scholarly journals Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells

Blood ◽  
2010 ◽  
Vol 115 (14) ◽  
pp. 2769-2776 ◽  
Author(s):  
Agamemnon E. Grigoriadis ◽  
Marion Kennedy ◽  
Aline Bozec ◽  
Fiona Brunton ◽  
Gudrun Stenbeck ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acid Phosphatase ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cathepsin K ◽  
Embryoid Bodies ◽  
Tartrate Resistant Acid Phosphatase ◽  
Αvβ3 Integrin ◽  
Directed Differentiation ◽  
Induced Pluripotent

Abstract The directed differentiation of human pluripotent stem cells offers the unique opportunity to generate a broad spectrum of human cell types and tissues for transplantation, drug discovery, and studying disease mechanisms. Here, we report the stepwise generation of bone-resorbing osteoclasts from human embryonic and induced pluripotent stem cells. Generation of a primitive streak-like population in embryoid bodies, followed by specification to hematopoiesis and myelopoiesis by vascular endothelial growth factor and hematopoietic cytokines in serum-free media, yielded a precursor population enriched for cells expressing the monocyte-macrophage lineage markers CD14, CD18, CD11b, and CD115. When plated in monolayer culture in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL), these precursors formed large, multinucleated osteoclasts that expressed tartrate-resistant acid phosphatase and were capable of resorption. No tartrate-resistant acid phosphatase-positive multinucleated cells or resorption pits were observed in the absence of RANKL. Molecular analyses confirmed the expression of the osteoclast marker genes NFATc1, cathepsin K, and calcitonin receptor in a RANKL-dependent manner, and confocal microscopy demonstrated the coexpression of the αvβ3 integrin, cathepsin K and F-actin rings characteristic of active osteoclasts. Generating hematopoietic and osteoclast populations from human embryonic and induced pluripotent stem cells will be invaluable for understanding embryonic bone development and postnatal bone disease.

scholarly journals Modeling Fanconi Anemia Using Human Induced Pluripotent Stem Cells By Reversible Complementation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3856-3856
Author(s):  
R. Grant Rowe ◽  
William Marion ◽  
Sonya Ruiz-Torres ◽  
Edroaldo Lummertz da Rocha ◽  
Yosra Girvan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Fanconi Anemia ◽  
Pluripotent Stem Cells ◽  
Erythroid Differentiation ◽  
Embryoid Bodies ◽  
Directed Differentiation ◽  
Sequencing Analysis ◽  
Hematopoietic Stem ◽  
Induced Pluripotent

Abstract Modeling of Fanconi anemia (FA) using human induced pluripotent stem cells (iPSCs) has been hindered by the requirement for an intact FA DNA repair pathway for effective reprogramming and maintenance of pluripotency in patient-derived iPSCs. Temporary complementation of FA pathway defects could permit effective reprogramming, with removal of complementation upon directed differentiation to hematopoietic stem and progenitor cells (HSPCs) to model the hematopoietic phenotypes of FA. In this study, we used three FA patient-derived iPSC lines engineered with doxycycline inducible complementation of disease-causing FANCA mutations. We maintained complementation with doxycycline exposure in the pluripotent state and during morphogen directed differentiation of hemogenic endothelium (HE) within embryoid bodies, which possesses the capacity to differentiate to HSPCs. Upon initiation of the endothelial-to-hematopoietic transition (EHT) in purified iPSC-derived HE, doxycycline was either removed or maintained in culture to either inactivate or sustain FANCA expression. Morphologic EHT and emergence of CD34+CD45+ immunophenotypic human HSPCs were not affected by the status of FANCA expression, which allowed for the isolation of otherwise isogenic FANCA-expressing and FANCA-deficient HSPCs for disease modeling. FANCA-deficient HSPCs were unable to form FANCD2/γH2AX foci relative to FANCA-expressing HSPCs in response to genotoxic stress, confirming impairment of the function of the FA pathway. We found that uncomplimented, FANCA-deficient HSPCs showed impaired cell cycle progression, increased apoptosis, and markedly decreased colony forming activity in methylcellulose compared to complemented, FANCA-expressing HSPCs. Unexpectedly, we also found that FANCA-deficient HSPCs showed accelerated erythroid differentiation compared to cells with an intact FA pathway. RNA sequencing analysis showed enrichment of signatures of normal HSPCs in complemented HSPCs, while uncomplimented HSPCs showed signatures of more mature hematopoietic cells and cells undergoing erythroid differentiation. Together, these findings demonstrate the utility of reversible complementation in modeling FA with patient-derived iPSCs, and provide an inexhaustible source of otherwise isongenic complimented and uncomplimented human FA HSPCs for use in the investigation of new therapies. Disclosures No relevant conflicts of interest to declare.

