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Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as β-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in β-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in β-thalassemia. We hypothesize that compensatory mechanisms are required in β-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2—a STAT5-dependent lipid binding protein downstream of erythropoietin—as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in β-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during β-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in β-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in β-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in β-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in β-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis., Maria Feola et al., elucidate the compensatory role of pleckstrin-2 in ineffective erythropoiesis in β-thalassemic mice by reducing cofilin-mediated apoptosis and enhancing enucleation by activating RacGTPases. These findings could support future therapeutic interventions.

© Author(s) (or their employer(s)) 2020., [Objective]: ATM serine/threonine kinase (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC)., [Design]: Combinational synergy screening was performed to endeavour a genotype-tailored targeted therapy., [Results]: Synergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance., [Conclusion]: Analysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition., Main funding is provided by the German Cancer Aid grant to AK (111879). Additional funding came from the Deutsche Forschungsgemeinschaft (DFG) ’Sachbeihilfe’ (KL 2544/1–1, 1–2, 5–1, 7-1) and ’Heisenberg-Programm’ (KL 2544/6–1), the Baden-Württemberg-Foundation ExPoChip and the INDIMEDVerbund PancChip. AK, FA, MI, SB, LW and TS are either Principal Investigators or students of HEIST RTG funded by the DFG GRK 2254/1. AK is an Else-KrönerFresenius Excellence fellow. LP received funds by the Bausteinprogramm of Ulm University. PCH is supported by a Max Eder Fellowship of the German Cancer Aid (111746), a German Cancer Aid Priority Program ’Translational Oncology’ 70112505 and by a Collaborative Research Centre grant (316249678 – SFB 1279) of the German Research Foundation. EH received funding from the German Cancer Aid (PiPAC, 70112505) and the Volkswagenstiftung/Ministry for Science and Culture in Lower Saxony (ZN3222). This work was also supported by the Deutsche Forschungsgemeinschaft (AZ.96/1–3) to NA, by the Deutsche Krebshilfe (111264) to AL and by the German Cancer Aid Priority Program Translational Oncology (70112504) to LW.

Objective ATM serine/threonine kinase (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC). Design Combinational synergy screening was performed to endeavour a genotype-tailored targeted therapy. Results Synergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance. Conclusion Analysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition., publishedVersion

Electrical stimulation (ES) of cells has been shown to induce a variety of responses, such as cytoskeleton rearrangements, migration, proliferation, and differentiation. In this study, we have investigated whether monophasic and biphasic pulsed ES could exert any effect on the proliferation and differentiation of human cardiac progenitor cells (hCPCs) isolated from human heart fragments. Cells were cultured under continuous exposure to monophasic or biphasic ES with fixed cycles for 1 or 3 days. Results indicate that neither stimulation protocol affected cell viability, while the cell shape became more elongated and reoriented more perpendicular to the electric field direction. Moreover, the biphasic ES clearly induced the upregulation of early cardiac transcription factors, MEF2D, GATA-4, and Nkx2.5, as well as the de novo expression of the late cardiac sarcomeric proteins, troponin T, cardiac alpha actinin, and SERCA 2a. Both treatments increased the expression of connexin 43 and its relocation to the cell membrane, but biphasic ES was faster and more effective. Finally, when hCPCs were exposed to both monophasic and biphasic ES, they expressed de novo the mRNA of the voltage-dependent calcium channel Cav 3.1(α1G) subunit, which is peculiar of the developing heart. Taken together, these results show that ES alone is able to set the conditions for early differentiation of adult hCPCs toward a cardiac phenotype.

The serine/threonine kinase Par1 is a core component of the machinery that sets up polarity in the embryo and regulates cell fate decisions but its role in the homeostasis of adult tissues is poorly understood. Inhibition of Par1 by the bacterium Helicobacter pylori (H. pylori) represents the only established pathology that affects Par1 function in an adult epithelium. Thus, during chronic H. pylori infection of the gastric mucosa Par1 is one of the targets of the non-obligate H.pylori cytotoxic protein and oncogene CagA, which stimulates inflammation and triggers morphological changes, both believed to contribute to the gastric cancer risk imposed by H. pylori infection. Based on Par1's role in cell polarity, it has been speculated that Par1 inhibition affects epithelial polarity. Here we report the unexpected finding that CagA-mediated Par1-inhibition promotes the generation of DNA Double Strand Breaks in primary gastric epithelial cells, which likely contributes to the reported accumulation of mutations in chronically infected mucosal cells. Abbreviations: AGS: human gastric adenocarcinoma cell line; CM: CagA Multimerization (and Par1 binding) domain; H. pylori: Helicobacter pylori; DSB: Double Strand Break; HGECs: human (primary) gastric epithelial cells; IB: immunoblot; IF: immunofluorescence; MOI: Multiplicity of Infection; ROS: reactive oxygen species; Par1: Partitioning Defective 1 kinase; WT: wild type.

