Stem Cell Responsiveness to Imatinib in Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease characterized by the presence of the BCR-ABL fusion gene, which results from the Philadelphia chromosome. Since the introduction of tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM), the clinical outcomes for patients with CML have improved significantly. However, IM resistance remains the major clinical challenge for many patients, underlining the need to develop new drugs for the treatment of CML. The basis of CML cell resistance to this drug is unclear, but the appearance of additional genetic alterations in leukemic stem cells (LSCs) is the most common cause of patient relapse. However, several groups have identified a rare subpopulation of CD34+ stem cells in adult patients that is present mainly in the bone marrow and is more immature and pluripotent; these cells are also known as very small embryonic-like stem cells (VSELs). The uncontrolled proliferation and a compromised differentiation possibly initiate their transformation to leukemic VSELs (LVSELs). Their nature and possible involvement in carcinogenesis suggest that they cannot be completely eradicated with IM treatment. In this study, we demonstrated that cells from CML patients with the VSELs phenotype (LVSELs) similarly harbor the fusion protein BCR-ABL and are less sensitive to apoptosis than leukemic HSCs after IM treatment. Thus, IM induces apoptosis and reduces the proliferation and mRNA expression of Ki67 more efficiently in LHSCs than in leukemic LVSELs. Finally, we found that the expression levels of some miRNAs are affected in LVSELs. In addition to the tumor suppressor miR-451, both miR-126 and miR-21, known to be responsible for LSC leukemia-initiating capacity, quiescence, and growth, appear to be involved in IM insensitivity of LVSELs CML cell population. Targeting IM-resistant CML leukemic stem cells by acting via the miRNA pathways may represent a promising therapeutic option.

Development of a potency assay for CD34+ cell-based therapy.

We have previously shown that intracardiac delivery of autologous CD34+ cells after acute myocardial infarction (AMI) is safe and leads to long term improvement. We are now conducting a multicenter, randomized, controlled Phase I/IIb study in post-AMI to investigate the safety and efficacy of intramyocardial injection of expanded autologous CD34+ cells (ProtheraCytes) (NCT02669810). Here, we conducted a series of in vitro studies characterizing the growth factor secretion, exosome secretion, gene expression, cell surface markers, differentiation potential, and angiogenic potential of ProtheraCytes clinical batches to develop a potency assay. We show that ProtheraCytes secrete vascular endothelial growth factor (VEGF) and its concentration is significantly correlated with the number of CD34+ cells obtained after expansion. ProtheraCytes also secrete exosomes containing proangiogenic miRNAs (126, 130a, 378, 26a), antiapoptotic miRNAs (21 and 146a), antifibrotic miRNAs (133a, 24, 29b, 132), and miRNAs promoting myocardial regeneration (199a and 590). We also show that ProtheraCytes have in vitro angiogenic activity, express surface markers of endothelial progenitor cells, and can differentiate in vitro into endothelial cells. After the in vitro characterization of multiple ProtheraCytes clinical batches, we established that measuring the concentration of VEGF provided the most practical, reliable, and consistent potency assay.

Deciphering the Cardiovascular Potential of Human CD34+ Stem Cells.

Ex vivo monitored human CD34+ stem cells (SCs) injected into myocardium scar tissue have shown real benefits for the recovery of patients with myocardial infarctions. They have been used previously in clinical trials with hopeful results and are expected to be promising for cardiac regenerative medicine following severe acute myocardial infarctions. However, some debates on their potential efficacy in cardiac regenerative therapies remain to be clarified. To elucidate the levels of CD34+ SC implication and contribution in cardiac regeneration, better identification of the main regulators, pathways, and genes involved in their potential cardiovascular differentiation and paracrine secretion needs to be determined. We first developed a protocol thought to commit human CD34+ SCs purified from cord blood toward an early cardiovascular lineage. Then, by using a microarray-based approach, we followed their gene expression during differentiation. We compared the transcriptome of undifferentiated CD34+ cells to those induced at two stages of differentiation (i.e., day three and day fourteen), with human cardiomyocyte progenitor cells (CMPCs), as well as cardiomyocytes as controls. Interestingly, in the treated cells, we observed an increase in the expressions of the main regulators usually present in cardiovascular cells. We identified cell surface markers of the cardiac mesoderm, such as kinase insert domain receptor (KDR) and the cardiogenic surface receptor Frizzled 4 (FZD4), induced in the differentiated cells in comparison to undifferentiated CD34+ cells. The Wnt and TGF-ß pathways appeared to be involved in this activation. This study underlined the real capacity of effectively stimulated CD34+ SCs to express cardiac markers and, once induced, allowed the identification of markers that are known to be involved in vascular and early cardiogenesis, demonstrating their potential priming towards cardiovascular cells. These findings could complement their paracrine positive effects known in cell therapy for heart disease and may help improve the efficacy and safety of using ex vivo expanded CD34+ SCs.

