scholarly journals Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2422
Author(s):  
Stoyan Petkov ◽  
Ralf Dressel ◽  
Ignacio Rodriguez-Polo ◽  
Rüdiger Behr
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Model ◽  
Common Marmoset ◽  
Animal Disease ◽  
Neural Lineage ◽  
Equine Encephalitis Virus ◽  
Somatic Cell Reprogramming ◽  
Induced Pluripotent

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of cell-based therapies; however, the safety and efficacy of potential iPSC-based treatments need to be verified in relevant animal disease models before their application in the clinic. Here, we report the derivation of iPSCs from common marmoset monkeys (Callithrix jacchus) using self-replicating mRNA vectors based on the Venezuelan equine encephalitis virus (VEE-mRNAs). By transfection of marmoset fibroblasts with VEE-mRNAs carrying the human OCT4, KLF4, SOX2, and c-MYC and culture in the presence of small molecule inhibitors CHIR99021 and SB431542, we first established intermediate primary colonies with neural progenitor-like properties. In the second reprogramming step, we converted these colonies into transgene-free pluripotent stem cells by further culturing them with customized marmoset iPSC medium in feeder-free conditions. Our experiments revealed a novel paradigm for flexible reprogramming of somatic cells, where primary colonies obtained by a single VEE-mRNA transfection can be directed either toward the neural lineage or further reprogrammed to pluripotency. These results (1) will further enhance the role of the common marmoset as animal disease model for preclinical testing of iPSC-based therapies and (2) establish an in vitro system to experimentally address developmental signal transduction pathways in primates.

scholarly journals Controlling the switch from neurogenesis to pluripotency during marmoset monkey somatic cell reprogramming with self-replicating mRNAs and small molecules

2020 ◽  
Author(s):  
Stoyan Petkov ◽  
Ralf Dressel ◽  
Ignacio Rodriguez-Polo ◽  
Rüdiger Behr
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Model ◽  
Common Marmoset ◽  
Animal Disease ◽  
Neural Lineage ◽  
Somatic Cell Reprogramming ◽  
Fetal Fibroblasts

SUMMARYInduced pluripotent stem cells (iPSCs) hold enormous potential for the development of cell-based therapies for many currently incurable diseases. However, the safety and efficacy of potential iPSC-based treatments need to be verified in relevant animal disease models before their application in the clinic. Moreover, in order to reduce possible risks for the patients, it is necessary to use reprogramming approaches that ensure to the greatest extent possible the genomic integrity of the cells. Here, we report the derivation of iPSCs from common marmoset monkeys (Callithrix jacchus) using self-replicating mRNA vectors based on the Venezuelan equine encephalitis virus (VEE-mRNAs). By transfection of marmoset fetal fibroblasts with Tomato-modified VEE-mRNAs carrying the human OCT4, KLF4, SOX2, and c-MYC (VEE-OKS-iM-iTomato) and culture in medium supplemented with two small molecule inhibitors, we first established intermediate primary colonies with neural progenitor-like properties. In the second reprogramming step, we converted these colonies into transgene-free pluripotent stem cells by further culturing them with customized marmoset iPSC medium in feeder-free conditions. The resulting cell lines possess pluripotency characteristics, such as expression of various pluripotency markers, long-term self-renewal, stable karyotype, and ability to differentiate into derivatives of the three primary germ layers in vitro and in vivo. Our experiments reveal a novel paradigm for flexible reprogramming of somatic cells, where primary colonies obtained by a single VEE-mRNA transfection can be directed either towards the neural lineage or further reprogrammed to pluripotency. These results (i) will further enhance the role of the common marmoset as animal disease model for preclinical testing of iPSC-based therapies and (ii) establish an in vitro system to experimentally address developmental signal transduction pathways in primates.

scholarly journals Characterization and Optimization of Chitosan-Coated Polybutylcyanoacrylate Nanoparticles for the Transfection-Guided Neural Differentiation of Mouse Induced Pluripotent Stem Cells

2021 ◽  
Vol 22 (16) ◽  
pp. 8741
Author(s):  
Martin Hsiu-Chu Lin ◽  
Ping-Shan Lai ◽  
Li-Ching Chang ◽  
Wei-Chao Huang ◽  
Ming-Hsueh Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Viral Vectors ◽  
Gene Transfection ◽  
Weight Ratio ◽  
Lineage Specification ◽  
Neural Lineage ◽  
Induced Pluripotent

