Patient-Derived Induced Pluripotent Stem Cells Reveal Distinct Hematopoietic Defects Conferred by Trisomy 21 and Truncated GATA-1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 911-911 ◽  
Author(s):  
Stella T Chou ◽  
Daniel VanDorn ◽  
Yu Yao ◽  
Deborah L. French ◽  
Mitchell J Weiss
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Trisomy 21 ◽  
Embryoid Bodies ◽  
Amino Terminus ◽  
Hematopoietic Progenitors ◽  
Megakaryocytic Differentiation ◽  
Colony Assays ◽  
Induced Pluripotent

Abstract Abstract 911 Infants with Down syndrome (DS, trisomy 21, T21) frequently exhibit hematological abnormalities including polycythemia and/or thrombocytopenia. About 10% of DS infants develop transient myeloproliferative disease (TMD), which usually self-resolves. However, approximately 30% of affected patients develop acute megakaryoblastic leukemia (AMKL) by age 4 years. Both TMD and AMKL are accompanied by somatic GATA1 gene mutations that give rise to GATA-1s (for “short”), an amino-truncated protein lacking amino acids 1–81. Thus, DS-associated AMKL requires at least three sequentially occurring genetic abnormalities in hematopoietic cells: germline T21, somatic GATA1 mutations in fetal progenitors, and postnatal mutations in additional, currently unidentified genes. To analyze this malignant progression step by step and better understand T21-associated hematopoietic abnormalities, we created induced pluripotent stem cells (iPSCs) from DS subjects (n=3), TMD blasts (n=5) and controls (n=3). All iPSC lines exhibited signature features of pluripotency and retained their relevant genotypes: T21, T21+GATA1s and normal euploid. We compared the blood-forming capacities of iPSC lines by generating embryoid bodies in defined medium containing hematopoietic cytokines. Stage-matched embryoid bodies of each genotype produced similar numbers CD41+/235+/43+ hematopoietic progenitors capable of erythroid, myeloid and megakaryocytic differentiation. However, in methylcellulose colony assays, progenitors from DS iPSCs contained 13.5-fold increased numbers of burst forming unit erythroid (BFU-E) progenitors compared to control iPSCs (p=.009) (Table). While the absolute numbers of colony forming unit-megakaryocytes (CFU-MK) were similar between DS and wild type iPSC-derived progenitors (p=0.21), the CFU-MK:CFU-myeloid ratio was increased in progenitors from DS iPSCs (p=0.014). Thus, DS iPSC-derived hematopoietic progenitors exhibit increased propensity for erythro-megakaryocytic differentiation, similar to what occurs in DS fetal liver (Chou et al; Tunstall-Pedoe et al, Blood v112, 2008). In contrast, CD41+/235+/43+ progenitors from all 5 DS TMD iPSC lines studied (T21/GATA1s) exhibited complete absence of erythroid developmental potential in liquid culture and methylcellulose colony assays (p<.001), despite robust production of myeloid and megakaryocytic cells. We confirmed this observation by comparing the hematopoietic potential of iPSCs generated from TMD blasts (T21/GATA1s) and normal blood cells (T21/GATA1wt) of the same DS patient (n = 2 different individuals). In each case, acquisition of the GATA1s mutation selectively blocked erythropoiesis and tended to increase megakaryopoiesis. Thus, the amino terminus of GATA-1, absent in GATA-1s, is required for primitive (yolk-sac type) erythropoiesis, the developmental stage that is recapitulated in our iPSC differentiation protocols. In agreement, loss of the GATA-1 amino terminus inhibits primitive erythropoiesis in mice (Li et al, Nature Genetics v37, 2005). Our findings illustrate distinct hematopoietic defects conferred by T21 and GATA-1s, and suggest how these might synergize in TMD and AMKL. More generally, our studies illustrate how analysis of patient-derived iPSCs can be used to analyze genetic blood disorders, particularly those that arise during fetal development.Table.Average number of colonies per 5000 CD41+/235+/43+ progenitors plated in methylcellulose assays for BFU-E and CFU-GM, and Megacult assay for CFU-MK. Standard deviation in parenthesesGenotype#patients#replicatesBFU-ECFU-GMCFU-MkEuploid31031 (+26)138 (+70)468 (+135)T2138417* (+201)52* (+32)574 (+211)T21+GATA1s5140** (+0)394** (+245)754 (+657)*p<0.05, euploid vs T21,**p<0.05 T21 vs T21+GATA1s. Disclosures: No relevant conflicts of interest to declare.

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).

