scholarly journals Genetic correction of β-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice

Cell Research ◽  
2012 ◽  
Vol 22 (4) ◽  
pp. 637-648 ◽  
Author(s):  
Yixuan Wang ◽  
Chen-Guang Zheng ◽  
Yonghua Jiang ◽  
Jiqin Zhang ◽  
Jiayu Chen ◽  
...  
Keyword(s):  
Ips Cells ◽  
Scid Mice ◽  
Patient Specific ◽  
Thalassemia Patient ◽  
Hemoglobin Production

Effects of the FGF2 aptamer on growth plate cartilage development of achondroplasia patient-specific iPS cells in a xenograft model

2019 ◽  
Author(s):  
Takeshi Kimura ◽  
Kie Yasuda ◽  
Yukako Nakano ◽  
Shinji Takeyari ◽  
Yasuji Kitabatake ◽  
...  
Keyword(s):  
Growth Plate ◽  
Xenograft Model ◽  
Ips Cells ◽  
Patient Specific ◽  
Growth Plate Cartilage ◽  
Cartilage Development

scholarly journals Cellular Reprogramming Employing Recombinant Sox2 Protein

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Marc Thier ◽  
Bernhard Münst ◽  
Stephanie Mielke ◽  
Frank Edenhofer
Keyword(s):  
Protein Delivery ◽  
Disease Modeling ◽  
Ips Cells ◽  
Cellular Reprogramming ◽  
Protein Stabilization ◽  
Biologically Active ◽  
Patient Specific ◽  
Tat Protein ◽  
Serum Replacement ◽  
Induced Pluripotent

Induced pluripotent stem (iPS) cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT) and Sox2 (Sox2-TAT) proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

scholarly journals Molecular Imaging, Pharmacokinetics, and Dosimetry of111In-AMBA in Human Prostate Tumor-Bearing Mice

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Chung-Li Ho ◽  
I-Hsiang Liu ◽  
Yu-Hsien Wu ◽  
Liang-Cheng Chen ◽  
Chun-Lin Chen ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Half Life ◽  
Biological Activities ◽  
Ct Imaging ◽  
Scid Mice ◽  
Prostate Tumor ◽  
Patient Specific ◽  
Specific Information ◽  
Tumor Bearing ◽  
Biodistribution Studies

Molecular imaging with promise of personalized medicine can provide patient-specific information noninvasively, thus enabling treatment to be tailored to the specific biological attributes of both the disease and the patient. This study was to investigate the characterization of DO3A-CH2CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH2(AMBA)in vitro, MicroSPECT/CT imaging, and biological activities of111In-AMBA in PC-3 prostate tumor-bearing SCID mice. The uptake of111In-AMBA reached highest with3.87±0.65% ID/g at 8 h. MicroSPECT/CT imaging studies suggested that the uptake of111In-AMBA was clearly visualized between 8 and 48 h postinjection. The distribution half-life (t1/2α) and the elimination half-life (t1/2β) of111In-AMBA in mice were 1.53 h and 30.7 h, respectively. TheCmax⁡and AUC of111In-AMBA were 7.57% ID/g and 66.39 h∗% ID/g, respectively. The effective dose appeared to be 0.11 mSv/MBq-1. We demonstrated a good uptake of111In-AMBA in the GRPR-overexpressed PC-3 tumor-bearing SCID mice.111In-AMBA is a safe, potential molecular image-guided diagnostic agent for human GRPR-positive tumors, ranging from simple and straightforward biodistribution studies to improve the efficacy of combined modality anticancer therapy.

scholarly journals Erratum: Corrigendum: Efficient derivation and inducible differentiation of expandable skeletal myogenic cells from human ES and patient-specific iPS cells

2015 ◽  
Vol 10 (9) ◽  
pp. 1457-1457 ◽  
Author(s):  
Sara M Maffioletti ◽  
Mattia F M Gerli ◽  
Martina Ragazzi ◽  
Sumitava Dastidar ◽  
Sara Benedetti ◽  
...  
Keyword(s):  
Ips Cells ◽  
Patient Specific ◽  
Myogenic Cells

scholarly journals Characterization of Hematopoietic Differentiation Profiles of MPN Patient-Derived Inducible Pluripotent Stem Cells Harboring Homozygous Vs Heterozygous Calreticulin Mutations

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3065-3065
Author(s):  
Lijuan Han ◽  
Marcelo A. Szymanski Toledo ◽  
Alexandre Theocharides ◽  
Angela Maurer ◽  
Tim H. Brümmendorf ◽  
...  
Keyword(s):  
Ips Cells ◽  
Patient Specific ◽  
Hematopoietic Differentiation ◽  
In Vitro Differentiation ◽  
Wild Type ◽  
Ips Cell ◽  
Cell Clones ◽  
Calr Mutations ◽  
The Impact

