scholarly journals Induced Pluripotent Stem Cells to Model Juvenile Myelomonocytic Leukemia: New Perspectives for Preclinical Research

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2335
Author(s):  
Zeinab Wehbe ◽  
Foued Ghanjati ◽  
Christian Flotho
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Progenitor Cells ◽  
Pluripotent Stem Cells ◽  
Treatment Modalities ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Hematopoietic Stem ◽  
Induced Pluripotent ◽  
Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a malignant myeloproliferative disorder arising in infants and young children. The origin of this neoplasm is attributed to an early deregulation of the Ras signaling pathway in multipotent hematopoietic stem/progenitor cells. Since JMML is notoriously refractory to conventional cytostatic therapy, allogeneic hematopoietic stem cell transplantation remains the mainstay of curative therapy for most cases. However, alternative therapeutic approaches with small epigenetic molecules have recently entered the stage and show surprising efficacy at least in specific subsets of patients. Hence, the establishment of preclinical models to test novel agents is a priority. Induced pluripotent stem cells (IPSCs) offer an opportunity to imitate JMML ex vivo, after attempts to generate immortalized cell lines from primary JMML material have largely failed in the past. Several research groups have previously generated patient-derived JMML IPSCs and successfully differentiated these into myeloid cells with extensive phenotypic similarities to primary JMML cells. With infinite self-renewal and the capability to differentiate into multiple cell types, JMML IPSCs are a promising resource to advance the development of treatment modalities targeting specific vulnerabilities. This review discusses current reprogramming techniques for JMML stem/progenitor cells, related clinical applications, and the challenges involved.

Patient-Derived Induced Pluripotent Stem Cells Recapitulate Hematopoietic Abnormalities of Juvenile Myelomonocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 637-637
Author(s):  
Gandre-Babbe Shilpa ◽  
Stella T Chou ◽  
Deborah L. French ◽  
Michelle Kang ◽  
Julie Weng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Vector System ◽  
Juvenile Myelomonocytic Leukemia ◽  
Malignant Cells ◽  
Gm Csf ◽  
Induced Pluripotent ◽  
Myelomonocytic Leukemia

Abstract Abstract 637 Juvenile Myelomonocytic Leukemia (JMML) is an aggressive childhood myeloproliferative disorder caused by NF1, NRAS, KRAS, PTPN11 or CBL gene mutations that induce Ras pathway activation with associated hypersensitivity to cytokine stimulation in myeloid progenitor cells. Understanding the pathophysiology of JMML and developing new treatments is constrained by limited access to relevant patient material. To address this problem, we generated induced pluripotent stem cells (iPSCs) from normal neonatal umbilical cord blood and two JMML patients with different mutations: 1) a somatic heterozygous E76K substitution in PTPN11 and 2) a CBL Y371H substitution arising from a germline mutation. We created iPSCs from the patient's heterozygous CBL Y371H newborn cord blood cells prior to the diagnosis of JMML and then from peripheral blood at age 7 months, when JMML ensued with outgrowth of CBL Y371H homozygous malignant cells. We reprogrammed control and malignant cells using the STEMCCA lentiviral vector system that expresses OCT4, KLF4, MYC and SOX2 in a doxycyline-inducible fashion. Resultant iPSCs exhibited hallmark features after more than twenty passages, including characteristic morphology, expression of endogenous pluripotency markers, silencing of viral reprogramming genes in the absence of doxycycline, normal karyotype and formation of endoderm-, ectoderm- and mesoderm-derived tissues in teratoma assays. Relevant PTPN11 and CBL genotypes of these clones were confirmed by DNA sequencing. Selected iPSC clones were differentiated into blood by inducing the formation of embryoid bodies in serum free medium with defined cytokines. By day 8–9, hematopoietic progenitors (CD43+, CD235+, CD41+) with erythroid, megakaryocytic and myeloid potential developed from both control (n = 2 lines) and JMML iPSCs (n= 2 lines from each patient). In methylcellulose colony assays, JMML progenitors exhibited relative hypersensitivity to GM-CSF, as reflected by increased numbers and larger size of myeloid colonies at limiting GM-CSF concentrations. In liquid cultures containing SCF, TPO, EPO, IL-3, IL-11 and IGF-1, with or without GM-CSF, the JMML progenitors produced increased proportions of CD33+CD14+ myelomonocytic cells compared to controls. Moreover, in GM-CSF dose-response assays, single cell phospho-flow cytometry analysis showed sustained STAT5 activation in JMML myeloid cells vs. controls. Thus, key pathological features of JMML, including propensity to myelomonocytic cell expansion and GM-CSF hypersensitivity, are recapitulated by in vitro differentiation of JMML iPSCs. Our findings illustrate the utility of iPSCs for modeling human blood disorders and more specifically, provide renewable sources of biologically relevant, patient-derived cells in which to explore the pathophysiology and therapy of JMML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Patient-derived induced pluripotent stem cells recapitulate hematopoietic abnormalities of juvenile myelomonocytic leukemia

Blood ◽  
2013 ◽  
Vol 121 (24) ◽  
pp. 4925-4929 ◽  
Author(s):  
Shilpa Gandre-Babbe ◽  
Prasuna Paluru ◽  
Chiaka Aribeana ◽  
Stella T. Chou ◽  
Silvia Bresolin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Juvenile Myelomonocytic Leukemia ◽  
Mek Inhibition ◽  
Gm Csf ◽  
Key Points ◽  
Induced Pluripotent ◽  
Myelomonocytic Leukemia

Key Points Patient-derived iPSCs recapitulate juvenile myelomonocytic leukemia. MEK inhibition normalizes GM-CSF independence and hypersensitivity in myeloid precursors from JMML iPSCs.

