Multiple sclerosis: getting personal with induced pluripotent stem cells

Abstract Human induced pluripotent stem (iPS) cells can be derived from lineage-restricted cells and represent an important tool to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. Recently, patient-derived iPS cells, containing donor genetic background, have offered a breakthrough approach to study human genetics of neurodegenerative diseases. By offering an unlimited source of patient-specific disease-relevant cells, iPS cells hold great promise for understanding disease mechanisms, identifying molecular targets and developing phenotypic screens for drug discovery. This review will discuss the potential impact of using iPS cell-derived models in multiple sclerosis (MS) research and highlight some of the current challenges and prospective for generating novel therapeutic treatments for MS patients.

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Extracellular Matrix-Dependent Generation of Integration- and Xeno-Free iPS Cells Using a Modified mRNA Transfection Method

Stem Cells International ◽

10.1155/2016/6853081 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Kang-In Lee ◽

Seo-Young Lee ◽

Dong-Youn Hwang

Keyword(s):

Extracellular Matrix ◽

Cell Therapy ◽

Culture System ◽

Ips Cells ◽

Great Promise ◽

Ips Cell ◽

Transfection Method ◽

Reprogramming Efficiency ◽

Mrna Transfection ◽

Induced Pluripotent

Human induced pluripotent stem cells (iPS cells) hold great promise in the field of regenerative medicine, especially immune-compatible cell therapy. The most important safety-related issues that must be resolved before the clinical use of iPS cells include the generation of “footprint-free” and “xeno-free” iPS cells. In this study, we sought to examine whether an extracellular matrix- (ECM-) based xeno-free culture system that we recently established could be used together with a microRNA-enhanced mRNA reprogramming method for the generation of clinically safe iPS cells. The notable features of this method are the use of a xeno-free/feeder-free culture system for the generation and expansion of iPS cells rather than the conventional labor-intensive culture systems using human feeder cells or human feeder-conditioned medium and the enhancement of mRNA-mediated reprogramming via the delivery of microRNAs. Strikingly, we observed the early appearance of iPS cell colonies (~11 days), substantial reprogramming efficiency (~0.2–0.3%), and a high percentage of ESC-like colonies among the total colonies (~87.5%), indicating enhanced kinetics and reprogramming efficiency. Therefore, the combined method established in this study provides a valuable platform for the generation and expansion of clinically safe (i.e., integration- and xeno-free) iPS cells, facilitating immune-matched cell therapy in the near future.

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Induced human pluripotent stem cells: promises and open questions

Biological Chemistry ◽

10.1515/bc.2009.103 ◽

2009 ◽

Vol 390 (9) ◽

Cited By ~ 30

Author(s):

Alexandra Rolletschek ◽

Anna M. Wobus

Keyword(s):

Pluripotent Stem Cells ◽

Viral Vectors ◽

Insertional Mutagenesis ◽

Ips Cells ◽

Patient Specific ◽

Ips Cell ◽

Open Questions ◽

Induced Pluripotent ◽

Selective Differentiation

Abstract Adult cells have been reprogrammed into induced pluripotent stem (iPS) cells by introducing pluripotency-associated transcription factors. Here, we discuss recent advances and challenges of in vitro reprogramming and future prospects of iPS cells for their use in diagnosis and cell therapy. The generation of patient-specific iPS cells for clinical application requires alternative strategies, because genome-integrating viral vectors may cause insertional mutagenesis. Moreover, when suitable iPS cell lines will be available, efficient and selective differentiation protocols are needed to generate transplantable grafts. Finally, we point to the requirement of a regulatory framework necessary for the commercial use of iPS cells.

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Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

Blood ◽

10.1182/blood-2009-04-217406 ◽

2009 ◽

Vol 114 (27) ◽

pp. 5473-5480 ◽

Cited By ~ 262

Author(s):

Zhaohui Ye ◽

Huichun Zhan ◽

Prashant Mali ◽

Sarah Dowey ◽

Donna M. Williams ◽

...