Abstract Introduction: Somatic calreticulin (CALR) mutations were discovered in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) and have been shown to be mutually exclusive with Janus kinase 2 (JAK2) and thrombopoietin receptor (MPL) mutations. Recent studies demonstrated that the binding of CALR mutant proteins to MPL induces constitutive activation of the JAK/STAT pathway, thus causing cellular transformation and abnormal megakaryopoiesis. Additionally, it has been reported that patients carrying homozygously mutated CALR ins5 exhibit myeloperoxidase (MPO) deficiency as a result of the absence of CALR chaperone function. However, the impact of CALR mutant homozygosity vs. heterozygosity in CALR del52 mutations as well as on hematopoietic differentiation has not yet been studied. Furthermore, clonal heterogeneity of hematopoietic stem/progenitor cell (HSPC) populations in a patient, together with technical limitations isolating single clones, are major challenges, when determining the impact of CALR mutant zygosity on clonal composition and diversity in MPN. To overcome these limitations, we generated patient-specific iPS cells carrying homozygous or heterozygous CALR mutations or their wild-type counterparts to study their roles in hematopoietic differentiation. Methods: iPS cells were generated by reprogramming peripheral blood-derived mononuclear cells from three patients carrying CALR del52, ins5, or del31 mutations using a CytoTune iPS 2.0 Sendai Reprogramming Kit. Individual colonies were picked and screened for CALR genotypes by PCR. Pluripotency of iPS cells was confirmed by immunofluorescences, and the clones were screened for additional mutations using panel-based next generation sequencing (NGS). Subsequently, CALR iPS cells were subjected to embryonic body formation, mesoderm commitment, and hematopoietic differentiation using our standard in vitro differentiation protocol. CD34+ HSPCs were MACS-sorted and characterized by flow cytometry, cytospins and RNA expression analysis on days 10, 15, and 20 during differentiation. Hematopoietic progenitors, erythrocytes, granulocytes, and megakaryocytes were identified by defined lineage markers. MPO expression was assessed by flow cytometry and cytochemical staining. Results: We established patient-specific iPS cells carrying CALR del52, ins5 or del31 mutation after written informed consent (Table 1). Pluripotency markers OCT4, Tra-1-60 and Tra-1-81 expression were confirmed in all iPS cell clones. In accordance with findings in peripheral blood cells, we detected MPO deficiency in homozygous iPS cell-derived CD15+ cells from CALRins5- and, in addition, also from CALRdel52-mutated patients (pMFI=0.0106 and pMFI=0.0187, resp.). Intriguingly, in vitro hematopoietic differentiation assays revealed additional abnormalities, such as decreased CD66b+ granulocytes derived from homozygous CALR del52 or ins5 iPS cells vs. heterozygous iPS cells on day 10 (pdel52=0.0303 and pins5=0.0253, resp.) and a trend towards increased KIThigh+CD45+ cells. Megakaryopoiesis, defined by CD41+CD42b+ cells, was increased in CALRins5 homozygous vs. heterozygous clones (p=0.0031). However, this bias was not observed in all clones, indicating clone-specific megakaryocytic differentiation potential. No phenotypic differences during hematopoietic differentiation were observed in iPS cell-derived progenitors carrying heterozygous CALRdel31 mutation and its isogenic unmutated CALR controls. Furthermore, our NGS data revealed patient-specific sets of co-occurring mutations in iPS cell clones, which may have contributed to the observed patient-specific phenotypes. As an example, the IDH2 R140Q mutation, reported to block cell differentiation, was found in approximately half of the CALRdel52 iPS clones, and these clones failed to differentiate into the hematopoietic lineage in vitro. Conclusions: We successfully generated patient-specific CALR mutant iPS cells. Upon in vitro differentiation, we detected MPO deficiency and aberrant granulocytic differentiation in CALR homozygous but not heterozygous or wild-type clones. Thus, it is now possible at the single stem cell level to further analyze the molecular mechanisms of CALR-mutant induced MPO deficiency and altered hematopoietic differentiation, in order to better understand disease biology in ET and PMF. Disclosures Brümmendorf: Merck: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Janssen: Consultancy; Takeda: Consultancy.

scholarly journals ALS patient-specific iPS cells

2012 ◽  
Vol 52 (11) ◽  
pp. 1137-1138 ◽  
Author(s):  
Haruhisa Inoue
Keyword(s):  
Ips Cells ◽  
Patient Specific

Telomere Elongation in Dyskeratosis Congenita Induced Pluripotent Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 497-497
Author(s):  
Suneet Agarwal ◽  
Yuin-Han Loh ◽  
Erin M McLoughlin ◽  
Junjiu Huang ◽  
In-Hyun Park ◽  
...  
Keyword(s):  
Telomere Length ◽  
Autosomal Dominant ◽  
Somatic Cells ◽  
Ips Cells ◽  
Dyskeratosis Congenita ◽  
Telomere Maintenance ◽  
Patient Specific ◽  
Self Renewal ◽  
Induced Pluripotent

