Hypoxia as a Driving Force of Pluripotent Stem Cell Reprogramming and Differentiation to Endothelial Cells

Inadequate supply of oxygen (O2) is a hallmark of many diseases, in particular those related to the cardiovascular system. On the other hand, tissue hypoxia is an important factor regulating (normal) embryogenesis and differentiation of stem cells at the early stages of embryonic development. In culture, hypoxic conditions may facilitate the derivation of embryonic stem cells (ESCs) and the generation of induced pluripotent stem cells (iPSCs), which may serve as a valuable tool for disease modeling. Endothelial cells (ECs), multifunctional components of vascular structures, may be obtained from iPSCs and subsequently used in various (hypoxia-related) disease models to investigate vascular dysfunctions. Although iPSC-ECs demonstrated functionality in vitro and in vivo, ongoing studies are conducted to increase the efficiency of differentiation and to establish the most productive protocols for the application of patient-derived cells in clinics. In this review, we highlight recent discoveries on the role of hypoxia in the derivation of ESCs and the generation of iPSCs. We also summarize the existing protocols of hypoxia-driven differentiation of iPSCs toward ECs and discuss their possible applications in disease modeling and treatment of hypoxia-related disorders.

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Abstract P482: Elucidating And Characterizing The Molecular Mechanistic Role Of Phospholamban L39 Stop In The Pathophysiology Of Cardiomyopathy Using Patient-derived Human Induced Pluripotent Stem Cells And Humanized Knock-in Mouse Model Systems

Circulation Research ◽

10.1161/res.129.suppl_1.p482 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Anichavezhi Devendran ◽

Rasheed Bailey ◽

Sumanta Kar ◽

Francesca Stillitano ◽

Irene Turnbull ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Mononuclear Cells ◽

Embryonic Stem ◽

Model Systems ◽

Patient Specific ◽

Induced Pluripotent

Background: Heart failure (HF) is a complex clinical condition associated with substantial morbidity and mortality worldwide. The contractile dysfunction and arrhythmogenesis related to HF has been linked to the remodelling of calcium (Ca ++ ) handling. Phospholamban (PLN) has emerged as a key regulator of intracellular Ca ++ concentration. Of the PLN mutations, L39X is intriguing as it has not been fully characterized. This mutation is believed to be functionally equivalent to PLN null (KO) but contrary to PLN KO mice, L39X carriers develop a lethal cardiomyopathy (CMP). Our study aims at using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from homozygous L39X carriers to elucidate the role of L39X in human pathophysiology. Our plan also involves the characterization of humanized L39X knock-in mice (KM), which we hypothesize will develop a CMP from mis-localization of PLN and disruption of Ca ++ signalling. Methodology and Results: Mononuclear cells from Hom L39X carriers were obtained to generate 11 integration-free patient-specific iPSC clones. The iPSC-CMs were derived using established protocols. Compared to the WT iPSC-CMs, the Hom L39X derived-CMs PLN had an abnormal cytoplasmic distribution and formed intracellular aggregates, with the loss of perinuclear localization. There was also a 70% and 50% reduction of mRNA and protein expression of PLN respectively in L39X compared to WT iPSC-CMs. These findings indicated that L39X PLN is both under-expressed and mis-localized within the cell. To validate this observation in-vivo, we genetically modified FVB mice to harbour the human L39X. Following electroporation, positively transfected mouse embryonic stem cells were injected into host blastocysts to make humanized KM that were subsequently used to generate either a protamine-Cre (endogenous PLN driven expression) or a cardiac TNT mouse (i.e., CMP specific). Conclusion: Our data confirm an abnormal intracellular distribution of PLN, with the loss of perinuclear accumulation and mis-localization, suggestive of ineffective targeting to or retention of L39X. The mouse model will be critically important to validate the in-vitro observations and provides an ideal platform for future studies centred on the development of novel therapeutic strategies including virally delivered CRISPR/Cas9 for in-vivo gene editing and testing of biochemical signalling pathways.