CagA is a multifunctional toxin of Helicobacter pylori that is secreted into host epithelial cells by a type IV secretion system. Following host cell translocation, CagA interferes with various host–cell signalling pathways. Most notably this toxin is involved in the disruption of apical–basolateral cell polarity and cell adhesion, as well as in the induction of cell proliferation, migration and cell morphological changes. These are processes that also play an important role in epithelial-to-mesenchymal transition and cancer cell invasion. In fact, CagA is considered as the only known bacterial oncoprotein. The cellular effects are triggered by a variety of CagA activities including the inhibition of serine–threonine kinase Par1b/MARK2 and the activation of tyrosine phosphatase SHP-2. Additionally, CagA was described to affect the activity of Src family kinases and C-terminal Src kinase (Csk) suggesting that interference with multiple cellular kinase- and phosphatase-associated signalling pathways is a major function of CagA. Here, we describe the effect of CagA on protein kinase C-related kinase 2 (PRK2), which acts downstream of Rho GTPases and is known to affect cytoskeletal rearrangements and cell polarity. CagA interacts with PRK2 and inhibits its kinase activity. Because PRK2 has been linked to cytoskeletal rearrangements and establishment of cell polarity, we suggest that CagA may hijack PRK2 to further manipulate cancer-related signalling pathways.

Adipose tissue has been shown to be particularly advantageous as source of mesenchymal stem cells (MSCs), because of its easy accessibility, and the possibility of obtaining stem cells in high yields. MSCs are obtained from the so-called Stromal Vascular Fraction, (SVF), exploiting their property of adhering to plastic surfaces and can be further purified by positive or negative immunomagnetic selection with appropriately chosen antibodies. These cells (Stromal Stem Cells, SSCs) can then be directly analyzed, frozen in liquid nitrogen, or expanded for further applications, e.g., for tissue engineering and regenerative medicine. The methodology described here in detail for SSCs isolated from mouse subcutaneous adipose tissue can be applied to human tissues, such as epicardium.

In tissue engineering, several factors play key roles in providing adequate stimuli for cells differentiation, in particular biochemical and physical stimuli, which try to mimic the physiological microenvironments. Since electrical stimuli are important in the developing heart, we have developed an easy-to-use, cost-effective cell culture platform, able to provide controlled electrical stimulation aimed at investigating the influence of the electric field in the stem cell differentiation process. This bioreactor consists of an electrical stimulator and 12 independent, petri-like culture chambers and a 3-D computational model was used to characterize the distribution and the intensity of the electric field generated in the cell culture volume. We explored the effects of monophasic and biphasic square wave pulse stimulation on a mouse adipose-derived stem cell line (m17.ASC) comparing cell viability, proliferation, protein, and gene expression. Both monophasic (8 V, 2 ms, 1 Hz) and biphasic (+4 V, 1 ms and -4 V, 1 ms; 1 Hz) stimulation were compatible with cell survival and proliferation. Biphasic stimulation induced the expression of Connexin 43, which was found to localize also at the cell membrane, which is its recognized functional mediating intercellular electrical coupling. Electrically stimulated cells showed an induced transcriptional profile more closely related to that of neonatal cadiomyocytes, particularly for biphasic stimulation. The developed platform thus allowed to set-up precise conditions to drive adult stem cells toward a myocardial phenotype solely by physical stimuli, in the absence of exogenously added expensive bioactive molecules, and can thus represent a valuable tool for translational applications for heart tissue engineering and regeneration.

