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 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.

Modeling Congenital Amegakaryocytic Thrombocytopenia Using Patient-Specific Induced Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 703-703
Author(s):  
Naoya Takayama ◽  
Shinji Hirata ◽  
Ryoko Jono-Ohnishi ◽  
Sou Nakamura ◽  
Sho-ichi Hirose ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Receptor Gene ◽  
Patient Specific ◽  
Gene Correction ◽  
Donor Skin ◽  
Induced Pluripotent

Abstract Abstract 703 Patient-specific, induced pluripotent stem cells (iPSCs) enable us to study disease mechanisms and drug screening. To clarify the phenotypic alterations caused by the loss of c-MPL, the thrombopoietin (TPO) receptor, we established iPSCs derived from skin fibroblasts of a patient who received curative bone marrow transplantation for congenital amegakarycytic thrombocytopenia (CAMT) caused by the loss of the TPO receptor gene, MPL. The resultant CAMT-iPSCs exhibited mutations corresponding to the original donor skin. Then using an in vitro culture system yielding hematopoietic progenitor cells (HPCs), we evaluated the role of MPL on the early and late phases of human hematopoiesis. Although CAMT-iPSCs generated CD34+ HPCs, per se, their colony formation capability was impaired, as compared to control CD34+ HPCs. Intriguingly, both Glycophorin A (GPA)+ erythrocyte development and CD41+ megakaryocyte yields from CAMT-iPSCs were also impaired, suggesting that MPL is indispensable for MEP (megakaryocyte erythrocyte progenitors) development. Prospective analysis along with the hematopoietic hierarchy revealed that, in CAMT-iPSCs but not control iPSCs expressing MPL, mRNA expression and phosphorylation of putative signaling molecules downstream of MPL are severely impaired, as is the transition from CD34+CD43+CD41-GPA- MPP (multipotent progenitors) to CD41+GPA+ MEP. Additional analysis also indicated that c-MPL is required for maintenance of a consistent supply of megakaryocytes and erythrocytes from MEPs. Conversely, complimentary transduction of MPL into CAMT-iPSCs using a retroviral vector restored the defective erythropoiesis and megakaryopoiesis; however, excessive MPL signaling appears to promote aberrant megakaryopoiesis with CD42b (GPIba)-null platelet generation and impaired erythrocyte production. Taken together, our findings demonstrate the usefulness of CAMT-iPSCs for validation of functionality in the human hematopoiesis system. For example, it appears that MPL is not indispensable for the emergence of HPCs, but is indispensible for their maintenance, and for subsequent MEP development. Our results also strongly indicate that an appropriate expression level of an administered gene is necessary to achieve curative gene correction / therapy using patient-derived iPSCs. Disclosures: No relevant conflicts of interest to declare.

Generation of HIV Resistant and Functional Macrophages From Hematopoietic Stem Cell Derived Induced Pluripotent Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 562-562
Author(s):  
Amal Kambal ◽  
Gaela Mitchell ◽  
Whitney Cary ◽  
William Gruenloh ◽  
Yunjoon Jung ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Patient Specific ◽  
Hematopoietic Stem ◽  
Induced Pluripotent ◽  
Anti Hiv ◽  
Hiv 1

Abstract Abstract 562 Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into HIV infected patients. In this study, we have utilized human hematopoietic stem cells (HSCs) to generate anti-HIV gene expressing iPSCs for HIV gene therapy. HSCs were de-differentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 shRNA and a human/rhesus chimeric TRIM5α gene. Upon directed differentiation of the anti-HIV iPSCs towards the hematopoietic lineage, a robust quantity (>35%) of colony forming CD133+ HSCs were obtained. These cells were further differentiated into functional end-stage macrophages which displayed a normal phenotypic profile. Upon viral challenge, the anti-HIV iPSC derived macrophages exhibited strong protection (>3 logs) from HIV-1 infection. Here we demonstrate the ability of iPSCs to develop into HIV-1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies. Disclosures: No relevant conflicts of interest to declare.