Gene transfection is a valuable tool for analyzing gene regulation and function, and providing an avenue for the genetic engineering of cells for therapeutic purposes. Though efficient, the potential concerns over viral vectors for gene transfection has led to research in non-viral alternatives. Cationic polyplexes such as those synthesized from chitosan offer distinct advantages such as enhanced polyplex stability, cellular uptake, endo-lysosomal escape, and release, but are limited by the poor solubility and viscosity of chitosan. In this study, the easily synthesized biocompatible and biodegradable polymeric polysorbate 80 polybutylcyanoacrylate nanoparticles (PS80 PBCA NP) are utilized as the backbone for surface modification with chitosan, in order to address the synthetic issues faced when using chitosan alone as a carrier. Plasmid DNA (pDNA) containing the brain-derived neurotrophic factor (BDNF) gene coupled to a hypoxia-responsive element and the cytomegalovirus promotor gene was selected as the genetic cargo for the in vitro transfection-guided neural-lineage specification of mouse induced pluripotent stem cells (iPSCs), which were assessed by immunofluorescence staining. The chitosan-coated PS80 PBCA NP/BDNF pDNA polyplex measured 163.8 ± 1.8 nm and zeta potential measured −34.8 ± 1.8 mV with 0.01% (w/v) high molecular weight chitosan (HMWC); the pDNA loading efficiency reached 90% at a nanoparticle to pDNA weight ratio of 15, which also corresponded to enhanced polyplex stability on the DNA stability assay. The HMWC-PS80 PBCA NP/BDNF pDNA polyplex was non-toxic to mouse iPSCs for up to 80 μg/mL (weight ratio = 40) and enhanced the expression of BDNF when compared with PS80 PBCA NP/BDNF pDNA polyplex. Evidence for neural-lineage specification of mouse iPSCs was observed by an increased expression of nestin, neurofilament heavy polypeptide, and beta III tubulin, and the effects appeared superior when transfection was performed with the chitosan-coated formulation. This study illustrates the versatility of the PS80 PBCA NP and that surface decoration with chitosan enabled this delivery platform to be used for the transfection-guided differentiation of mouse iPSCs.

Induced Pluripotent Stem Cells From a Patient with Reticular Dysgenesis Recapitulate Defective Myelopoiesis in-Vitro: A Disease Model to Enhance Our Understanding of a Rare Disease.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2142-2142
Author(s):  
Katja G. Weinacht ◽  
Kerstin Felgentreff ◽  
Alex Devine ◽  
Axel Schambach ◽  
Waleed Al-herz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Rare Disease ◽  
Pluripotent Stem Cells ◽  
Adenylate Kinase ◽  
Genetic Defect ◽  
Disease Model ◽  
Maturation Arrest ◽  
Induced Pluripotent

Abstract Abstract 2142 Reticular dysgenesis (RD) is one of the most severe forms of combined immunodeficiency characterized by severe congenital neutropenia (SCN) and impaired lymphocyte development. Origin of the neutropenia is thought to be an early differentiation arrest at the promyelocte stage. Recently the disease has been mapped to mutations in the gene encoding for Adenylate Kinase 2 (AK2). Adenylate kinase 2 is a phosphokinase implicated in mitochondrial energy homeostasis. The exact mechanism, however, how a defect in this ubiquitously expressed enzyme selectively manifests in defective granulopoiesis and lymphopoiesis remains poorly understood. A significant obstacle to the study of rare diseases like Reticular Dysgenesis has been the inadequacy of animal models and limited availability of patient specimens. We have successfully generated induced pluripotent stem cells (iPSCs) from a patient with Reticular Dysgenesis and proven genetic defect in Adenylate Kinase 2 (AK2). Myeloid precursors derived from the AK2-deficient iPSCs morphologically mimic the myeloid maturation arrest seen in the bone marrow of patients with this condition (Figure 1). We are currently using this in-vitro model as a platform to study the mechanisms underlying the maturation arrest on a cellular and molecular level. Summary: Here we report a pluripotent stem cell (iPSC)-based disease model for Reticular Dysgenesis. This model does not only accurately recapitulate the myeloid disease phenotype observed in vivo but enables us to study previously unknown disease pathology. This is an example how iPSC technology enhances our understanding of a rare disease that has thus far been limited by the availability of study material. Disclosures: No relevant conflicts of interest to declare.

Mini Review; Differentiation of Human Pluripotent Stem Cells into Oocytes

2020 ◽  
Vol 15 (4) ◽  
pp. 301-307 ◽  
Author(s):  
Gaifang Wang ◽  
Maryam Farzaneh
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Human Pluripotent Stem Cells ◽  
Human Oocyte ◽  
Differentiation Potential ◽  
Cell Lineages ◽  
Cell Based Therapy ◽  
Induced Pluripotent

Primary Ovarian Insufficiency (POI) is one of the main diseases causing female infertility that occurs in about 1% of women between 30-40 years of age. There are few effective methods for the treatment of women with POI. In the past few years, stem cell-based therapy as one of the most highly investigated new therapies has emerged as a promising strategy for the treatment of POI. Human pluripotent stem cells (hPSCs) can self-renew indefinitely and differentiate into any type of cell. Human Embryonic Stem Cells (hESCs) as a type of pluripotent stem cells are the most powerful candidate for the treatment of POI. Human-induced Pluripotent Stem Cells (hiPSCs) are derived from adult somatic cells by the treatment with exogenous defined factors to create an embryonic-like pluripotent state. Both hiPSCs and hESCs can proliferate and give rise to ectodermal, mesodermal, endodermal, and germ cell lineages. After ovarian stimulation, the number of available oocytes is limited and the yield of total oocytes with high quality is low. Therefore, a robust and reproducible in-vitro culture system that supports the differentiation of human oocytes from PSCs is necessary. Very few studies have focused on the derivation of oocyte-like cells from hiPSCs and the details of hPSCs differentiation into oocytes have not been fully investigated. Therefore, in this review, we focus on the differentiation potential of hPSCs into human oocyte-like cells.

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

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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