Recent genomic characterization of PDA reveals that between 20-25 % of PDA harbor recurrent mutations in genes, including BRCA1/2, PALB2, and ATM, which are critical for homologous recombination (HR), an important form of DNA repair. In many patients, these may be germline mutations. This subgroup of PDAs, termed HR-deficient PDA, has emerged as a defined biological entity associated with increased chemoresistance and a more aggressive disease course. The defects in HR observed in these tumors impart cells with a specific vulnerability to PARP inhibitors and platinum-containing therapy. Still, as observed in the case of many other targeted therapies, only a fraction of HR-defective patient tumors respond to PARP inhibition. More so, many patients that initially respond eventually often develop resistance and progress. Therefore, novel therapies which can be effective against HR-defective PDA, alone or in combination with PARP inhibitors or other combinatorial regimens, are urgently needed. We have recently determined that inactivation of the HR pathway is associated with overexpression of polymerase theta (PolO–, also known as POLQ) in PDA. POLQ is a key enzyme that regulates an alternative pathway of DNA repair, known as the alternative non-homologous end-joining (Alt-NHEJ) pathway. In the setting of defective HR, Alt-NHEJ becomes a critical pathway responsible for the repair of DNA breaks and POLQ inhibition in HR-defective tumor cells demonstrates a synthetic lethality phenotype, not observed in cells with intact HR. Furthermore, POLQ knockdown significantly upregulated the cGAS-STING pathway in HR-deficient PDAs linking the DNA damage response to the immune response. Overall, targeting POLQ may represent a novel and in a valuable therapeutic strategy in HR-defective pancreatic cancer, as POLQ inhibitors are currently in development for clinical use. Citation Format: Annie Wang, Andrea Zamperone, Mohammad Sohail, Lidong Wang, Fiyinfolu Balogun, Jiufeng Li, Ende Zhao, Daniel Diolaiti, Agnel Sfeir, Diane M. Simeone. Polymerase theta synthetic lethal interaction in homologous recombination-deficient pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I14.

Hepatocyte growth factor (HGF), a pleiotropic cytokine with mitogenic, motogenic, morphogenic, and antiapoptotic effects in various cell types, is a cardioprotective growth factor that can counteract the loss of cardiomyocytes usually observed in cardiac diseases. HGF is a quite unstable molecule in its biologically active heterodimeric form. Since all HGF-induced biological responses are mediated by its high-affinity tyrosine kinase receptor (Met/HGF-R) encoded by the Met gene, we asked whether a monoclonal antibody (MAb) that displays receptor full agonist activity could protect cardiac muscle cell lines from hydrogen peroxide-induced apoptosis. We report that the MAb efficiently inhibited hydrogen peroxide-induced cell shrinkage, DNA fragmentation, annexin V positivity, mitochondrial translocation of bax, and caspase activation. The MAb was thus able to counteract apoptosis evaluated by both morphological and biochemical criteria. The agonist activity of the MAb was mediated by Met/HGF-R, since a Met/HGF-R-specific short hairpin RNA (shRNA) inhibited both activation of transduction pathways and motility triggered by MAb DO-24. The protective antiapoptotic effect of MAb DO-24 was dependent on activation of the ras-MAPK Erk1/2 and phosphatidylinositol 3-kinase (PI3-kinase)-Akt transduction pathways, since it was abrogated by treatments with their specific pharmacological inhibitors, PD-98059 and wortmannin. Moreover, the MAb induced a motogenic, but not mitogenic, response in these cells, mimicking in all aspects the natural ligand HGF but displaying a significant higher stability than HGF in culture. This MAb may thus be a valuable substitute for HGF, being more easily available in a biologically active, highly stable, and purified form.

CagA is a multifunctional toxin of Helicobacter pylori that is secreted into host epithelial cells by a type IV secretion system. Following host cell translocation, CagA interferes with various host–cell signalling pathways. Most notably this toxin is involved in the disruption of apical–basolateral cell polarity and cell adhesion, as well as in the induction of cell proliferation, migration and cell morphological changes. These are processes that also play an important role in epithelial-to-mesenchymal transition and cancer cell invasion. In fact, CagA is considered as the only known bacterial oncoprotein. The cellular effects are triggered by a variety of CagA activities including the inhibition of serine–threonine kinase Par1b/MARK2 and the activation of tyrosine phosphatase SHP-2. Additionally, CagA was described to affect the activity of Src family kinases and C-terminal Src kinase (Csk) suggesting that interference with multiple cellular kinase- and phosphatase-associated signalling pathways is a major function of CagA. Here, we describe the effect of CagA on protein kinase C-related kinase 2 (PRK2), which acts downstream of Rho GTPases and is known to affect cytoskeletal rearrangements and cell polarity. CagA interacts with PRK2 and inhibits its kinase activity. Because PRK2 has been linked to cytoskeletal rearrangements and establishment of cell polarity, we suggest that CagA may hijack PRK2 to further manipulate cancer-related signalling pathways.