Keyword(s):

Stem Cells ◽

Cell Lines ◽

Blood Cells ◽

Ips Cells ◽

Patient Specific ◽

Hematopoietic Differentiation ◽

Ips Cell ◽

Healthy Donors ◽

Cd34 Cells ◽

Induced Pluripotent

Abstract Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34+ cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34+CD45+) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

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338 IN VITRO GENERATION OF LENTOID BODIES FROM INDUCED PLURIPOTENT STEM CELLS OF TRANSGENIC MICE

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab338 ◽

2015 ◽

Vol 27 (1) ◽

pp. 257

Author(s):

T. Anand ◽

D. Kumar ◽

T. R. Talluri ◽

H. Niemann ◽

W. A. Kues

Keyword(s):

Stem Cells ◽

Cell Types ◽

Ips Cells ◽

Sleeping Beauty ◽

Specific Cell ◽

Fluorescence Excitation ◽

Ips Cell ◽

Developmental Potential ◽

Induced Pluripotent

Pluripotent cells have the developmental potential to generate all adult cell types, so ocular diseases resulting from the failure of specific cell types could be potentially treatable through the transplantation of differentiated cells derived from stem cells. The present study was conducted with the aim of generating a cataract model. We attempted to derive the induced pluripotent stem (iPS) cells from fibroblast cells of transgenic (crytom) mice carrying a transgenic construct-alphaA crystallin promoter driving the tandem dimer (td) Tomato marker transgene, integrated in the genome. The 4- to 6-week-old female crytom mice were selected, superovulated, and mated. The fetuses were recovered and examined on various different days (10.5 to 15.5 days postfertilization), and the reporter expression was found to be initiated 12.5 days postfertilization and the intensity was increased thereafter. The expression of tdTomato was confirmed in the fetuses by Western blotting. Murine embryonic fibroblast (MEF) cultures were generated and electroporated with a reprogramming transposon cassette carrying Yamanaka factors (OCT4, SOX2, KLF4, and MYC) and Sleeping Beauty transposase to generate iPS cells which were picked up and clonally expanded. The cells were confirmed by PCR for tdTomato in the genome and characterised for the expression of Oct4 and cryAB by immunofluorescence. The iPS cells were also injected into the nude CD1 mice to test for teratoma formation. The generated cells were allowed to differentiate spontaneously on 3 different types (viz. P19, NTERA, and STO) of cell lines as feeders, in the absence of LIF, and cells were expected to fluoresce if differentiated to eye lens lineage. After long-term cultures, the iPS cells were found to differentiate and form lentoid bodies which expressed tdTomato. Thus, alphaA crystallin-tdTomato construct was allowed following lens cell formation by specific fluorescence excitation in a spatial and temporal manner. The employment of cell type-specific reporters for establishing and optimizing targeted differentiation in vitro seems to be an efficient and generally applicable approach for developing differentiation protocols for desired cell populations. Hence a transgenic murine iPS cell line was generated which exhibited potential to be used as a model for eye cataracts and other eye abnormalities.

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Reduced Production of Mature Neutrophils From Induced Pluripotent Stem Cells Derived From a Severe Congenital Neutropenia Patient with HAX1 Gene Deficiency

Blood ◽

10.1182/blood.v118.21.2402.2402 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2402-2402 ◽

Cited By ~ 1

Author(s):

Tatsuya Morishima ◽

Ken-ichiro Watanabe ◽

Akira Niwa ◽

Takayuki Tanaka ◽

Katsutsugu Umeda ◽

...

