Search

Background: Acute myocardial infarction (AMI) results from obstruction of a coronary artery and affects around 126 million individuals globally. We have shown in a small pilot study that intracardiac delivery of autologous CD34+ cells after myocardial infarction is safe and leads to long term improvement. The improvement was such that three patients initially recommended for early heart transplantation in this study, no longer required it years after the cell injection. After the successful results of this pilot study, we started 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). While patient recruitment is ongoing, we have developed a potency assay for the batch release of ProtheraCytes®. Methods: The proposed mechanisms of action by which ProtheraCytes® promote cardiac regeneration and revascularisation of damaged myocardial tissue is via the secretion of angiogenic factors and endothelial differentiation. 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. Results: Here 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 (Pearson r = 0.7484; p-value = 0.0009). 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 and cardiomyocyte progenitor cells, and can differentiate in vitro into endothelial cells. Conclusions: The potency assay should represent the product's mechanism of action, quantitatively measure the relevant biological product attribute, and have lot to lot consistency. Developing a potency assay is a required step before commencing the pivotal Phase 3 clinical studies. 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.

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.

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.

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. Electronic supplementary material The online version of this article (10.1007/s12015-018-9821-1) contains supplementary material, which is available to authorized users.

SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. Here we identify the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and show that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, our observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression.

Confocal laser microspectrofluorometry was used to investigate restoration of nuclear pirarubicin (THP-DOX) accumulation and sensitivity by verapamil, quinine and S9788 in 2 variants of the Chinese hamster ovary cell lines LR73, selected for resistance to doxorubicin (LR73D) or transfected with the mdr1 gene (LR73R). The 2 resistant cell lines present a multidrug-resistance phenotype (MDR). Verapamil and S9788, which interact with P-glycoprotein (P-gp), were able to restore nuclear THP-DOX accumulation in LR73R and LR73D cells to a level equivalent to that in sensitive cells. On the other hand, quinine was unable to increase nuclear THP-DOX accumulation significantly even at a concentration of 50 μM. All modulators completely restored THP-DOX sensitivity in resistant cell lines. Our results also show that verapamil and S9788 allow high nuclear drug accumulation, whereas quinine did not affect nuclear accumulation. The effect of quinine on the mdr1 gene expression was determined by the use of reverse transcription coupled with polymerase chain reaction. After a 2 hr treatment with 20 μM of quinine, mdr1 gene expression increased slightly. Int. J. Cancer 73:600–606, 1997. © 1997 Wiley-Liss, Inc.

Tumor cells, and particularly leukemic cells, can be considered as maturation-arrested cells which have escaped some normal control and continue to proliferate. This maturation arrest can be reversed by differentiation agents such as antitumor drugs currently used in conventional cytotoxic chemotherapy. In this respect, anthracyclines have been shown to trigger the differentiation of leukemic and solid tumor cells, but the molecular mechanisms by which such drugs lead to the differentiating phenotype are still poorly understood. Using human leukemic multipotent K562 cells, we have demonstrated that subtoxic concentrations of aclacinomycin (ACLA) and doxorubicin (DOX) preferentially stimulate the hemoglobinic pathway (globins and heme synthesis) and the expression of mRNAs of globins and of porphobilinogen deaminase (PBGD). However, our results indicate that both drugs exert this differentiating effect along distinct regulatory pathways. Indeed, only ACLA and not DOX induces the expression of erythropoietin receptor (EpoR) mRNAs and of membrane EpoR, as well as an overexpression of the erythroid transcription factors GATA-1 and NF-E2 known to play a central role in erythroid gene regulation. Similarly, using transfection assays, ACLA but not DOX activates the regulatory regions (promoters and enhancers) of GATA-1, EpoR, PBGD, epsilon- and gamma-globin genes. Finally, results of run-on assays indicate that ACLA induces an enhancement of the transcription rate of these erythroid genes whereas DOX preferentially increases stability of GATA-1, NF-E2 and PBGD mRNAs. In conclusion, ACLA mainly acts at the transcriptional level via specific activation of erythroid regulatory regions whereas DOX rather acts at the posttranscriptional level by increasing the half-lives of erythroid mRNAs.