283 GENERATION AND CHARACTERIZATION OF BOVINE INDUCED PLURIPOTENT STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 289
Author(s):  
O. J. Koo ◽  
H. S. Kwon ◽  
D. K. Kwon ◽  
K. S. Kang ◽  
B. C. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Transgenic Animals ◽  
Mouse Embryonic Fibroblast ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Serum Replacement ◽  
Induced Pluripotent ◽  
Large Animals

Stem cells in large animals are an excellent model for cell therapy research and fine resources for producing transgenic animals. However, there are only few reports of stem cells in large animals because of technical differences between species. In this report, we successfully generate bovine induced pluripotent stem cells (iPSC) using 4 human reprogramming factors (Oct4, Sox2, Klf4, and c-myc) under control of PiggyBac transposition vector. Fibroblasts derived from bovine fetuses were transfected using FugeneHD agent. After 21 days, colony-shaped structures on the culture plates were mechanically detached and then seeded on a mouse embryonic fibroblast (MEF) feeder layer pretreated with mitomycin C. The culture medium was DMEM/F12 supplemented with 20% serum replacement, 5 ng mL–1 basic fibroblast growth factor (bFGF), 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin antibiotics. The iPSC colonies showed alkaline phosphatase activity and expressed several pluripotency markers (Oct4, Sox2, SSEA1, and SSEA4). To confirm differentiation potential, the iPSC were cultured as embryoid bodies and then plated again. βIII-tubulin (ectoderm) and GFAP or α-SMA (mesoderm) were well expressed on the attached cells. The results revealed that the bovine fibroblasts were well inducted to iPSC that had potential of multilineage differentiation. We hope this technology contributes to improving transgenic cattle production. This study was financially supported by IPET (grant # 109023-05-3-CG000, 111078-03-1-CG000) and the BK21 program for Veterinary Science.

scholarly journals Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Damián Hernández ◽  
Rodney Millard ◽  
Priyadharshini Sivakumaran ◽  
Raymond C. B. Wong ◽  
Duncan E. Crombie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Electrical Stimulation ◽  
Induced Pluripotent Stem Cells ◽  
Cell Line ◽  
Pluripotent Stem Cells ◽  
Embryoid Bodies ◽  
Cardiac Differentiation ◽  
Dependent Manner ◽  
Human Ipscs ◽  
Induced Pluripotent

Background.Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes.Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days.Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression ofACTC1,TNNT2,MYH7, andMYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner.Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used.

Generation of Induced Pluripotent Stem Cells From Primary Chronic Myelogenous Leukemia Patient Sample.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1206-1206
Author(s):  
Keiki Kumano ◽  
Shunya Arai ◽  
Koki Ueda ◽  
Kumi Nakazaki ◽  
Yasuhiko Kamikubo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Induced Pluripotent Stem Cells ◽  
Chronic Myelogenous Leukemia ◽  
Pluripotent Stem Cells ◽  
Hematological Malignancies ◽  
Hematological Malignancy ◽  
Myelogenous Leukemia ◽  
Hematopoietic Progenitors ◽  
Induced Pluripotent

Abstract Abstract 1206 Introduction: Induced pluripotent stem cells (iPSCs) can be generated from various cell types by the expression of defined transcription factors. In addition to the regenerative medicine, iPSCs have been used for the study of the pathogenesis of inherited genetic disease. Recently, it was reported that iPSCs were generated not only from normal tissue, but also from malignant cells. In those cases, cancer cells themselves must be the starting material from which iPSCs are derived. However, in almost all the cases, they used the established cell lines (chronic myelogenous leukemia (CML), gastrointestinal cancers, and melanoma) except for the JAK2-V617F mutation (+) polycythemia vera (PV) patient. In this study, we established the iPSCs from primary CML patient sample. Results: After obtaining informed consent, bone marrow cells from CML patient were reprogrammed by introducing the transcription factors Oct3/4, Sox2, KLF4, and c-myc. To improve the efficiency of the development of iPSCs, we added valproic acid (VPA), a histone deacetylase inhibitor, to the culture. Two CML derived iPSCs (CML-iPSCs) were generated. CML-iPSCs expressed the pluripotency markers such as SSEA-4 and Tra-1-60, and the endogenous expression of embryonic stem cell (ESC) characteristic transcripts (Oct3/4, Sox2, KLF4, Nanog, LIN28, REX1) was confirmed by RT-PCR. Oct4 and Nanog promoter regions were demetylated in the CML-iPSCs. Although CML-iPSCs expressed bcr-abl, they were resistant to the imatinib. Then we differentiated them into hematopoietic progenitors within the ‘unique sac-like structures’ (iPS-sacs). This method was reported to be able to produce the hematopoietic progenitors with higher efficiency than the usual embryoid body formation method using human ESCs (Takayama et al., Blood, 111, 5298–306, 2008). The hematopoietic progenitors showed the hematopoietic marker CD45 and immature marker CD34, and recovered the sensitivity to the imatinib, which recapitulated the feature of initial CML disease. Then we investigated the mechanism of the resistance to the imatinib in CML-iPSCs. The phosphorylation state of ERK1/2, AKT, and STAT5, which are the essential for the survival of bcr-abl (+) hematopoietic progenitors, were evaluated after imatinib treatment in CML-iPSCs. The phosphorylation of ERK1/2 and AKT, which were also essential for the maintenance of iPSCs, were unchanged after treatment, although STAT5 was not activated both before and after treatment. These results showed that the signaling for iPSCs maintenance compensated for the inhibition of bcr-abl in CML-iPSCs and that the oncogene addiction was lost in CML-iPSCs. Conclusion: We generated the iPSCs from primary CML patient samples, re-differentiated them into hematopoietic lineage and showed the recapitulation of the features of initial disease. Primary samples of hematological malignancy are usually difficult to be expanded. However, if once they are reprogrammed to iPSCs, they can expand unlimitedly. As a result, we can obtain the genetically abnormal hematopoietic cells continuously by re-differentiating them into hematopoietic cells and use them for the studies which require the large number of living cells such as the analysis for leukemia stem cells or drug screening. Thus iPSCs technology would be useful for the study of hematological malignancy, especially for which animal model was not established such as myelodysplastic syndrome and be applicable for other cancers than hematological malignancies. We are now trying to establish the iPSCs derived from other hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

Differentiation of Mature Eosinophils From Human Embryonic and Induced Pluripotent Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1594-1594
Author(s):  
Wenyu Yang ◽  
Feng Ma ◽  
Kenji Matsumoto ◽  
Natsumi Nishihama ◽  
Hiroshi Sagara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Allergic Diseases ◽  
Time Dependent ◽  
Dependent Manner ◽  
Hematopoietic Progenitors ◽  
Culture Cells ◽  
Induced Pluripotent

Abstract Abstract 1594 Eosinophils are multifunctional leukocytes implicated in the pathogenesis of numerous inflammatory processes. As the major effectors, eosinophils function in a variety of biological responses, allergic diseases and helminth infections. It is generally accepted human eosinophils develop through a pathway initially sharing common feature with basophils. However, there lacks a clear chart for early development of human eosinophils, such as during embryonic or fetal stages. We recently established an efficient method for producing eosinophils from human embryonic and induced pluripotent stem cells (hESC/iPSCs). By a two-step induction, we first generated multipotential hematopoietic progenitors by co-culturing hESC/iPSCs with mouse AGM-derived stromal cells for 2 weeks. Then, total co-culture cells were transferred into suspension culture favoring eosinophil development with addition of IL-3 and other factors (SCF, IL-6, TPO, Flt-3 ligand) . The maturation of hESC/iPSC -derived eosinophils was shown in a time-dependent manner, first co-expressing eosinophil-and basophil-specific markers [eosinophil peroxidase (EPO), and 2D7, respectively], then the portion of eosinophil markers gradually increased while that of basophil markers decreased (EPO+ cells from 56.4% at day 7 to 94.4% at day 21, while 2D7+ cells from 62.8% to 25.7%, respectively), typically mimicking the development of eosinophils from human adult hematopoietic progenitors. By flowcytometric analysis, an eosinophil-specific surface marker, Siglec-8, was also expressed on these hESC/iPSC-derived eosinophils in a time-dependent manner (from 10.8% at day 7 to 91.3% at day 21), paralleling to those with EPO. The expression of eosinophil-specific granule cationic proteins (EPO, MBP, ECP, EDN) and IL-5 receptor mRNA was also detected by RT-PCR. Furthermore, transmission electron microscopy (TEM) observation confirmed the eosinophil property. Eosinophils derived from hiPSCs hold similar characteristics as those from hESCs. The function of hES/hiPSC-derived eosinophils is being under investigation. Our study provides an experimental model for exploring early genesis of eosinophils, especially in uncovering the mechanisms controlling the development of the initial innate immune system of human being in normal and diseased individuals. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Reprogramming of COPD lung fibroblasts through formation of induced pluripotent stem cells

2014 ◽  
Vol 306 (6) ◽  
pp. L552-L565 ◽  
Author(s):  
Hesham Basma ◽  
Yoko Gunji ◽  
Shunichiro Iwasawa ◽  
Amy Nelson ◽  
Maha Farid ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Bodies ◽  
Differentially Expressed ◽  
Lung Fibroblasts ◽  
Chronic Obstructive ◽  
Collagen Gels ◽  
Primary Fibroblasts ◽  
Induced Pluripotent

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study, we tested whether functional differences between chronic obstructive pulmonary disease (COPD) and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4, nanog, and sox2, formed embryoid bodies in vitro, and induced teratomas in nonobese diabetic/severe combined immunodeficient mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared with their corresponding primary fibroblasts. Primary COPD fibroblasts contracted three-dimensional collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast, redifferentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1,881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts, with 605 genes differing by more than twofold. After redifferentiation, 112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than twofold. Similar findings were observed with microRNA (miRNA) expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation, only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly, of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts, 293 genes were changed toward control after redifferentiation. In conclusion, functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs.

Sign in / Sign up

Export Citation Format

Share Document