Keyword(s):

Cell Lines ◽

Normal Control ◽

Annexin V ◽

Ips Cells ◽

Patient Specific ◽

Es Cell ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Ips Cell ◽

Induced Pluripotent

Abstract Abstract 2402 Induced pluripotent stem (iPS) cells are reprogrammed somatic cells with embryonic stem (ES) cell-like characteristics. As iPS cells can be generated from somatic cells of patients with a certain disease, they are expected to be a novel model to study pathogenesis of various diseases. Recently, we established a neutrophil differentiation system from human iPS cells (Morishima T, et al. J Cell Physiol. 2011). In an attempt to apply the system to investigate pathophysiology of neutrophil-affected disorders, we generated iPS cells from a severe congenital neutropenia (SCN) patient with HAX1 gene deficiency. The patient was an 11-year-old boy with severe congenital neutropenia as well as developmental delay and epilepsy. DNA sequence analysis revealed HAX1 gene mutation in exon 2 (Matsubara K, et al. Haematologica. 2007). Four iPS cell lines were generated from skin fibroblasts of the patient by retroviral overexpression of the three or four transcription factors Oct3/4, Sox2, and Klf4, with or without c-Myc. These patient-derived iPS cell lines showed human ES cell like morphology and could be maintained under human ES cell culture condition. They also expressed typical human ES cell markers and were capable of differentiating into the cell lineages and tissues representing three germ layers by teratoma formation in vivo. These cells had normal karyotype and short tandem repeat analysis indicated that they were derived from parental skin fibroblast. DNA sequencing analysis of the iPS cell lines identified the same mutation carried in the parental skin fibroblasts, thus confirmed that we had established the HAX1 deficiency patient-specific iPS cells (HAX1-iPSCs). Next these HAX1-iPSCs and the healthy-person derived iPS cells were differentiated into neutrophils in vitro using feeder-free culture protocols established in our laboratory. In this culture system, small human iPS cell clumps were cultured on the matrigel-coated dish with recombinant cytokines and without any feeder cells and fetal calf serum. Around day 25 of culture, mature neutrophils were obtained as floating cells. Morphologically, the majority of HAX1-iPSCs-derived cells were classified into myeloblast or promyelocyte stage and there were only a few mature neutrophils. The proportion of mature neutrophils was only less than 10% in HAX1-iPSCs-derived cells whereas more than 40% in normal control. Flow cytometric analysis revealed that the percentage of immature CD34 positive cells was significantly higher and that of myeloid-committed CD11b positive cells was lower in the HAX1-iPSCs-derived cells than normal control. Immunocytochemical analysis for neutrophil specific granules showed that lactoferrin- and gelatinase-positive cells decreased in the HAX1-iPSCs-derived cells compared with normal control, confirming that HAX1-iPSCs-derived cells contained less mature neutrophils than normal control. Apoptosis assay by Annexin V staining revealed that HAX1-iPSCs-derived cells showed higher percentage of Annexin V-positive cells compared with normal control. Overall, these HAX1 deficiency patient-specific iPS cell lines recapitulate the hematological phenotype in the patient. These results indicate that patient-derived iPS cells provide us a novel disease model and make a contribution to the understanding of the pathophysiology of the diseases that affect neutrophils. Disclosures: No relevant conflicts of interest to declare.

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HUMAN INDUCED PLURIPOTENT STEM CELL REGION DETECTION IN BRIGHT-FIELD MICROSCOPY IMAGES USING CONVOLUTIONAL NEURAL NETWORKS

Biomedical Engineering Applications Basis and Communications ◽

10.4015/s1016237219500091 ◽

2019 ◽

Vol 31 (02) ◽

pp. 1950009 ◽

Cited By ~ 3

Author(s):

Yuan-Hsiang Chang ◽

Kuniya Abe ◽

Hideo Yokota ◽

Kazuhiro Sudo ◽

Yukio Nakamura ◽

...