In this work we present the design procedure of an electrical stimulation-based bioreactor capable of delivering highly controlled electrical stimulation to the culture environment. The device was validated through in vitro tests aimed at examining the role of different electrical stimulation patterns in enhancing differentiation of adipose-derived stem cells and human cardiac progenitor cells toward the cardiac phenotype

In this work, we present the design of an electrical stimulation-based bioreactor capable of delivering different and highly controlled electrical stimuli to the culture environment. The device represents an in vitro platform allowing examining selectively the effects of different stimulation patterns on stem cells behaviour. In the design stage, we used a computational approach, aimed at establishing operating conditions delivering a desired uniform electric field distribution within the cell- seeded surface of the culture chamber. The device was validated through in vitro tests exploiting the possibility to promote commitment of adipose-derived stem cellsand human cardiac progentiro cells toward the cardiac phenotype.

Cocoa contains phenolic compounds with known antioxidant and antiradical properties beneficial in different pathologies, including cardiovascular diseases. Herein, we have evaluated the protective effects of clovamide, a minor cocoa component, against oxidative stress induced in the rat cardiomyoblast cell line, also comparing it to its bio-isosteric form, rosmarinic acid, and to the main monomeric flavan-3-ol from low-molecular-weight polyphenol in cocoa, i.e. epicatechin. At nano–micro-molar concentrations, the three compounds inhibited the production of reactive oxygen species and apoptosis, evaluated under different aspects, namely, annexin V positivity, DNA fragmentation, caspase release and activation. These molecules can, thus, be considered for their bioactive beneficial activity in the context of cardiovascular pathologies and, particularly, in the protection towards oxidative stress that follows ischemic injury. Clovamide may, thus, be the primary compound for the development of innovative nutraceutical strategies towards cardiovascular diseases.

In tissue engineering, several factors play key roles in providing adequate stimuli for cells differentiation, in particular biochemical and physical stimuli, which try to mimic the physiological microenvironments. Since electrical stimuli are important in the developing heart, we have developed an easy-to-use, cost-effective cell culture platform, able to provide controlled electrical stimulation aimed at investigating the influence of the electric field in the stem cell differentiation process. This bioreactor consists of an electrical stimulator and 12 independent, petri-like culture chambers and a 3-D computational model was used to characterize the distribution and the intensity of the electric field generated in the cell culture volume. We explored the effects of monophasic and biphasic square wave pulse stimulation on a mouse adipose-derived stem cell line (m17.ASC) comparing cell viability, proliferation, protein, and gene expression. Both monophasic (8 V, 2 ms, 1 Hz) and biphasic (+4 V, 1 ms and -4 V, 1 ms; 1 Hz) stimulation were compatible with cell survival and proliferation. Biphasic stimulation induced the expression of Connexin 43, which was found to localize also at the cell membrane, which is its recognized functional mediating intercellular electrical coupling. Electrically stimulated cells showed an induced transcriptional profile more closely related to that of neonatal cadiomyocytes, particularly for biphasic stimulation. The developed platform thus allowed to set-up precise conditions to drive adult stem cells toward a myocardial phenotype solely by physical stimuli, in the absence of exogenously added expensive bioactive molecules, and can thus represent a valuable tool for translational applications for heart tissue engineering and regeneration.

The emerging field of tissue engineering and regenerative medicine is a multidisciplinary science that is based on the combination of a reliable source of stem cells, biomaterial scaffolds, and cytokine growth factors. Adult mesenchymal stem cells are considered important cells for applications in this field, and adipose tissue has revealed to be an excellent source of them. Indeed, adipose-derived stem cells (ASCs) can be easily isolated from the stromal vascular fraction (SVF) of adipose tissue. During the isolation and propagation of murine ASCs, we observed the appearance of a spontaneously immortalized cell clone, named m17.ASC. This clone has been propagated for more than 180 passages and stably expresses a variety of stemness markers, such as Sca-1, c-kit/CD117, CD44, CD106, islet-1, nestin, and nucleostemin. Furthermore, these cells can be induced to differentiate toward osteogenic, chondrogenic, adipogenic, and cardiogenic phenotypes. m17.ASC clone displays a normal karyotype and stable telomeres; it neither proliferates when plated in soft agar nor gives rise to tumors when injected subcutaneously in NOD/SCID-γ (null) mice. The analysis of gene expression highlighted transcriptional traits of SVF cells. m17.ASCs were genetically modified by lentiviral vectors carrying green fluorescent protein (GFP) as a marker transgene and efficiently engrafted in the liver, when injected in the spleen of NOD/SCID-γ (null) monocrotaline-treated mice. These results suggest that this non-tumorigenic spontaneously immortalized ASC line may represent a useful tool (cell model) for studying the differentiation mechanisms involved in tissue repair as well as a model for pharmacological/toxicological studies.