The setup of tumorigenesis processes is generally associated with various events leading to abnormal expression of oncogenes and/or tumor suppressor genes. Recently, the expression and/or activity of a range of molecules involved in these processes were reported to require proteolytic processing of their precursor proteins by the serine pro-protein convertases (PCs) in order to mediate their biological functions. These include adhesion molecules, proteases, growth factors, cytokines and their receptors. Since their discovery, the identification of new PCs substrates and specific PCs inhibitors became an attractive strategy in cancer therapy. In this review, we will report the implication of these enzymes and the processing of their substrates in tumor progression and metastasis. Newly reported studies on the potential use of the PCs as new therapeutic targets will be also discussed.

Gene expression programs are established by networks of interacting transcription factors. The basic helix-loop-helix factor SCL and the LIM-only protein LMO2 are components of transcription factor complexes that are essential for hematopoiesis. Here we show that LMO2 and SCL are predominant interaction partners in hematopoietic cells and that this interaction occurs through a conserved interface residing in the loop and helix 2 of SCL. This interaction nucleates the assembly of SCL complexes on DNA and is required for target gene induction and for the stimulation of erythroid and megakaryocytic differentiation. We also demonstrate that SCL determines LMO2 protein levels in hematopoietic cells and reveal that interaction with SCL prevents LMO2 degradation by the proteasome. We propose that the SCL-LMO2 interaction couples protein stabilization with higher order protein complex assembly, thus providing a powerful means of modulating the stoichiometry and spatiotemporal activity of SCL complexes. This interaction likely provides a rate-limiting step in the transcriptional control of hematopoiesis and leukemia, and similar mechanisms may operate to control the assembly of diverse protein modules.

During development, hematopoiesis initiates in the yolk sac through a process that depends on VEGF/Flk1 signaling and on the function of the SCL/Tal1 transcription factor. Here we show that VEGF modifies the developmental potential of primitive erythroid progenitors and prolongs their life span. Furthermore, the survival of yolk sac erythrocytes in vivo depends on the dose of VEGF. Interestingly, in Vegflo/lo embryos carrying a hypomorph allele, Flk1-positive cells reach the yolk sac at E8.5,but are severely compromised in their ability to generate primitive erythroid precursors. These observations indicate that during embryonic development,different thresholds of VEGF are required for the migration and clonal expansion of hematopoietic precursors. The near absence of primitive erythroid precursors in Vegflo/lo embryos correlates with low levels of Scl in the yolk sac. Strikingly, gain-of-function of SCL partially complements the hematopoietic defect caused by the hypomorph Vegflo allele, and re-establishes the survival of erythroid cells and the expression of erythroid genes (Gata1 andβH1). This indicates that SCL functions downstream of VEGF to ensure an expansion of the hematopoietic compartment.

Human erythroleukemic K 562 cells were induced to were induced to differentiate along the erythroid lineage by anthracycline antitumor drugs, such as aclacinomycin (ACLA) and doxorubicin (DOX). Subsequent stimulation of heme and globin synthesis led to a differential quantitative expression of hemoglobins. Gower 1 (epsilon2, zeta2) was the major type for ACLA and X (epsilon2, gamma2) for DOX. Although ACLA and DOX increased both the expression of gamma-globin and porphobilinogen deaminase mRNAs, striking differences were observed in the expression of erythropoietin receptor mRNAs and in erythroid transcription factors GATA-1 and NF-E2, known to play a key role in erythroid gene regulation. Indeed, ACLA induces an increase either in the binding capacity of GATA-1 and NF-E2 or in the accumulation of erythropoietin receptor, GATA-1 and NF-E2 transcripts. In contrast, their expression with DOX was not significantly modified compared to uninduced cells, except for a slight decrease in NF-E2 expression on day 3. In conclusion, these data show that: 1. increased expression of erythroid transcription factors and erythroid genes are associated only with ACLA treatment, and 2. although cytotoxicity of both ACLA and DOX is certainly dependent on DNA intercalation, regulation of differentiation processes by these two drugs involves distinct mechanisms.