Keyword(s):

Regenerative Medicine ◽

Ips Cells ◽

Patient Specific ◽

Specific Cell ◽

Bright Field ◽

Differentiated Cells ◽

Number Of Patients ◽

Bright Field Microscopy ◽

Induced Pluripotent ◽

Microscopy Images

Human induced pluripotent stem (iPS) cells represent an ideal source for patient specific cell-based regenerative medicine. For practical uses of iPS cells, large-scale, cost- and time-effective production of fully reprogrammed iPS cells from a number of patients should be achieved. To achieve this goal, culture protocols for inducing iPS cells as well as methods for selecting fully reprogrammed iPS cells in a mixture of cells which are still in reprogramming and non-iPS differentiated cells, should be improved. This paper proposes a convolutional neural network (CNN) structure to classify a bright-field microscopy image as respective probability images. Each probability image represents regions of differentiated cells, fully reprogrammed iPS cells or cells still in reprogramming, respectively. The CNN classifier was trained by multiple types of image patches which represent differentiated, reprogramming and reprogrammed iPS cells, etc. Classification of an image containing the confirmed iPS cells by the trained CNN classifier shows that high classification accuracy can be achieved. Classifications of sets of time-lapse microscopy images show that growth and transition from CD34[Formula: see text] human cord blood cells through reprogramming to reprogrammed iPS cells can be visualized and quantitatively analyzed by the output time-series probability images. These experiment results show our CNN structure yields a potential tool to detect the differentiated cells that possibly undergo reprogramming to iPS cells for screening reagents or culture conditions in human iPS induction, and ultimately further understand the ideal culturing conditions for practical use in regenerative medicine.

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Abstract 006: An Advance toward Making A Heart: Engineering Functional Human Heart Constructs with Induced Pluripotent Stem Cells and Decellularized Mouse Hearts

Circulation Research ◽

10.1161/res.113.suppl_1.a006 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Lei Yang ◽

Tung-Ying Lu ◽

Bo Lin ◽

Jong Kim ◽

Kimimasa Tobita ◽

...

Keyword(s):

Tissue Engineering ◽

Heart Development ◽

Human Heart ◽

Heart Tissue ◽

Patient Specific ◽

Great Promise ◽

Ips Cell ◽

Rat Cardiomyocytes ◽

Novel Strategy ◽

Induced Pluripotent

Background: Heart disease is the leading cause of death in the world, requiring the development of novel and personalized therapeutic strategies for disease treatment. Heart tissue engineering holds a great promise for those therapies based on the reconstruction of patient-specific cardiac muscle. A bioartificial heart was reported by reseeding rat cardiomyocytes ( CMs ) into decellularized rat heart. Whether such method could be used to make human heart constructs remained unknown. Objectives: We sought to rebuild functional heart constructs using human induced pluripotent stem ( iPS) cells and decellularized whole mouse hearts. Methods: We repopulated decellularized mouse hearts with human iPS cell derived multipotential cardiovascular progenitors ( MCPs ). MCPs represent the earliest heart progenitors during human cardiogenesis and can give rise to multiple cardiovascular lineage cells. Results: The seeded MCPs differentiated in situ into CMs, smooth muscle and endothelial cells, which reconstructed the decellularized mouse hearts. The engineered constructs exhibited myocardium and vessel-like structures, contracted spontaneously with a rate of 40~50 beats/min, exhibited intracellular Ca2+ transients and responded as expected to various drug interventions. In addition, we found heart ECM could promote proliferation and myofilament formation of CMs from the repopulated human MCPs. Therefore this study established a novel strategy of human heart tissue engineering, which could be beneficial to study heart development, and future preclinical applications.

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Human DC3 Antigen Presenting Dendritic Cells from Induced Pluripotent Stem Cells

10.1101/2021.06.22.449451 ◽

2021 ◽

Author(s):

Taiki Satoh ◽

Marcelo A Szymanski de Toledo ◽

Janik Boehnke ◽

Kathrin Olschok ◽

Niclas Flosdorf ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Cd8 T Cells ◽