Directed differentiation of mouse-induced pluripotent stem cells generates dopaminergic nerve

2010 ◽  
Vol 34 (8) ◽  
pp. S36-S36
Author(s):  
Ping Duan ◽  
Xuelin Ren ◽  
Wenhai Yan ◽  
Xuefei Han ◽  
Xu Yan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Directed Differentiation ◽  
Induced Pluripotent

scholarly journals Directed differentiation of human induced pluripotent stem cells into mature kidney podocytes and establishment of a Glomerulus Chip

2018 ◽  
Vol 13 (7) ◽  
pp. 1662-1685 ◽  
Author(s):  
Samira Musah ◽  
Nikolaos Dimitrakakis ◽  
Diogo M. Camacho ◽  
George M. Church ◽  
Donald E. Ingber
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Directed Differentiation ◽  
Induced Pluripotent

scholarly journals Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Laís Vicari de Figueiredo Pessôa ◽  
Pedro Ratto Lisboa Pires ◽  
Maite del Collado ◽  
Naira Caroline Godoy Pieri ◽  
Kaiana Recchia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Mirna Profile ◽  
Patient Specific ◽  
Induced Pluripotent

Introduction. Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objectives. We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Methods. Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results. Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions. We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

308 Tet1 PLAYS IMPORTANT ROLES IN MAINTAINING CHARACTERIZATIONS OF PORCINE INDUCED PLURIPOTENT STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 301
Author(s):  
A. R. Fan ◽  
K. Y. Ma ◽  
T. C. Zhao ◽  
P. P. An ◽  
B. Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Self Renewal ◽  
Qrt Pcr ◽  
Post Transfection ◽  
Fetal Fibroblasts ◽  
Induced Pluripotent

It was recently found that the ten-eleven-translocation (TET) family of Fe(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and thus promotes active demethylation of genomes. Tet1 is highly expressed in mouse embryonic stem cells (mESC) and has been demonstrated to involve in mESC maintenance. Here we used small interference RNA (siRNA) to transiently knockdown expression of Tet1 in porcine induced pluripotent stem cells (iPSC) in order to identify its functions. The fetal fibroblasts were isolated from a 30-day-old porcine fetus and induced into iPSC with defined transcription factors, namely Oct-4, Sox-2, Klf-4, and C-myc. The colonies appeared on Day 12 and were picked up on Day 14. These colonies had normal ES-like morphology and alkaline phosphatase activity. Specifically, they were positively stained for pluripotency-specific markers, including Oct4, Sox2, Nanog, Rex1, and SSEA1. When cultured in vitro, the cells formed embryoid bodies (EB), and all 3 germ layer markers (endoderm: AFP, alphaAT; mesoderm: BMP4, Enolase; ectoderm: GFAP, Neurod) were detected positively in EB. For siRNA transfections, iPSC from the colonies were transfected with 40 pmol of siRNA and 2 µL of Lipofectamine 2000 in 1 well of a 24-well plate. After transfection, iPSC were subjected to fluorescence-activated cell sorting to determine the fraction of FAM-positive cells in order to confirm transfection efficiency; the percentage of positive cells reached 48 ± 4.96. We observed obvious knockdown of Tet1 after short-term transfection of siRNA, and the knockdown efficiency was confirmed using qRT-PCR and immunofluorescence staining. Notably, knockdown of Tet1 resulted in morphological abnormality and loss of undifferentiated state of porcine iPSC. However, no obvious morphological changes were observed in the negative control (transfected with nonsense-siRNA), positive control (transfected with GAPDH-siRNA), or mock control (transfected with DEPC-treated water). To gain insight into the molecular mechanism underlying the self-renewal defect, we analysed the effects of Tet1 knockdown on the expression of key stem cell factors and differentiation markers of different embryonic layers using qRT-PCR. We found that knockdown of Tet1 resulted in downregulated expression of pluripotency-related genes, such as Lefty-2, Klf-2, and Sox-2 (the expression ratios of post-transfection to pre-transfection were 0.31 ± 0.21, 0.48 ± 0.072, and 0.65 ± 0.046, respectively), and upregulated expression of differentiation-related genes, including Pitx-2, Hand-1, Gata-6, and Lef-1 (the expression ratios of post-transfection to pre-transfection were 4.35 ± 1.36, 2.56 ± 0.68, 2.91 ± 1.47, and 2.33 ± 1.11, respectively). However, Oct-4, C-myc, Klf-4, and Nanog were not downregulated (the expression ratios of post-transfection to pre-transfection were 0.91 ± 0.15, 1.12 ± 0.26, 1.15 ± 0.21, and 1.08 ± 0.08, respectively). Taken together, Tet1 plays important roles in porcine iPSC self-renewal and characterization maintenance. This study was financed by National Basic Research Program of China (NO.2009CB941001).

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