Induced Pluripotent Stem Cells and Erythrocyte Production

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-38-SCI-38
Author(s):  
Igor Slukvin
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Embryonic Stem ◽  
Erythroid Cells ◽  
Hematopoietic Progenitors ◽  
Adult Hemoglobin ◽  
Equity Ownership ◽  
Induced Pluripotent

Abstract Abstract SCI-38 Induced pluripotent stem cells (iPSCs) are somatic cells that have been turned into embryonic-like stem cells by forced expression of factors critical for establishing pluripotency. Because iPSCs can be differentiated into any type of cell in the human body, including hematopoietic cells, they are seen as a logical alternative source of red blood cells (RBCs) for transfusion. In addition, the unlimited expansion potential of iPSCs makes it easy to adopt iPSC technology for RBC biomanufacturing. iPSCs can be generated from any type of donor, including O/Rh-negative universal donors and donors with very rare blood phenotypes, which makes it possible to generate blood products to accommodate virtually all patient groups. We have developed an approach for generating large quantities of RBCs from iPSCs by inducing them to differentiate into CD34+CD43+ hematopoietic progenitors in coculture with OP9 stromal cells, followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. Erythroid cultures produced by this approach consist of leukocyte-free populations of CD235a+ RBCs with robust expansion potential and long (up to 90 days) life spans. In these cultures, up to 1.8×105 RBCs can be generated from a single iPSC. Similar to embryonic stem cells, iPSC-derived RBCs express predominantly embryonic and fetal hemoglobin, with very little adult hemoglobin. It is already feasible to adopt iPSC technologies for producing cGMP-grade RBCs using defined animal-product-free differentiation conditions. However, the induction of the complete switch from embryonic to fetal and adult hemoglobin, as well as the terminal maturation and enucleation of iPSC-derived erythroid cells, remains a significant challenge. We recently identified at least three distinct waves of hematopoietic progenitors with erythroid potential in iPSC differentiation cultures. The characterization of erythroid cells produced from these waves of hematopoiesis may help to define populations with definitive erythroid potential and facilitate the production of erythrocytes from iPSCs. Additional critical steps toward translating iPSC-based RBC technologies to the clinic include the development of bioreactor-based-technology for further scaling-up of cell production, and evaluation of the therapeutic potential and safety of human pluripotent stem cell-derived blood cells in animal models. Overall, the manufacturing of RBCs provides several advantages. It can improve the continuity of the blood supply, minimize/eliminate the risk of infection transmission, reduce the incidence of hemolytic and nonhemolytic transfusion reactions, and provide an opportunity to generate RBCs that fit specific clinical needs by using genetically engineered iPSCs or iPSCs with rare blood groups. Disclosures: Slukvin: CDI: Consultancy, Equity Ownership; Cynata: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Dysregulation of the TGF β Pathway in Induced Pluripotent Stem Cells (iPSCs) Generated from Patients with Diamond Blackfan Anemia (DBA)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 254-254
Author(s):  
Jingping Ge ◽  
Marisa Apicella ◽  
Jason A. Mills ◽  
Loic Garcon ◽  
Deborah L. French ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ribosome Biogenesis ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ectopic Expression ◽  
Canonical Pathway ◽  
Hematopoietic Stem ◽  
Induced Pluripotent

Abstract DBA is an inherited bone marrow failure syndrome that usually develops in the first year of life with clinical features of red cell aplasia and sometimes variable developmental abnormalities. Most affected patients have heterozygous loss of function mutations of one of the 11 ribosomal proteins (RPs) or mutations in the GATA1 gene which encodes an erythroid specific transcription factor. We have previously demonstrated that induced pluripotent stem cells (iPSCs) from fibroblast of DBA patients with RPS19 or RPL5 mutations recapitulate the pathogenesis of DBA, with the mutant lines showing abnormal ribosome biogenesis and altered erythropoiesis. The mechanism whereby haploinsufficiency for RPs causes failure of erythropoiesis and the other DBA features is still unknown. We investigated the pathways that are affected in these DBA iPSCs using an Affymetrix human exon array, and we observed the striking dysregulation of the TGF β pathway in DBA lines. The TGF β downstream target genes, such as DKK1, BAMBI, FN1, COL3A1, COLA1A1 and PAI-1 significantly increased in the DBA iPSCs. The TGF β signaling is complex and can occur via a canonical pathway or by a number of non-canonical pathways. We measured levels of a number of intermediates in these pathways by western blot, and observed a significant increase in the levels of p-JNK, a mediator of a non-canonical pathway, in the DBA iPSCs. Moreover, when the mutant cells were corrected by ectopic expression of WT RPS19 or RPL5, levels of p-JNK returned to normal. We also investigated the SMAD family, which are mediators of the TGF β canonical pathway and are known to negatively regulate the regeneration of hematopoietic stem cells. We observed a drastic decrease in SMAD4, but no change in p-SMAD2. Again corrected lines showed normal expression levels of SMAD4. Our data suggests that the activation of a non-canonical TGF β pathway in the DBA iPSCs may lead increased expression of the downstream genes; and the decrease of anti-proliferative factor SMAD4 may explain how DBA iPSCs maintain their growth. We conclude that the mutations of RPS19 or RPL5 both affect ribosome biogenesis and TGF β signaling, which can cause the failure of erythropoiesis at the stem cell stage. We further suggest that the suppression of SMAD4 may be used as a therapeutic target for DBA treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Emergence of CD43-Expressing Hematopoietic Progenitors from Human Induced Pluripotent Stem Cells