Abstract Abstract 497 Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer premature degeneration of multiple tissues. Bone marrow failure is the principal cause of mortality, and allogeneic stem cell transplantation is limited by increased treatment-related mortality. Somatic cells can be reprogrammed using defined genetic and chemical factors, yielding “induced pluripotent stem” (iPS) cell lines which have the capacity to differentiate into any tissue. Patient-specific iPS cells therefore hold promise as therapeutic agents and disease models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, and we have found that telomere length is increased in human iPS cells relative to the normal primary somatic cells from which they are derived. Here we investigated whether defects in telomerase function would limit derivation or self-renewal of iPS cells from patients with DC. We reprogrammed primary fibroblasts from patients with X-linked and autosomal dominant DC, caused by mutations in the genes encoding dyskerin and telomerase RNA component (TERC), respectively. We were able to establish multiple DC-specific iPS lines showing all hallmarks of pluripotency, including the formation of hematopoietic progenitors in vitro. Unexpectedly, DC-specific iPS cells were able to sustain continual proliferation in vitro, in contrast to the premature senescence displayed by the DC fibroblasts. Although early passage DC iPS cells had shorter telomeres than donor fibroblasts, we found that telomere length in DC iPS cells increased with continued passage in culture. To explain this finding, we discovered that steady state levels of TERC, which are critically limiting in several forms of DC, are upregulated in normal and DC iPS cells. We found that TERC upregulation is a feature of the pluripotent state, that the TERC locus is a target of pluripotency-associated transcription factors, and that transcriptional silencing accompanies a 3' deletion at the TERC locus in autosomal dominant DC. Our results demonstrate that reprogramming restores self-renewal capacity in DC cells despite genetic lesions affecting telomerase, and suggest that strategies to enhance endogenous TERC expression may be feasible and therapeutically beneficial in DC patients. The studies demonstrate the value of patient-specific iPS cells for basic and translational discovery, and further the rationale for autologous iPS based cellular therapy of genetic hematologic disorders. Disclosures: Daley: MPM Capital: Consultancy; Solasia: Consultancy; Epizyme: Consultancy; iPierian: Consultancy, Equity Ownership.

Genomic Safe Harbors in Human iPS Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-47-SCI-47
Author(s):  
Eirini P. Papapetrou ◽  
Michel Sadelain ◽  
Frederic Bushman
Keyword(s):  
Genetic Engineering ◽  
Human Genome ◽  
Ips Cells ◽  
Human Cells ◽  
Patient Specific ◽  
Safe Harbor ◽  
Ips Cell ◽  
Hematopoietic Stem ◽  
Cell Clones ◽  
Human Ips Cells

Abstract Abstract SCI-47 Current hematopoietic stem cell (HSC) gene therapy relies on randomly integrated retroviral vectors and is hampered by the risk of insertional oncogenesis often leading to leukemia. This risk would be minimized if therapeutic transgenes could be inserted in selected sites of the genome that permit appropriate function without disruption or dysregulation of endogenous genes — referred to as “genomic safe harbors.” The advent of induced pluripotent stem (iPS) cell technology offered unprecedented opportunities for the genetic engineering of human cells. iPS cells, unlike HSC, can be extensively cultured in vitro, enabling the selection and study of unique sites of transgene integration for the first time in a relevant setting. We proposed a definition of safe harbor sites, based on their topology in the genome with relation to coding genes and other genomic landmarks, using five criteria: (i) distance of at least 50 kb from the 5’ end of any gene, (ii) distance of at least 300 kb from any cancer-related gene, (iii) distance of at least 300 kb from any microRNA, (iv) location outside a transcription unit, and (v) location outside ultraconserved regions of the human genome (1). To test them, we developed a strategy to select iPS cell clones harboring a single copy of a randomly integrating vector at sites that meet our safe harbor criteria. In a recent proof-of-principle study, using a model for genetic correction of β-thalassemia major, we demonstrated that erythroid progeny of patient-specific iPS cell clones harboring a lentivirally encoded β-globin transgene in a safe harbor site express therapeutic levels of β-globin without perturbing neighboring genes. This approach, entailing the prospective screening and selection of integration sites, based on combined bioinformatics and functional analyses, provides a robust and dependable strategy for the genetic engineering of human iPS cells. iPS cell-derived cell products used in regenerative medicine will need to be genetically engineered to correct a genetic disease or permit in vivo cell tracking or the elimination of residual undifferentiated cells or progeny gone astray. Our strategy should be broadly applicable to introducing reporter, suicide, or therapeutic genes in a clinically relevant manner. We are currently exploiting this strategy to express a conditional HSV-tk suicide gene for purging of iPS cell progeny from teratoma-initiating cells. With the emergence of improved technologies for homologous recombination into human cells, targeted gene addition may soon become a realistic option if predefined validated safe harbor sites in the human genome are identified. We are utilizing our selection strategy in iPS cells (using lentiviral vectors with reporter cassettes that can be exchanged using Cre recombinase-mediated cassette exchange) as a platform for the de novo discovery and characterization of putative universal safe harbor sites that can be broadly used for the genetic engineering of multiple human cell types. Disclosures: No relevant conflicts of interest to declare.

Induced human pluripotent stem cells: promises and open questions

2009 ◽  
Vol 390 (9) ◽  
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.

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

Blood ◽  
2009 ◽  
Vol 114 (27) ◽  
pp. 5473-5480 ◽  
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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