CRISPR/Cas9-mediated genome editing has demonstrated significant promise for genetic correction in autologous hematopoietic stem/progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs)

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Induced Pluripotent Stem Cells ◽  
Progenitor Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Genetic Disorders ◽  
Hematopoietic Stem ◽  
Hematological Disorders ◽  
Induced Pluripotent

According to current research, CRISPR/Cas9-mediated genome editing has shown enormous potential in the correction of genetic defects in autologous hematopoietic stem/progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). Furthermore, the advancement of iPSC reprogramming technology as well as the CRISPR/Cas9 system has opened the door to new possibilities in the field of gene and cell therapy combinations. Despite the fact that there are a number of technological obstacles to overcome, CRISPR/Cas9 remains a promising therapeutic method with a great deal of potential for future gene therapy applications. Early success in treating hereditary hematological disorders opens the door to new options for treating other genetic disorders and constitutes a significant step forward in the development of gene therapy.

scholarly journals Investigation of New Therapeutic Compounds for Juvenile Myelomonocytic Leukemia Using Induced Pluripotent Stem Cells with Stably Activated Ras Pathway

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4651-4651
Author(s):  
Lisa Maria Kuhn ◽  
Cyrill Schipp ◽  
Daniel Hein ◽  
Bianca Killing ◽  
Nan Qin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Hematopoietic Stem ◽  
Ras Pathway ◽  
Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a chronic, poor prognostic myeloid neoplasm of childhood that is characterized by malignant expansion of monocytic cells. Chemo- and radiotherapy are not effective in JMML, therefore allogeneic hematopoietic stem cell transplantation is the only therapy option for most affected children. Relapse is the most frequent cause of treatment failure and event-free-survival at five years is low (approximately 50%). Recent studies showed that in 90% of JMML patients the proliferation of monocytic tumor cells is driven by mutations in a confined set of genes (KRAS, NRAS, PTPN11, NF1 or CBL) that activate the RAS signalling pathway. Drugs specifically targeting this pathway are therefore attractive candidates for therapy of JMML patients. As in vitro models of JMML, we generated inducible pluripotent stem cells (iPSC) stably expressing wildtype or activating oncogenic versions of KRAS (G12D) or NRAS (G13D) as well as iPSCs with CRISPR interference mediated downregulated NF1 expression. Manipulation of KRAS, NRAS, and NF1 expression and activation of downstream signaling targets (MEK, ERK) of the Ras pathway were confirmed by RT-PCR and western blot analyses, respectively. After transduction iPSCs retained typical pluripotency markers and could be differentiated into CD34+ and CD45+ cells of the hematopoietic lineage. We then carried out a screen to test the response of these iPSC cell lines to experimental and clinical drugs targeting the Ras signaling pathway, as well as to other compounds suggested to be promising candidate drugs or drugs already in clinical trial for JMML. In our screen the model cell lines were resistant to all tested MEK-inhibitors, including Selumetinib and Trametinib. The broad receptor tyrosine kinase inhibitor Dovitinib and the DNA methyltransferase inhibitor Azacytidine elicited strong responses in all iPSC cell lines regardless of their KRAS, NRAS or NF1 state. This underlines their extensive, but non-targeted killing potential. In our screen, an experimental small molecule drug induced significantly more cell death in KRAS-G12D iPSCs (IC50 1.5 µM) than in comparable wildtype cells (IC50 3.3 µM, p<0.0001), which could be validated in independent assays. In addition to targeted cell death activation, the drug has been suggested to promote differentiation of hematopoietic cells, which could potentially increase its anti-tumor efficiency. Experimental studies analyzing the underlying mechanism of its differential effect on KRAS wildtype compared to KRAS-G12D cells are currently carried out and will be presented. Our results suggest, that iPSCs with RAS pathway activation due to stable expression of oncogenic KRAS or NRAS or downregulation of NF1 expression are valuable tools for preclinical testing and may identify promising novel lead compounds for JMML treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Transgene-free hematopoietic stem and progenitor cells from human induced pluripotent stem cells

2017 ◽  
Author(s):  
Laurence Guyonneau-Harmand ◽  
Bruno L’Homme ◽  
Brigitte Birebent ◽  
Christophe Desterke ◽  
Nathalie Chevallier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Progenitor Cells ◽  
Cell Population ◽  
Pluripotent Stem Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Bona Fide ◽  
Transgene Delivery ◽  
Induced Pluripotent

Introductory paragraphThe successful production of Hematopoietic Stem and Progenitor Cells (HSPCs) from human pluripotent sources is conditioned by transgene delivery1-5. We describe here a dedicated and tractable one step, GMP-grade, transgene-free and stroma-free protocol to produce HSPCs from human induced pluripotent stem cells (hiPSCs). This procedure, applied to several sources of hiPSCs with equal efficiency, is based on a directed differentiation with morphogens and cytokines to generate a cell population close to nascent HSPCs or their immediate forerunners i.e., hemogenic endothelial cells6-9. Following engraftment into immunocompromised recipients, this cell population was proved capable of a robust myeloid, lymphoid and definitive red blood cell production in sequential recipients for at least 40 weeks. Further identification of the repopulating cells show that they express the G protein–coupled receptor APELIN (APLNR) and the homing receptor CXCR4. This demonstrates that the generation of bona fide HSPCs from hiPSCs without transgenes is possible and passes through an early endo-hematopoietic intermediate. This work opens the way to the generation of clinical grade HSPCs for the treatment of hematological diseases and holds promise for the analysis of HSPC development in the human species.

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