Industrialized GMP Production of CD34+ Cells (ProtheraCytes®) at Clinical Scale for Treatment of Ischemic Cardiac Diseases Is Feasible and Safe.

Regenerative medicine now needs to pass a crucial turning point, from academic research to the market. Several sources/types of cells have been experimented with, more or less successfully. CD34+ cells have demonstrated multipotent or even pluripotent capacities, making them good candidates for regenerative medicine, particularly for treating heart diseases. Strongly encouraged by the results we achieved in a pilot study using CD34+ stem cells in patients with poor-prognosis acute myocardial infarcts (AMIs), we soon began the development of an industrialized platform making use of a closed automated device (StemXpand®) and a disposable kit (StemPack®) for the large-scale expansion of CD34+ cells with reproducible good manufacturing practice (GMP). This scalable platform can produce expanded CD34+ cells (ProtheraCytes®) of sufficient quality that, interestingly, express early markers of the cardiac and endothelial pathways and early cardiac-mesoderm markers. They also contain CD34+ pluripotent cells characterized as very small embryonic-like stem cells (VSELs), capable of differentiating under appropriate stimuli into different tissue lineages, including endothelial and cardiomyocytic ones.

Differential Expression of the Tetraspanin CD9 in Normal and Leukemic Stem Cells.

CD9 plays a crucial role in cellular growth, mobility, and signal transduction, as well as in hematological malignancy. In myeloid neoplasms, CD9 is involved in the altered interactions between leukemic and stromal cells. However, apart from its role in CD34+ progenitors and myeloid and megakaryocytic differentiation, its function in normal and leukemic pluripotent cells has not yet been determined. Very small embryonic-like stem cells (VSELs) are promising pluripotent stem cells found in adult tissues that can be developed for safe and efficient regenerative medicine. VSELs express different surface receptors of the highest importance in cell functioning, including CD9, and can be effectively mobilized after organ injury or in leukemic patients. In the present study, we observed that CD9 is among the most expressed receptors in VSELs under steady-state conditions; however, once the VSELs are expanded, CD9+ VSELs decrease and are more apoptotic. CD9- VSELs had no proliferative improvement in vitro compared to those that were CD9+. Interestingly, the addition of SDF-1 induced CD9 expression on the surface of VSELs, as observed by flow cytometry, and improved their migration. In addition, we observed, in the phenotypically identical VSELs present in the peripheral blood of patients with myeloproliferative neoplasms, compared to healthy subjects, a significantly higher number of CD9+ cells. However, in their hematopoietic stem cell (HSC) counterparts, the expression remained comparable. These results indicate that, likewise, in progenitors and mature cells, CD9 may play an important function in normal and malignant VSELs. This could explain the refractoriness observed by some groups of expanded stem cells to repairing efficiently damaged tissue when used as a source in cell therapies. Understanding the function of the CD9 receptor in normal and malignant CD34+ and VSELs, along with its relationship with the CXCR4/SDF-1 pathway, will enable advances in the field of adult pluripotent cell usage in regenerative medicine and in their role in leukemia.

Design and Validation of an Automated Process for the Expansion of Peripheral Blood-Derived CD34+ Cells for Clinical Use After Myocardial Infarction.

We previously demonstrated that intracardiac delivery of autologous peripheral blood-derived CD34+ stem cells (SCs), mobilized by granulocyte-colony stimulating factor (G-CSF) and collected by leukapheresis after myocardial infarction, structurally and functionally repaired the damaged myocardial area. When used for cardiac indication, CD34+ cells are now considered as Advanced Therapy Medicinal Products (ATMPs). We have industrialized their production by developing an automated device for ex vivo CD34+ -SC expansion, starting from a whole blood (WB) sample. Blood samples were collected from healthy donors after G-CSF mobilization. Manufacturing procedures included: (a) isolation of total nuclear cells, (b) CD34+ immunoselection, (c) expansion and cell culture recovery in the device, and (d) expanded CD34+ cell immunoselection and formulation. The assessment of CD34+ cell counts, viability, and immunophenotype and sterility tests were performed as quality tests. We established graft acceptance criteria and performed validation processes in three cell therapy centers. 59.4 × 106 ± 36.8 × 106 viable CD34+ cells were reproducibly generated as the final product from 220 ml WB containing 17.1 × 106 ± 8.1 × 106 viable CD34+ cells. CD34+ identity, genetic stability, and telomere length were consistent with those of basal CD34+ cells. Gram staining and mycoplasma and endotoxin analyses were negative in all cases. We confirmed the therapeutic efficacy of both CD34+ -cell categories in experimental acute myocardial infarct (AMI) in immunodeficient rats during preclinical studies. This reproducible, automated, and standardized expansion process produces high numbers of CD34+ cells corresponding to the approved ATMP and paves the way for a phase I/IIb study in AMI, which is currently recruiting patients. Stem Cells Translational Medicine 2019;8:822&832.