2017 ◽  
Vol 44 (3) ◽  
pp. 143-150 ◽  
Author(s):  
Katharina U. Kessel ◽  
Anika Bluemke ◽  
Hans R. Schöler ◽  
Holm Zaehres ◽  
Peter Schlenke ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Hematopoietic Progenitors ◽  
Induced Pluripotent

Hematopoiesis of Human Induced Pluripotent Stem Cells Derived From Patients with Down Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 881-881
Author(s):  
Natsumi Nishihama ◽  
Yasuhiro Ebihara ◽  
Feng Ma ◽  
Wenyu Yang ◽  
Daisuke Tomizawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Down Syndrome ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Colony Formation ◽  
Trisomy 21 ◽  
Conflicts Of Interest ◽  
P Value ◽  
Increased Risk ◽  
Induced Pluripotent

Abstract Abstract 881 Trisomy 21, genetic hallmark of Down syndrome, is the most frequent human chromosomal abnormality. Infants and children with Down Syndrome (DS) are known to have some hematological disorders with an increased risk of developing leukemia. Ten to 20% of newborn with DS are diagnosed as neonatal preleukemic status, Transient Myeloproliferative Disorder (TMD), and approximately 30% of TMD patients are predisposed to acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in the N-terminal activation domain of the GATA1 gene, leading to expression of a shorter GATA1 isoform (GATA1s), have been reported in AMKL and TMD (Wechsler et al., 2002; Mundschau et al., 2003), but neither patients nor mice with germline mutations leading to expression of GATA1s developed AMKL and TMD in the absent of trisomy 21. These findings suggested that trisomy 21 itself directly contributes to the development of AMKL and TMD. However, the role of trisomy 21 in hematopoiesis, particularly in the human fetus remains poorly understood. To better understand the effects of trisomy 21 on hematopoiesis in embryonic stage and leukemogenesis, we employed human induced pluripotent stem cells (hiPSCs) derived from patients with DS (DS-hiPSCs). Six DS-hiPS and 5 hiPS cell lines (control) from healthy donors, which we used here, were all created from skin fibroblasts and reprogrammed by the defined 3 or 4 reprogramming factors (OCT3/4, KLF4, and SOX2, or c-MYC in addition to the 3 factors, respectively). We generated blood cells from DS-hiPSCs and controls with coculture system using murine aorta-gonad-mesonephros (AGM)-derived stromal cell line (Ma et al., 2009). The cells from hiPSCs were harvested at D11 or D12 of coculture and analyzed the presence of hematopoietic markers and the potentials of hematopoietic colony formation. In the experiments using hiPSCs reprogrammed by 3 factors, human CD34 expression in harvested cells from DS-hiPSCs or controls were detected 10.06 ± 4.35% and 3.04%, respectively. CD45 expression of CD34+ cells was small proportion in both DS-hiPSCs and controls. We next examined the hematopoietic colony formation. Both myeloid and erythroid colonies were detected. Number of colonies formed from DS-hiPSCs was 43.7±11.1 to 74.3±11.2 per an iPSC colony. It's approximately 2 to 3.5 folds numbers of control (p-value<0.05). Similar results were obtained in the experiments using hiPSCs reprogrammed by 4 factors. These results indicated that hiPSCs derived from patients with Down syndrome could differentiate into multiple hematopoietic cell lineages and the differentiation into hematopoietic lineage was promoted in DS patients. Further researches are under investigation to identify the responsible genes in trisomy 21 for acceleration of hematopoiesis with microarray analysis. Our study may contribute to understanding of the effects of trisomy 21 on hematopoiesis and effective use of patients derived hiPSCs in research and clinical application. Disclosures: No relevant conflicts of interest to declare.

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