipscs ◽

Novel Method ◽

Vitro Model ◽

Induced Pluripotent

Similar to embryonic stem cells (ESCs), iPSCs have the ability to differentiate into all three cell lineages, while not being subjected to the ethical complications attributed to ESCs. Our project goal was to develop a novel method of culturing murine iPSCs, reprogrammed from mouse embryonic fibroblasts, in large-scale quantities while maintaining their pluripotent characteristics and genomic integrity over a long term period. Stirred Suspension Bioreactors (SSBs) propose several benefits over static culture systems and facilitate the large-scale, economical expansion required for clinical studies. In our work with the SSB system, various techniques were used to promote and analyze the continued pluripotency of the murine iPSCs. Our results showed that iPSCs maintained their pluripotent state in SSBs and retained their ability to differentiate into different cell lineages after long term culture. miPSC were induced to differentiate into heart, bone and cartilage tissues at the end of maintenance period. The cumulative cell-fold expansion of iPSCs in suspension culture was 8.8×1010 cells, with 75% mean pluripotency at each passage. The cells expressed the major pluripotency markers as detected by RT-PCR. This study confirms the utility of the SSB system as a tool to cultivate large quantities of functional mouse iPSCs. SSBs have tremendous potential in future clinical applications and work has now begun to applying this system towards culturing human iPSCs. One potential application is the derivation of disease and patient derived iPSCs, which can recapitulate the disease phenotype for use as an in vitro model.

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