VSELs Maintain their Pluripotency and Competence to Differentiate after Enhanced Ex Vivo Expansion.

The very small embryonic-like stem cells (VSELs) are known as a subset of adult pluripotent stem cells able to differentiate to all three germ layers. However, their small number and quiescence restrict the possibility of their use in cell therapy. In the present study, we first delineate different subpopulation of VSELs from human cord blood CD34+ cells to define their purity. We next determine genes expression levels in the whole transcriptome of VSELs expressing the pluripotent marker NANOG and control cells under the steady state condition. We found that more than a thousand of genes are downregulated in VSELs, as well as many membrane receptors, cells signaling molecules and CDKs mRNAs. In addition, we observed discordance in some pluripotent genes expression levels with embryonic stem cells (ESCs), which could explain VSELs quiescence. We then evaluate VSELs capacity to expand and differentiate in vitro in specific and appropriate media. After 12 days culture in specific medium containing a pyrimidoindole derivative (UM171), VSELs were significantly expanded for the first time without feeder cells and importantly preserve their capacities to differentiate into hematopoietic and endothelial cells. Interestingly, this stimulation of VSELs self-renewal restores the expression of some downregulated genes known as key regulators of cell proliferation and differentiation. The properties of such pluripotent expanded cells make them a potential candidate in regenerative medicine.

Apelin: an antithrombotic factor that inhibits platelet function.

Apelin peptide and its receptor APJ are directly implicated in various physiological processes ranging from cardiovascular homeostasis to immune signaling. Here, we show that apelin is a key player in hemostasis with an ability to inhibit thrombin- and collagen-mediated platelet activation. Mice lacking apelin displayed a shorter bleeding time and a prothrombotic profile. Their platelets exhibited increased adhesion and a reduced occlusion time in venules, and displayed a higher aggregation rate after their activation by thrombin compared with wild-type platelets. Consequently, human and mouse platelets express apelin and its receptor APJ. Apelin directly interferes with thrombin-mediated signaling pathways and platelet activation, secretion, and aggregation, but not with ADP and thromboxane A2-mediated pathways. IV apelin administration induced excessive bleeding and prevented thrombosis in mice. Taken together, these findings suggest that apelin and/or APJ agonists could potentially be useful adducts in antiplatelet therapies and may provide a promising perspective for patients who continue to display adverse thrombotic events with current antiplatelet therapies.

Ageing is a risk factor in imatinib mesylate cardiotoxicity.

AIMS: Chemotherapy-induced heart failure is increasingly recognized as a major clinical challenge. Cardiotoxicity of imatinib mesylate, a highly selective and effective anticancer drug belonging to the new class of tyrosine kinase inhibitors, is being reported in patients, some progressing to congestive heart failure. This represents an unanticipated challenge that could limit effective drug use. Understanding the mechanisms and risk factors of imatinib mesylate cardiotoxicity is crucial for prevention of cardiovascular complications in cancer patients. METHODS AND RESULTS: We used genetically engineered mice and primary rat neonatal cardiomyocytes to analyse the action of imatinib on the heart. We found that treatment with imatinib (200 mg/kg/day for 5 weeks) leads to mitochondrial-dependent myocyte loss and cardiac dysfunction, as confirmed by electron microscopy, RNA analysis, and echocardiography. Imatinib cardiotoxicity was more severe in older mice, in part due to an age-dependent increase in oxidative stress. Mechanistically, depletion of the transcription factor GATA4 resulting in decreased levels of its prosurvival targets Bcl-2 and Bcl-XL was an underlying cause of imatinib toxicity. Consistent with this, GATA4 haploinsufficient mice were more susceptible to imatinib, and myocyte-specific up-regulation of GATA4 or Bcl-2 protected against drug-induced cardiotoxicity. CONCLUSION: The results indicate that imatinib action on the heart targets cardiomyocytes and involves mitochondrial impairment and cell death that can be further aggravated by oxidative stress. This in turn offers a possible explanation for the current conflicting data regarding imatinib cardiotoxicity in cancer patients and suggests that cardiac monitoring of older patients receiving imatinib therapy may be especially warranted.

Cyclin D2 rescues size and function of GATA4 haplo-insufficient hearts.