Background In zebrafish, vascular endothelial growth factor-C precursor (proVEGF-C) processing occurs within the dibasic motif HSIIRR(214) suggesting the involvement of one or more basic amino acid-specific proprotein convertases (PCs) in this process. In the present study, we examined zebrafish proVEGF-C expression and processing and the effect of unprocessed proVEGF-C on caudal fin regeneration. Methodology/principal findings Cell transfection assays revealed that the cleavage of proVEGF-C, mainly mediated by the proprotein convertases Furin and PC5 and to a less degree by PACE4 and PC7, is abolished by PCs inhibitors or by mutation of its cleavage site (HSIIRR(214) into HSIISS(214)). In vitro, unprocessed proVEGF-C failed to activate its signaling proteins Akt and ERK and to induce cell proliferation. In vivo, following caudal fin amputation, the induction of VEGF-C, Furin and PC5 expression occurs as early as 2 days post-amputation (dpa) with a maximum levels at 4-7 dpa. Using immunofluorescence staining we localized high expression of VEGF-C and the convertases Furin and PC5 surrounding the apical growth zone of the regenerating fin. While expression of wild-type proVEGF-C in this area had no effect, unprocessed proVEGF-C inhibited fin regeneration. Conclusions/significances Taken together, these data indicate that zebrafish fin regeneration is associated with up-regulation of VEGF-C and the convertases Furin and PC5 and highlight the inhibitory effect of unprocessed proVEGF-C on fin regeneration.

The differentiation of hematopoietic cells is tightly controlled by transcription factor complexes, composed of hemato-specific and ubiquitous proteins. The bHLH factor SCL and the LIM-only protein LMO2 are central components of transcription factor complexes and are essential for hematopoiesis. However, the mechanism regulating the assembly of SCL-complexes is unknown. Here we show that SCL, in contrast to LMO, GATA and E proteins, cannot be replaced by other members of its family in hematopoietic gene transactivation and in gel shift assays. Furthermore, we show by GST pull-down assays and by co-immunoprecipitation that interaction with LMO2 is a unique property of SCL, as the neurogenic bHLH NSCL1 related to SCL cannot bind LMO2. By generating SCL-NSCL1 chimeras, and by phylogenetic alignment, we identified the SCL interface that confers transcriptional specificity to the complex. Strikingly, this interface is also necessary for the interaction with LMO2. In contrast with the wild type SCL, the mutant without this interface is not able to enhance erythroid differentiation when overexpressed in hematopoietic cells, as assessed by glycophorin A gene activation, benzidine staining and methylcellulose cultures. Interestingly, we also demonstrate in vivo and in vitro that LMO2 protein levels are greatly increased in the presence of SCL, while mRNA levels remain constant. When the SCL interface described above is mutated, LMO2 protein level is no longer increased, suggesting that the accumulation of LMO2 is mediated by a direct interaction with SCL. In primary hematopoietic cells, when SCL protein levels are genetically reduced by LacZ insertion into one allele in the SCL locus, we observe a dramatic decrease in LMO2 protein levels. In addition, in the TF-1 hematopoietic cell line, most of the LMO2 protein (90%) is found associated with SCL and/or Ldb1, suggesting that free LMO2 is rapidly degraded. Thus, SCL levels determine LMO2 levels in hematopoietic cells. Next, we provide direct evidence that LMO2 is a target for proteasomal degradation. First, we show that LMO2 is ubiquitinated in vivo, by GST purification. Second, by using the ts20 cell line expressing a temperature-sensitive ubiquitin-activating E1 enzyme, we show that LMO2 degradation requires a functional ubiquitin conjugation system, since LMO2 is not degraded when E1 is inactive. Third, we show that the half-life of LMO2 is very short, and it can be increased with the proteasome inhibitor MG132. Finally, a similar increase in LMO2 half-life can be observed when SCL is co-expressed with LMO2. These data indicate that SCL stabilizes LMO2, which is otherwise rapidly degraded by the ubiquitin-proteasome pathway in absence of its interacting partners. Taken together, our results strongly suggest that SCL, by binding and stabilizing LMO2, is a critical determinant of the hematopoietic transcriptional specificity. The interaction between SCL and LMO2 is an essential nucleation step for the assembly of SCL-complexes on DNA, where the regulation of LMO2 levels appears to be the rate-limiting step. We propose that protein stability is a new mechanism of regulation in the formation of SCL complexes, required for proper gene activation during eryhtroid differentiation.