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Liver Organoids: Updates on Disease Modeling and Biomedical Applications

Biology ◽

10.3390/biology10090835 ◽

2021 ◽

Vol 10 (9) ◽

pp. 835

Author(s):

Carmen Caiazza ◽

Silvia Parisi ◽

Massimiliano Caiazzo

Keyword(s):

Stem Cells ◽

Disease Modeling ◽

Embryonic Stem ◽

Low Complexity ◽

In Vivo Models ◽

Innovative Tool ◽

Induced Pluripotent ◽

Liver Organoids

Liver organoids are stem cell-derived 3D structures that are generated by liver differentiation signals in the presence of a supporting extracellular matrix. Liver organoids overcome low complexity grade of bidimensional culture and high costs of in vivo models thus representing a turning point for studying liver disease modeling. Liver organoids can be established from different sources as induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hepatoblasts and tissue-derived cells. This novel in vitro system represents an innovative tool to deeper understand the physiology and pathological mechanisms affecting the liver. In this review, we discuss the current advances in the field focusing on their application in modeling diseases, regenerative medicine and drug discovery.

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Opportunities for Antibody Discovery Using Human Pluripotent Stem Cells: Conservation of Oncofetal Targets

International Journal of Molecular Sciences ◽

10.3390/ijms20225752 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5752 ◽

Cited By ~ 1

Author(s):

Heng Liang Tan ◽

Andre Choo

Keyword(s):

Stem Cells ◽

Cancer Cells ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Embryonic Stem ◽

Oncofetal Antigens ◽

Antibody Discovery ◽

Induced Pluripotent ◽

New Biomarkers

Pluripotent stem cells (PSCs) comprise both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The application of pluripotent stem cells is divided into four main areas, namely: (i) regenerative therapy, (ii) the study and understanding of developmental biology, (iii) drug screening and toxicology and (iv) disease modeling. In this review, we describe a new opportunity for PSCs, the discovery of new biomarkers and generating antibodies against these biomarkers. PSCs are good sources of immunogen for raising monoclonal antibodies (mAbs) because of the conservation of oncofetal antigens between PSCs and cancer cells. Hence mAbs generated using PSCs can potentially be applied in two different fields. First, these mAbs can be used in regenerative cell therapy to characterize the PSCs. In addition, the mAbs can be used to separate or eliminate contaminating or residual undifferentiated PSCs from the differentiated cell product. This step is critical as undifferentiated PSCs can form teratomas in vivo. The mAbs generated against PSCs can also be used in the field of oncology. Here, novel targets can be identified and the mAbs developed as targeted therapy to kill the cancer cells. Conversely, as new and novel oncofetal biomarkers are discovered on PSCs, cancer mAbs that are already approved by the FDA can be repurposed for regenerative medicine, thus expediting the route to the clinics.

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Application of biomaterials to in vitro pluripotent stem cell disease modeling of the skeletal system

Journal of Materials Chemistry B ◽

10.1039/c5tb02645h ◽

2016 ◽

Vol 4 (20) ◽

pp. 3482-3489 ◽

Cited By ~ 6

Author(s):

Giuliana E. Salazar-Noratto ◽

Frank P. Barry ◽

Robert E. Guldberg

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Genetic Manipulation ◽

Embryonic Stem ◽

Cell Disease ◽

Skeletal System ◽

System P ◽

Induced Pluripotent

Disease-specific pluripotent stem cells can be derived through genetic manipulation of embryonic stem cells or by reprogramming somatic cells (induced pluripotent stem cells).

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MicroRNAs: Crucial Players in the Differentiation of Human Pluripotent and Multipotent Stem Cells into Functional Hepatocyte-Like Cells

Current Stem Cell Research & Therapy ◽

10.2174/1574888x16666211006102039 ◽

2021 ◽

Vol 16 ◽

Author(s):

Hao Xu ◽

Liying Wu ◽

Guojia Yuan ◽

Xiaolu Liang ◽

Xiaoguang Liu ◽

...

Keyword(s):

Stem Cells ◽

Liver Disease ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Critical Role ◽

Ectopic Expression ◽

Embryonic Stem ◽

The Body

: Hepatic disease negatively impacts liver function and metabolism. Primary human hepatocytes are the gold standard for the prediction and successful treatment of liver disease. However, the sources of hepatocytes for drug toxicity testing and disease modeling are limited. To overcome this issue, pluripotent stem cells (PSCs) have emerged as an alternative strategy for liver disease therapy. Human PSCs, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can self-renew and give rise to all cells of the body. Human PSCs are attractive cell sources for regenerative medicine, tissue engineering, drug discovery, and developmental studies. Several recent studies have shown that mesenchymal stem cells (MSCs) can also differentiate (or trans-differentiate) into hepatocytes. Differentiation of human PSCs and MSCs into functional hepatocyte-like cells (HLCs) opens new strategies to study genetic diseases, hepatotoxicity, infection of hepatotropic viruses, and analyze hepatic biology. Numerous in vitro and in vivo differentiation protocols have been established to obtain human PSCs/MSCs-derived HLCs and mimic their characteristics. It was recently discovered that microRNAs (miRNAs) play a critical role in controlling the ectopic expression of transcription factors and governing the hepatocyte differentiation of human PSCs and MSCs. In this review, we focused on the role of miRNAs in the differentiation of human PSCs and MSCs into hepatocytes.

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Identification of Molecular Signatures in Neural Differentiation and Neurological Diseases Using Digital Color-Coded Molecular Barcoding

Stem Cells International ◽

10.1155/2020/8852313 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Debora Salerno ◽

Alessandro Rosa

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Neural Differentiation ◽

Disease Modeling ◽

Neurological Diseases ◽

Embryonic Stem ◽

Molecular Signatures ◽

Molecular Barcoding ◽

Induced Pluripotent

Human pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells, represent powerful tools for disease modeling and for therapeutic applications. PSCs are particularly useful for the study of development and diseases of the nervous system. However, generating in vitro models that recapitulate the architecture and the full variety of subtypes of cells that make the complexity of our brain remains a challenge. In order to fully exploit the potential of PSCs, advanced methods that facilitate the identification of molecular signatures in neural differentiation and neurological diseases are highly demanded. Here, we review the literature on the development and application of digital color-coded molecular barcoding as a potential tool for standardizing PSC research and applications in neuroscience. We will also describe relevant examples of the use of this technique for the characterization of the heterogeneous composition of the brain tumor glioblastoma multiforme.

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OncogenicPIK3CApromotes cellular stemness in an allele dose-dependent manner

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1821093116 ◽

2019 ◽

Vol 116 (17) ◽

pp. 8380-8389 ◽

Cited By ~ 16

Author(s):

Ralitsa R. Madsen ◽

Rachel G. Knox ◽

Wayne Pearce ◽

Saioa Lopez ◽

Betania Mahler-Araujo ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Dependent Manner ◽

Developmental Context ◽

Epithelial Morphology ◽

Pi3k Signaling Pathway ◽

Induced Pluripotent

ThePIK3CAgene, which encodes the p110α catalytic subunit of PI3 kinase (PI3K), is mutationally activated in cancer and in overgrowth disorders known asPIK3CA-related overgrowth spectrum (PROS). To determine the consequences of geneticPIK3CAactivation in a developmental context of relevance to both PROS and cancer, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous knockin ofPIK3CAH1047R. While heterozygous iPSCs remained largely similar to wild-type cells, homozygosity forPIK3CAH1047Rcaused widespread, cancer-like transcriptional remodeling, partial loss of epithelial morphology, up-regulation of stemness markers, and impaired differentiation to all three germ layers in vitro and in vivo. Genetic analysis ofPIK3CA-associated cancers revealed that 64% had multiple oncogenicPIK3CAcopies (39%) or additional PI3K signaling pathway-activating “hits” (25%). This contrasts with the prevailing view thatPIK3CAmutations occur heterozygously in cancer. Our findings suggest that a PI3K activity threshold determines pathological consequences of oncogenicPIK3CAactivation and provide insight into the specific role of this pathway in human pluripotent stem cells.

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Induced Pluripotent Stem Cells as Vasculature Forming Entities

Journal of Clinical Medicine ◽

10.3390/jcm8111782 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1782 ◽

Cited By ~ 4

Author(s):

Antonio Palladino ◽

Isabella Mavaro ◽

Carmela Pizzoleo ◽

Elena De Felice ◽

Carla Lucini ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Regenerative Medicine ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Tissue Growth ◽

Safety Issues ◽

Induced Pluripotent

Tissue engineering (TE) pursues the ambitious goal to heal damaged tissues. One of the most successful TE approaches relies on the use of scaffolds specifically designed and fabricated to promote tissue growth. During regeneration the guidance of biological events may be essential to sustain vasculature neoformation inside the engineered scaffold. In this context, one of the most effective strategies includes the incorporation of vasculature forming cells, namely endothelial cells (EC), into engineered constructs. However, the most common EC sources currently available, intended as primary cells, are affected by several limitations that make them inappropriate to personalized medicine. Human induced Pluripotent Stem Cells (hiPSC), since the time of their discovery, represent an unprecedented opportunity for regenerative medicine applications. Unfortunately, human induced Pluripotent Stem Cells-Endothelial Cells (hiPSC-ECs) still display significant safety issues. In this work, we reviewed the most effective protocols to induce pluripotency, to generate cells displaying the endothelial phenotype and to perform an efficient and safe cell selection. We also provide noteworthy examples of both in vitro and in vivo applications of hiPSC-ECs in order to highlight their ability to form functional blood vessels. In conclusion, we propose hiPSC-ECs as the preferred source of endothelial cells currently available in the field of personalized regenerative medicine.

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Development and Application of Endothelial Cells Derived From Pluripotent Stem Cells in Microphysiological Systems Models

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.625016 ◽

2021 ◽

Vol 8 ◽

Author(s):

Crystal C. Kennedy ◽

Erin E. Brown ◽

Nadia O. Abutaleb ◽

George A. Truskey

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Blood Vessels ◽

Pluripotent Stem Cells ◽

The Body ◽

Organ Systems ◽

Temporal Waveform ◽

Induced Pluripotent

The vascular endothelium is present in all organs and blood vessels, facilitates the exchange of nutrients and waste throughout different organ systems in the body, and sets the tone for healthy vessel function. Mechanosensitive in nature, the endothelium responds to the magnitude and temporal waveform of shear stress in the vessels. Endothelial dysfunction can lead to atherosclerosis and other diseases. Modeling endothelial function and dysfunction in organ systems in vitro, such as the blood–brain barrier and tissue-engineered blood vessels, requires sourcing endothelial cells (ECs) for these biomedical engineering applications. It can be difficult to source primary, easily renewable ECs that possess the function or dysfunction in question. In contrast, human pluripotent stem cells (hPSCs) can be sourced from donors of interest and renewed almost indefinitely. In this review, we highlight how knowledge of vascular EC development in vivo is used to differentiate induced pluripotent stem cells (iPSC) into ECs. We then describe how iPSC-derived ECs are being used currently in in vitro models of organ function and disease and in vivo applications.

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In Vitro Generation of Oocyte Like Cells and Their In Vivo Efficacy: How Far We have been Succeeded

Cells ◽

10.3390/cells9030557 ◽

2020 ◽

Vol 9 (3) ◽

pp. 557 ◽

Cited By ~ 2

Author(s):

Dinesh Bharti ◽

Si-Jung Jang ◽

Sang-Yun Lee ◽

Sung-Lim Lee ◽

Gyu-Jin Rho

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem ◽

In Vivo Studies ◽

Special Focus ◽

Morphological Alterations ◽

Cell Sources ◽

Induced Pluripotent

In the last few decades, stem cell therapy has grown as a boon for many pathological complications including female reproductive disorders. In this review, a brief description of available strategies that are related to stem cell-based in vitro oocyte-like cell (OLC) development are given. We have tried to cover all the aspects and latest updates of the in vitro OLC developmental methodologies, marker profiling, available disease models, and in vivo efficacies, with a special focus on mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and embryonic stem cells (ESCs) usage. The differentiation abilities of both the ovarian and non-ovarian stem cell sources under various induction conditions have shown different effects on morphological alterations, proliferation- and size-associated developments, hormonal secretions under gonadotropic stimulations, and their neo-oogenesis or folliculogenesis abilities after in vivo transplantations. The attainment of characters like oocyte-like morphology, size expansion, and meiosis initiation have been found to be major obstacles during in vitro oogenesis. A number of reports have either lacked in vivo studies or have shown their functional incapability to produce viable and healthy offspring. Though researchers have gained many valuable insights regarding in vitro gametogenesis, still there are many things to do to make stem cell-derived OLCs fully functional.

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