Erythropoiesis involves stem cell differentiation to mature red blood cells (RBCs). Erythropoietin (Epo) is essential for erythroipoiesis, and Epo binding to Epo receptor triggers a complicated and incompletely understood set of potentially related molecular signals influencing cell survival, differentiation, and enucleation. Although Epo is associated with increased survival of erythroid precursors, it induces reactive oxygen species (ROS), and high Epo concentration has an anti-enucleation effect in vitro.i Furthermore, diseases of ineffective erythropoiesis, e.g. β-thalassemia, are associated with increased Epo and ROS concentrations implicated in the expansion of and damage to erythroid precursors, respectively. Treating erythroblasts with low dose ROS scavenger promotes enucleation, but high dose ROS scavenger leads to cell death,i suggesting that an optimal ROS concentration is integral to effective erythropoiesis. We and others have shown that ROS is increased in β-thalassemic erythroid precursors, but despite increased ROS, erythroid precursor apoptosis is not increased. We hypothesize that compensatory mechanisms prevent the ill-effects of increased ROS on erythroid precursors. Our prior experiments demonstrate disordered erythropoiesis in β-thalassemic (th1/th1) mice, restored in transferrin-treated th1/th1 mice,ii despite which, ROS remained increased in erythroid precursor from transferrin-treated th1/th1 mice. To identify mechanisms responsible for transferrin's effect, we performed RNA seq analysis of erythroblasts from wild type (WT), th1/th1, and transferrin-treated th1/th1 mice. We identified increased pleckstrin-2 (plek2) in th1/th1 relative to WT mice, normalized in transferrin-treated th1/th1 mice. We hypothesize that plek2 activation counteracts the ill effects of ROS and promotes enucleation in β-thalassemia. Using confocal microscopy, we demonstrate that 1) plek2 co-localizes with actin on the cell membrane after the pro-erythroblast stage but is in the nucleus throughout terminal erythropoiesis in WT mice; 2) membrane-associated plek2 is present earlier, in pro-erythroblasts, and remains membrane-associated until orthochromatophilic stage in th1/th1 mice; and 3) plek2 localization is normalized in transferrin-treated th1/th1 mice. Because plek2 activation leads to its association with the cell membrane and plek2 activation is increased in th1/th1 erythroblasts, we set out to explore the role of plek2 activation on ineffective erythropoiesis in transferrin-treated th1/th1 mice. Prior publications propose that plek2 interacts with and results in the phosphorylation of cofilin, preventing cofilin's translocation to the mitochondria as part of the apoptosis pathway in response to increased ROS.iii We demonstrate decreased in mitochondria cofilin localization and increased cellular p-cofilin in th1/th1 erythroblasts, normalized after transferrin treatment. These data suggest that despite an increase in ROS, plek2 and its induction of p-cofilin inhibit apoptosis in β-thalassemic erythroid precursors. Furthermore, in light of a prior report of an anti-enucleation effect of plek2 in vitroiii and the known regulation of enucleation by RacGTPasesiv, we hypothesize that plek2 activation triggers RacGTPase and prevents enucleation in th1/th1 mice. We demonstrate that in addition to changes in erythroblast RacGTPase concentration, membrane co-localization between plek2 and RacGTPase is enhanced and occurs earlier in th1/th1 erythroid differentiation relative to WT, normalized after transferrin treatment. Lastly, cleavage of Rho-associated kinase, Rock1, associated with enucleation,v is also decreased in th1/th1 erythroblasts, enhanced after transferrin treatment. Taken together, we speculate that plek2 haplo-insufficiency benefits β-thalassemic mice by enabling apoptosis of ineffective erythroblasts, or result in worsening, possibly lethal, phenotype in light of the direct or indirect (through effects on RacGTPase or Rock1) role of plek2 in enucleation. Presently, we are testing this hypothesis by generating plek2+/- and plek-/- β-thalassemic mice. In conclusion, we demonstrate the important compensatory role of plek2 in β-thalassemic erythropoiesis. i Zhao Exp Hematol 2016 ii Liu Blood 2013 iii Zhao haematol 2014 iv Ji Nat Cell Bio 2008 v Gabet Cell Death Diff 2011 Disclosures No relevant conflicts of interest to declare.