Pluripotent Stem Cells ◽

Jak2 V617f ◽

Ips Cells ◽

Patient Specific ◽

Ips Cell ◽

Induced Pluripotent ◽

Antigen Presenting

Dendritic cells (DC) are professional antigen-presenting cells that develop from hematopoietic stem cells. Different DC subsets exist based on ontogeny, location and function, including the recently identified proinflammatory DC3 subset. DC3 have the prominent activity to polarize CD8+ T cells into CD8+ CD103+ tissue resident T cells. Here we describe human DC3 differentiated from induced pluripotent stem cells (iPS cells). iPS cell-derived DC3 have the gene expression and surface marker make-up of blood DC3 and polarize CD8+ T cells into CD8+ CD103+ tissue-resident memory T cells in vitro. To test the impact of malignant JAK2 V617F mutation on DC3, we differentiated patient-specific iPS cells with JAK2 V617Fhet and JAK2 V617Fhom mutations into JAK2 V617Fhet and JAK2 V617Fhom DC3. The JAK2 V617F mutation enhanced DC3 production and caused a bias towards erythrocytes and megakaryocytes. The patient-specific iPS cell-derived DC3 are expected to allow studying DC3 in human diseases and developing novel therapeutics.

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Cell Culture, iPS Cells and Neurodegenerative Diseases

10.1093/med/9780190233563.003.0013 ◽

2016 ◽

Author(s):

Roxana Nat ◽

Andreas Eigentler

Keyword(s):

Cell Culture ◽

Muscular Atrophy ◽

Ips Cells ◽

Patient Specific ◽

Neural Cells ◽

Differentiation Potential ◽

Ips Cell ◽

Human Neurons ◽

Culture Models ◽

Induced Pluripotent

Somatic reprogramming technology, which enables the conversion of adult human non-neural cells into neurons, has progressed rapidly in recent years. The derivation of patient-specific induced pluripotent stem (iPS) cells has become routine. The inherent broad differentiation potential of iPS cells makes possible the generation of diverse types of human neurons. This constitutes a remarkable step in facilitating the development of more appropriate and comprehensive preclinical human disease models, as well as for high throughput drug screenings and cell therapy. This chapter reviews recent progress in the human iPS cell culture models related to common and rare NDDs, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, spinal muscular atrophy, and degenerative ataxias. It focuses on the pathophysiological features revealed in cell cultures, and the neuronal subtypes most affected in NDDs. The chapter discusses the validity, limitation, and improvements of this system in faithfully and reproducibly recapitulating disease pathology.

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iPS-Cell Technology and the Problem of Genetic Instability—Can It Ever Be Safe for Clinical Use?

Journal of Clinical Medicine ◽

10.3390/jcm8030288 ◽

2019 ◽

Vol 8 (3) ◽

pp. 288 ◽

Cited By ~ 24

Author(s):

Stephen Attwood ◽

Michael Edel

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Great Promise ◽

Clinical Use ◽

Medicinal Products ◽

Ips Cell ◽

Factors Affecting ◽

Induced Pluripotent ◽

Stem Cell Therapeutics

The use of induced Pluripotent Stem Cells (iPSC) as a source of autologous tissues shows great promise in regenerative medicine. Nevertheless, several major challenges remain to be addressed before iPSC-derived cells can be used in therapy, and experience of their clinical use is extremely limited. In this review, the factors affecting the safe translation of iPSC to the clinic are considered, together with an account of efforts being made to overcome these issues. The review draws upon experiences with pluripotent stem-cell therapeutics, including clinical trials involving human embryonic stem cells and the widely transplanted mesenchymal stem cells. The discussion covers concerns relating to: (i) the reprogramming process; (ii) the detection and removal of incompletely differentiated and pluripotent cells from the resulting medicinal products; and (iii) genomic and epigenetic changes, and the evolutionary and selective processes occurring during culture expansion, associated with production of iPSC-therapeutics. In addition, (iv) methods for the practical culture-at-scale and standardization required for routine clinical use are considered. Finally, (v) the potential of iPSC in the treatment of human disease is evaluated in the light of what is known about the reprogramming process, the behavior of cells in culture, and the performance of iPSC in pre-clinical studies.

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