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.

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.

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 Generation of HIV-1 Resistant and Functional Macrophages From Hematopoietic Stem Cell–derived Induced Pluripotent Stem Cells

2011 ◽  
Vol 19 (3) ◽  
pp. 584-593 ◽  
Author(s):  
Amal Kambal ◽  
Gaela Mitchell ◽  
Whitney Cary ◽  
William Gruenloh ◽  
Yunjoon Jung ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Pluripotent Stem Cells ◽  
Hematopoietic Stem ◽  
Induced Pluripotent ◽  
Hiv 1

scholarly journals A High-Throughput Method as a Diagnostic Tool for HIV Detection in Patient-Specific Induced Pluripotent Stem Cells Generated by Different Reprogramming Methods

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Daniela Hübscher ◽  
Sabine Rebs ◽  
Luis Haupt ◽  
Thomas Borchert ◽  
Celina Isabell Guessoum ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
High Throughput ◽  
Pluripotent Stem Cells ◽  
Mononuclear Cells ◽  
Sendai Virus ◽  
Patient Specific ◽  
Pathogenic Viruses ◽  
Induced Pluripotent ◽  
Hiv 1

Induced pluripotent stem cells (iPSCs) provide a unique opportunity for generation of patient-specific cells for use in translational purposes. We aimed to compare iPSCs generated by different reprogramming methods regarding their reprogramming efficiency, pluripotency capacity, and the possibility to use high-throughput PCR-based methods for detection of human pathogenic viruses. iPSCs from skin fibroblasts (FB), peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells (MSCs) were generated by using three different reprogramming systems including chromosomal integrating and nonintegrating methods. Reprogramming efficiencies were in accordance with the literature, indicating that the parental cell type and the reprogramming method play a major role for the reprogramming efficiencies (FB: STEMCCA: 1.30±0.18, Sendai virus: 1.37±0.01, and episomal plasmids: 0.04±0.02; PBMCs: Sendai virus: 0.002±0.001, episomal plasmids: 0) but result in the same characteristics of pluripotency. We found the highest reprogramming efficiencies for MSC with 3.32±1.2 by using episomal plasmids. Since GMP standard working procedures and screening units need virus contamination-free cell lines, we studied HIV-1 contamination in the generated iPSCs. We used the high-throughput cobas® 6800/8800 system, which is normally used for detection of HIV-1 in plasma of patients, and found that footprint-free reprogramming methods as episomal plasmids and Sendai virus are useful for the described virus detection method. This fast, cost-effective, robust, and reliable assay demonstrates the feasibility to use high-throughput PCR-based methods for detection of human pathogenic viruses in ps-iPSC lines that were generated with nongenome integrating reprogramming methods.

Derivation of Hematopoietic Progenitor Cells From Vector-Free Human Induced Pluripotent Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4746-4746
Author(s):  
Friedrich Schuening ◽  
Michail Zaboikin ◽  
Tatiana Zaboikina ◽  
Narasimhachar Srinivasakumar
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Dermal Fibroblasts ◽  
Embryoid Bodies ◽  
Chromosomal Anomalies ◽  
Hematopoietic Stem ◽  
Induced Pluripotent

Abstract Abstract 4746 Induced pluripotent stem cells (iPSCs), due to their self-renewal and differentiation capability, have tremendous potential in regenerative medicine. Differentiation of IPSCs in vitro to obtain sufficient number of hematopoietic stem cells (HSCs) and their progenitors (HPCs) from iPSCs for therapeutic purposes is a holy grail of cellular therapy. To this end, we are comparing different in vitro differentiation approaches for generation of HSCs/HPCs from IPSCs. We have generated iPSCs from human adult dermal fibroblasts using two different reprogramming methods: 1) Transduction with retroviral vectors encoding human Klf4, Oct3/4, Sox2 and cMyc or 2) Electroporation with Epstein–Barr virus (EBV) based episomal plasmid vectors encoding Klf4, Oct3/4, Sox2, L-Myc and p53 targeting shRNA. The transduced/electroporated cells were reprogrammed on SNL5 mouse feeder cells. Putative iPSC-like colonies were cloned and adapted to grow under feeder-free conditions on Matrigel (BD) in mTeSR1 (Stem Cell Technologies) medium. From over 30 individual clones isolated, six were further characterized for: 1) expression of pluripotency markers (Tra-1–60, SSEA-3, SSEA-4, Nanog and Oct3/4) by immunofluorescence; 2) endogenous and total mRNA expression by quantitative real-time reverse-transcriptase PCR (RT-qPCR) for Klf4, Oct3/4, Sox2 and cMyc to distinguish between cellular and vector derived expression of reprogramming factors; 3) RT-qPCR to determine expression of other markers of pluripotency such as Nanog and DNA methyl transferease; 4) karyotype analysis to determine chromosomal anomalies. The vector-free IPSC clones were also tested for residual integrated EBV plasmid DNA by qPCR. Trilineage differentiation ability of the clones was determined through embryoid body formation in suspension cultures, and subsequent staining of resulting embryoid bodies after adherence to gelatin coated dishes for makers of ectoderm, mesoderm and endoderm. HSCs/HPCs were obtained from IPSCs by 1) coculture with OP9 stromal cells, or 2) step-wise differentiation in feeder-free conditions on Matrigel under defined conditions in the presence of appropriate growth factors [Niwa A et al. PLoS One. (2011); 6(7):e22261.]. The resultant HSCs/HPCs were subjected to colony forming assays in semi-solid medium containing hematopoietic cytokines. Both erythroid and myelomonocytic colonies could be readily identified. The influence of ambient oxygen concentration on the HSC/HPC derivation procedure is being investigated. The results of these studies will be presented. Disclosures: No relevant conflicts of interest to declare.

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