Transcription factor GATA4 is a key regulator of cardiomyocyte growth, and differentiation and 50% reduction in GATA4 levels results in hypoplastic hearts. Search for GATA4 targets/effectors revealed cyclin D(2) (CD2), a member of the D-type cyclins (D(1), D(2), and D(3)) that play a vital role in cell growth and differentiation as a direct transcriptional target and a mediator of GATA4 growth in postnatal cardiomyocytes. GATA4 associates with the CD2 promoter in cardiomyocytes and is sufficient to induce endogenous CD2 transcription and to dose-dependently activate the CD2 promoter in heterologous cells. Cardiomyocyte-specific overexpression of CD2 results in enhanced postnatal cardiac growth because of increased cardiomyocyte proliferation. When these transgenic mice are crossed with Gata4 heterozygote mice, they rescue the hypoplastic cardiac phenotype of Gata4(+/-) mice and enhance cardiomyocyte survival and heart function. The data uncover a role for CD2 in the postnatal heart as an effector of GATA4 in myocyte growth and survival. The finding that postnatal upregulation of a cell-cycle gene in GATA4 haplo-insufficient hearts may be protective opens new avenues for maintaining or restoring cardiac function in GATA4-dependent cardiac disease.

Mechanisms of angiotensin II-dependent progression to heart failure.

Up-regulation of angiotensin II (AII) signalling plays an important role in the pathogenesis of cardiac hypertrophy and failure as evidenced by the efficacy of AII receptor blockers or inhibitors of AII biosynthesis in reversing ventricular hypertrophy and preventing human heart failure. The mechanisms underlying AII action in the heart remain undefined. Myocardial-specific expression of the AII type 1 receptor (AT1R) in mice is sufficient for inducing progressive myocyte hypertrophy and cardiac remodelling leading to adult heart failure with a disease progression course reminiscent of work overload-induced human heart failure. We examined the functional, structural and genetic changes associated with disease progression in this model. The results reveal that AT1R-dependent interaction between myocytes and non-myocytes is critical in cardiac remodelling. At the level of cardiomyocytes, decreased mitochondrial function is one of the earliest events of AII action leading to mitochondrial depletion and increased apoptosis. Up-regulation of cardiac Bcl-2 prevents mitochondrial deterioration, cardiomyocyte loss and pathologic remodelling. Importantly, Bcl-2 completely rescues premature death due to heart failure and maintains the 'compensated' state. The data suggest that targeting Bcl-2 or interfering with mitochondrial dysfunction may offer new therapeutic opportunities for preventing transition from compensated hypertrophy to heart failure.

Convergence of protein kinase C and JAK-STAT signaling on transcription factor GATA-4.

Angiotensin II (AII), a potent vasoactive hormone, acts on numerous organs via G-protein-coupled receptors and elicits cell-specific responses. At the level of the heart, AII stimulation alters gene transcription and leads to cardiomyocyte hypertrophy. Numerous intracellular signaling pathways are activated in this process; however, which of these directly link receptor activation to transcriptional regulation remains undefined. We used the atrial natriuretic factor (ANF) gene (NPPA) as a marker to elucidate the signaling cascades involved in AII transcriptional responses. We show that ANF transcription is activated directly by the AII type 1 receptor and precedes the development of myocyte hypertrophy. This response maps to STAT and GATA binding sites, and the two elements transcriptionally cooperate to mediate signaling through the JAK-STAT and protein kinase C (PKC)-GATA-4 pathways. PKC phosphorylation enhances GATA-4 DNA binding activity, and STAT-1 functionally and physically interacts with GATA-4 to synergistically activate AII and other growth factor-inducible promoters. Moreover, GATA factors are able to recruit STAT proteins to target promoters via GATA binding sites, which are sufficient to support synergy. Thus, STAT proteins can act as growth factor-inducible coactivators of tissue-specific transcription factors. Interactions between STAT and GATA proteins may provide a general paradigm for understanding cell specificity of cytokine and growth factor signaling.

Essential role of GATA-4 in cell survival and drug-induced cardiotoxicity.

In recent years, significant progress has been made in understanding cardiomyocyte differentiation. However, little is known about the regulation of myocyte survival despite the fact that myocyte apoptosis is a leading cause of heart failure. Here we report that transcription factor GATA-4 is a survival factor for differentiated, postnatal cardiomyocytes and an upstream activator of the antiapoptotic gene Bcl-X. An early event in the cardiotoxic effect of the antitumor drug doxorubicin is GATA-4 depletion, which in turn causes cardiomyocyte apoptosis. Mouse heterozygotes for a null Gata4 allele have enhanced susceptibility to doxorubicin cardiotoxicity. Genetic or pharmacologic enhancement of GATA-4 prevents cardiomyocyte apoptosis and drug-induced cardiotoxicity. The results indicate that GATA-4 is an antiapoptotic factor required for the adaptive stress response of the adult heart. Modulation of survival/apoptosis genes by tissue-specific transcription factors may be a general paradigm that can be exploited effectively for cell-specific regulation of apoptosis in disease states.