Erythroid cell development critically depends on the SCL/Tal1 transcription factor and on erythropoietin signalling. In the present study, we have taken several approaches to show that the two genes operate within the same pathway to consolidate the erythroid lineage. Signaling through the erythropoietin receptor (EpoR) upregulates SCL protein levels in a clonal cell line (TF-1) in vitro, and in murine fetal liver cells in vivo, when Epor−/− cells were compared to those of wild type littermates at E12.5. In addition, we provide functional evidence for a linear pathway from EpoR to SCL that regulates erythropoiesis. Interfering with SCL induction or SCL function prevents the anti-apoptotic effect of Epo in TF-1 cells and conversely, ectopic SCL expression is sufficient to substitute for Epo to transiently maintain cell survival. In vivo, SCL gain of function complements the cellular defects in Epor−/− embryos to support cell survival and maturation during primitive and definitive erythropoiesis, as assessed by cellular and histological analyses of Epor−/− SCLtg embryos. Moreover, several erythroid specific genes that are decreased in Epor−/− embryos are rescued by the SCL transgene including glycophorinA, bH1 and bmaj globin, providing molecular confirmation of the functional and genetic interaction between Epor and SCL. Conversely, erythropoiesis becomes deficient in compound Epor+/−SCL+/− heterozygote mice, indicating that the genetic interaction between EpoR and SCL is synthetic. Finally, using EpoR mutants that harbour well defined signalling deficiencies, combined with gain and loss of function approaches for specific kinases, we identify MAPK as the major signal transduction pathway downstream of EpoR that upregulates SCL function, necessary for erythroid cell survival and differentiation. Taken together, our observations are consistent with the view that cytokines can influence cell fate by altering the dosage of lineage transcriptional regulators.

// Abdel-Majid Khatib 1,2 , Rachid Lahlil 3 , Martin Hagedorn 1,2 , Claudine Delomenie 4 , Olivier Christophe 3 , Cecile Denis 3 and Geraldine Siegfried 1,2 1 Universite Bordeaux, Pessac, France 2 INSERM, LAMC, UMR 1029, Pessac, France 3 INSERM U 770, Kremlin-Bicetre, France 4 IFR141 IPSIT, Universite Paris Sud, Chatenay-Malabry, France Correspondence to: Geraldine Siegfried, email: // Abdel-Majid Khatib, email: // Keywords : HIF, CoCl2, angiogenesis, regeneration, gene ontology, functional enrichment, Pathology Section Received : July 02, 2015 Accepted : February 02, 2016 Published : February 25, 2016 Abstract Hypoxia Inducible Factor (HIF) is the main transcription factor that mediates cell response to hypoxia. Howeverthe complex factor cascades induced by HIF during regenerative angiogenesis are currently incompletely mapped and the biological outcome mediated by chronic HIF induction during vessel regeneration are not well known. Here, we investigated the biological impact of HIF induction on vascular regeneration and identified the differentially regulated genes during regeneration, HIF induction and hypoxic regeneration. The use of the fin zebrafish regeneration model revealed that exposure to HIF inducer (cobalt chloride) prevents vessel differentiation by maintaining their vascular plexuses in an immature state. The regenerated fins are easily breakable, lacking completely endochondral ossification. Gene expression arrays combined to gene functional enrichment analysis revealed that regenerative process and HIF induction shared the regulation of common genes mainly involved in DNA replication and proteasome complex. HIF induction during regeneration affected the expression of exclusive genes involved in cell differentiation and communication, consistent with the observed immature vascular plexuses of the regenerated fins during HIF induction. The use of morpholino (MO) knockdown strategy revealed that the expression of some of these genes such as tubulin and col10a1 are required for fin regeneration. Taken together, this study revealed the impact of HIF induction on regenerative angiogenesis and provided a framework to develop a gene network leading to regenerative process during HIF expression.