Human heart harbors a population of resident progenitor cells that can be isolated by stem cell antigen-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, less than 1%-5% survive and differentiate. Among the major causes of this failure are the distressing protocols used to culture in vitro and implant progenitor cells into damaged hearts. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and were embedded in self-produced extracellular matrix preserving their phenotype and multipotency in the absence of significant apoptosis. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the murine myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such an approach to deliver stem cells to the myocardium. Interestingly, the successful delivery of cells in murine healthy hearts suggests that myocardium displays a continued cell cupidity that is strictly regulated by the limited release of progenitor cells by the adopted source. When an unregulated cell source is added to the system, cells are delivered to the myocardium. The exploitation of this novel concept may pave the way to the setup of new protocols in cardiac cell therapy.

Erythropoiesis is a process during which multipotent hematopoietic stem cells proliferate, differentiate and ultimately produce enucleated reticulocytes. Terminal erythroid differentiation begins at the morphologically recognizable pro-erythroblast (pro-E) stage and is completed when orthochromatic erythroblasts (ortho-E) expel their nuclei to produce reticulocytes. Progressive differentiation between these stages occurs in homologous cell division progressively doubling proportions of pro-E, basophilic (baso-E), polychromatophilic (poly-E), and ortho-E, and multiple signaling pathways are involved in the generation of enucleated erythroid cells, including multiple steps requiring actin cytoskeleton reorganization. We have previously shown that β-thalassemic mice (th1/th1) demonstrate a disordered progression from pro-E to baso-E and that exogenous transferrin therapy restores normal proportion of early stage erythroid precursors in th1/th1 mice (Liu Blood 2013). To identify genes that play novel function in different stages of terminal erythropoiesis, we performed RNA seq analysis of sorted bone marrow pro-E from WT, th1/th1, and transferrin-treated th1/th1 mice. We identify pleckstrin-2 (plek2) as a gene of interest with a 15-fold increase in plek2 mRNA expression in th1/th1 relative to WT mice, normalized in transferrin-treated th1/th1 mice. Plek2 is an actin binding protein, like pleckstrin-1, contains a central DEP domain known to bind RacGTPase, is Epo dependent, and is expressed in all stages of terminal erythropoiesis. We evaluate plek2 mRNA and protein expression in sorted bone marrow erythroid precursors from WT, th1/th1, and transferrin-treated th1/th1 mice. Our data demonstrates a statistically significant increase in plek2 mRNA in th1/th1 relative to WT mice, with the highest expression of plek2 in poly-E, normalized in transferrin-treated th1/th1 mice (Figure 1A). A similar pattern of increased protein concentration in th1/th1 relative to WT mice and normalization in transferrin-treated th1/th1 mice is evident in sorted bone marrow samples (Figure 1B). Prior in vitro studies demonstrate that membrane localization of plek2 is required for erythroid differentiation. Thus, we performed sub-cellular fractionation in bone marrow erythroid precursors and determined for the first time that in sorted erythroblasts from WT bone marrow, plek2 is found exclusively in the cytoplasm in pro-E and in both cytoplasm and membrane from baso-E to ortho-E (Figure 2), co-localized with actin filaments in the membrane (data not shown). In contrast, sorted erythroblasts from th1/th1 bone marrow reveal membrane-associated plek2 starting from pro-E, demonstrating earlier co-localization with actin filaments (data not shown) and suggesting an earlier activation of plek2 and consequent actin cytoskeleton reorganization during erythroid differentiation in th1/th1 mice, normalized in transferrin-treated th1/th1 mice (Figure 2). Erythropoiesis involves a complicated and incompletely understood set of potentially related molecular signals influencing cell survival, differentiation, enucleation, and release into the circulation. For example, although Epo increases survival, Epo signaling also activates RacGTPases, inhibiting enucleation. Recent in vitro data demonstrates that knockdown of plek2 affected enucleation with significantly lower reticulocyte count. Although the involvement of RacGTPase in plek2-mediated erythroid differentiation has not been explored, we hypothesize that plek2 activation triggers RacGTPase and prevents enucleation in th1/th1 mice. Our data demonstrates that RacGTPase concentration is increased in sorted bone marrow erythroid precursors from th1/th1 relative to WT mice and normalized in transferrin-treated th1/th1 mice (Figure 1B). These results suggest that plek2 plays an important role in erythropoiesis likely as a key factor in the improved enucleation of transferrin-treated th1/th1 mice. Disclosures No relevant conflicts of interest to declare.

A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM). We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs) onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol.