Study of Bernard–Soulier Syndrome Megakaryocytes and Platelets Using Patient-Derived Induced Pluripotent Stem Cells

AbstractBernard–Soulier syndrome (BSS) is a hereditary macrothrombocytopenia caused by defects in the glycoprotein (GP) Ib-IX-V complex. The mechanism of giant platelet formation remains undefined. Currently, megakaryocytes (MKs) can be generated from induced pluripotent stem cells (iPSCs) to study platelet production under pharmacological or genetic manipulations. Here, we generated iPSC lines from two BSS patients with mutations in different genes (GP1BA and GP1BB: termed BSS-A and BSS-B, respectively). The iPSC-derived MKs and platelets were examined under electron microscopy and stained by immunofluorescence to observe proplatelet formation and measure platelet diameters which were defined by circumferential tubulin. BSS-iPSCs produced abnormal proplatelets with thick shafts and tips. In addition, compared with the normal iPSCs, the diameters were larger in platelets derived from BSS-A and BSS-B with the means ± standard deviations of 4.34 ± 0.043 and 3.88 ± 0.045 µm, respectively (wild-type iPSCs 2.61 ± 0.025 µm, p < 0.001). Electron microscopy revealed giant platelets with the abnormal demarcation membrane system. Correction of BSS-A and BSS-B-iPSCs using lentiviral vectors containing respective GP1BA and GP1BB genes improved proplatelet structures and platelet ultrastructures as well as reduced platelets sizes. In conclusion, the iPSC model can be used to explore molecular mechanisms and potential therapy for BSS.

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Abstract TP473: Investigating the Pathogenesis of Moyamoya Disease Using Patient Derived Induced Pluripotent Stem Cells

Stroke ◽

10.1161/str.51.suppl_1.tp473 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Shailaja Rao ◽

Qian Zhang ◽

Haruto Uchino ◽

Arjun Pendharkar ◽

Michelle Cheng ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Moyamoya Disease ◽

Pluripotent Stem Cells ◽

Molecular Mechanisms ◽

Mononuclear Cells ◽

Brdu Incorporation ◽

Branch Points ◽

Scratch Assay ◽

Induced Pluripotent

Background: Moyamoya disease (MMD) is a rare, progressive steno-occlusive cerebrovascular disorder of the internal carotid artery, leading to stroke. Affected arteries exhibit thickened intima with depleted elastic lamina and media, indicating a dysfunction of the vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). However the pathogenesis of the disease is still unclear. We aim to address this gap in knowledge by using patient derived induced pluripotent stem cells (iPSCs), to generate VSMCs and ECs. Methods: Peripheral blood mononuclear cells (PBMCs) from controls and MMD patients (n=3 per group) were used for generating iPSCs. VSMC functionality was measured by collagen gel contraction assay and scratch assay. EC proliferative function was assessed by BrDU incorporation assay, and its migration capacity was evaluated by scratch assay and in vitro tube formation. VSMCs and ECs were also exposed to either hydrogen peroxide (H2O2) or normoxia/ hypoxia model (1%O 2 ) to investigate how cells respond to these insults. Hypoxia inducible factor 1α (HIF1α) activation was determined using western blot. Results: MMD VSMCs trended towards being more contractile and migrating faster than control VSMCs, in response to 10%FBS or SDF1α. On the other hand, MMD ECs migrated slower than control ECs in response to 10%FBS (p=0.0081) or VEGF (p=0.0072). MMD ECs also formed lesser tubes and exhibited fewer branch points when compared to controls. The rate of EC proliferation was similar between both groups. Cell death assays indicate that MMD VSMCs and ECs were more sensitive to the deleterious effects of H2O2 exposure when compared to control cells. Interestingly, MMD VSMCs had elevated HIF1α protein expression in normoxia, which was further increased after hypoxia. Conclusions: Our preliminary results indicate that both MMD VSMCs and ECs are dysfunctional and may be related to the elevated basal expression of HIF1α, possibly contributing to MMD pathology. We are currently investigating the interactions between VSMCs and ECs in MMD compared with controls using co-cultures. Ongoing studies also include transcriptome analysis of these differentiated cells, which will advance the understanding of the cellular and molecular mechanisms underlying MMD.

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Modeling Parkinson’s Disease Using Induced Pluripotent Stem Cells

Stem Cells International ◽

10.1155/2020/1061470 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15

Author(s):

Xinchao Hu ◽

Chengyuan Mao ◽

Liyuan Fan ◽

Haiyang Luo ◽

Zhengwei Hu ◽

...

Keyword(s):

Parkinson’S Disease ◽

Stem Cells ◽

Parkinson's Disease ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Molecular Mechanisms ◽

Cellular Level ◽

Patient Specific ◽

Ubiquitin Protein Ligase ◽

Induced Pluripotent

Parkinson’s disease (PD) is the second most common neurodegenerative disease. The molecular mechanisms of PD at the cellular level involve oxidative stress, mitochondrial dysfunction, autophagy, axonal transport, and neuroinflammation. Induced pluripotent stem cells (iPSCs) with patient-specific genetic background are capable of directed differentiation into dopaminergic neurons. Cell models based on iPSCs are powerful tools for studying the molecular mechanisms of PD. The iPSCs used for PD studies were mainly from patients carrying mutations in synuclein alpha (SNCA), leucine-rich repeat kinase 2 (LRRK2), PTEN-induced putative kinase 1 (PINK1), parkin RBR E3 ubiquitin protein ligase (PARK2), cytoplasmic protein sorting 35 (VPS35), and variants in glucosidase beta acid (GBA). In this review, we summarized the advances in molecular mechanisms of Parkinson’s disease using iPSC models.

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Harnessing the Power of Induced Pluripotent Stem Cells and Gene Editing Technology: Therapeutic Implications in Hematological Malignancies

Cells ◽

10.3390/cells10102698 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2698

Author(s):

Ishnoor Sidhu ◽

Sonali P. Barwe ◽

Raju K. Pillai ◽

Anilkumar Gopalakrishnapillai

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Gene Editing ◽

Molecular Mechanisms ◽

Hematological Malignancies ◽

Disease Modeling ◽

Clonal Evolution ◽

Induced Pluripotent ◽

Ipsc Disease Modeling

In vitro modeling of hematological malignancies not only provides insights into the influence of genetic aberrations on cellular and molecular mechanisms involved in disease progression but also aids development and evaluation of therapeutic agents. Owing to their self-renewal and differentiation capacity, induced pluripotent stem cells (iPSCs) have emerged as a potential source of short in supply disease-specific human cells of the hematopoietic lineage. Patient-derived iPSCs can recapitulate the disease severity and spectrum of prognosis dictated by the genetic variation among patients and can be used for drug screening and studying clonal evolution. However, this approach lacks the ability to model the early phases of the disease leading to cancer. The advent of genetic editing technology has promoted the generation of precise isogenic iPSC disease models to address questions regarding the underlying genetic mechanism of disease initiation and progression. In this review, we discuss the use of iPSC disease modeling in hematological diseases, where there is lack of patient sample availability and/or difficulty of engraftment to generate animal models. Furthermore, we describe the power of combining iPSC and precise gene editing to elucidate the underlying mechanism of initiation and progression of various hematological malignancies. Finally, we discuss the power of iPSC disease modeling in developing and testing novel therapies in a high throughput setting.

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The Application of Induced Pluripotent Stem Cells Against Liver Diseases: An Update and a Review

Frontiers in Medicine ◽

10.3389/fmed.2021.644594 ◽

2021 ◽

Vol 8 ◽

Author(s):

Lei Zhang ◽

Ke Pu ◽

Xiaojun Liu ◽

Sarah Da Won Bae ◽

Romario Nguyen ◽

...

Keyword(s):

Stem Cells ◽

Liver Disease ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Liver Diseases ◽

Molecular Mechanisms ◽

Disease Modeling ◽

Health Concern ◽

Promising Alternative ◽

Induced Pluripotent

Liver diseases are a major health concern globally, and are associated with poor survival and prognosis of patients. This creates the need for patients to accept the main alternative treatment of liver transplantation to prevent progression to end-stage liver disease. Investigation of the molecular mechanisms underpinning complex liver diseases and their pathology is an emerging goal of stem cell scope. Human induced pluripotent stem cells (hiPSCs) derived from somatic cells are a promising alternative approach to the treatment of liver disease, and a prospective model for studying complex liver diseases. Here, we review hiPSC technology of cell reprogramming and differentiation, and discuss the potential application of hiPSC-derived liver cells, such as hepatocytes and cholangiocytes, in refractory liver-disease modeling and treatment, and drug screening and toxicity testing. We also consider hiPSC safety in clinical applications, based on genomic and epigenetic alterations, tumorigenicity, and immunogenicity.

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Hepatic Differentiation of Murine Disease-Specific Induced Pluripotent Stem Cells Allows Disease ModellingIn Vitro

Stem Cells International ◽

10.4061/2011/924782 ◽

2011 ◽

Vol 2011 ◽

pp. 1-11 ◽

Cited By ~ 5

Author(s):

Reto Eggenschwiler ◽

Komal Loya ◽

Malte Sgodda ◽

Francoise André ◽

Tobias Cantz

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Molecular Mechanisms ◽

Functional Characterization ◽

Hepatic Differentiation ◽

Fumarylacetoacetate Hydrolase ◽

Induced Pluripotent ◽

Disease Specific

Direct reprogramming of somatic cells into pluripotent cells by retrovirus-mediated expression of OCT4, SOX2, KLF4, and C-MYC is a promising approach to derive disease-specific induced pluripotent stem cells (iPSCs). In this study, we focused on three murine models for metabolic liver disorders: the copper storage disorder Wilson's disease (toxic-milk mice), tyrosinemia type 1 (fumarylacetoacetate-hydrolase deficiency, FAH−/−mice), and alpha1-antitrypsin deficiency (PiZ mice). Colonies of iPSCs emerged 2-3 weeks after transduction of fibroblasts, prepared from each mouse strain, and were maintained as individual iPSC lines. RT-PCR and immunofluorescence analyses demonstrated the expression of endogenous pluripotency markers. Hepatic precursor cells could be derived from these disease-specific iPSCs applying anin vitrodifferentiation protocol and could be visualized after transduction of a lentiviral albumin-GFP reporter construct. Functional characterization of these cells allowed the recapitulation of the disease phenotype for further studies of underlying molecular mechanisms of the respective disease.

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Histone Deacetylases in Neural Stem Cells and Induced Pluripotent Stem Cells

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/835968 ◽

2011 ◽

Vol 2011 ◽

pp. 1-6 ◽

Cited By ~ 17

Author(s):

Guoqiang Sun ◽

Chelsea Fu ◽

Caroline Shen ◽

Yanhong Shi

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Neural Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Histone Deacetylases ◽

Molecular Mechanisms ◽

Self Renewal ◽

Stem Cell Pluripotency ◽

Induced Pluripotent

Stem cells have provided great hope for the treatment of a variety of human diseases. However, the molecular mechanisms underlying stem cell pluripotency, self-renewal, and differentiation remain to be unveiled. Epigenetic regulators, including histone deacetylases (HDACs), have been shown to coordinate with cell-intrinsic transcription factors and various signaling pathways to regulate stem cell pluripotency, self-renewal, and fate determination. This paper focuses on the role of HDACs in the proliferation and neuronal differentiation of neural stem cells and the application of HDAC inhibitors in reprogramming somatic cells to induced pluripotent stem cells (iPSCs). It promises to be an active area of future research.

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