ES2666351T3 - Composition and method of stem cells for the preservation of heart tissue - Google Patents

Abstract

Method to improve the survival of hematopoietic stem cells in hypoxic and serum-deprived conditions by culturing the cells in a medium containing lysophosphatidic acid.

Description

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DESCRIPTION

Composition and method of stem cells for the preservation of heart tissue Technical field

The disclosure refers to a composition and method of human hematopoietic stem cells treated with lysophosphatidic acid that improves survival in hypoxia and deprived serum conditions and preserves heart tissue after myocardial infarction.

Background of the invention

Heart disease is the leading cause of death and disability in both industrialized nations and in the developing world, and accounts for approximately 40% of all human mortality. Many surviving patients develop a chronic form of heart disease called congestive heart failure (CHF), which is associated with progressive deterioration of the heart muscle, scar formation, dilation and left ventricular dysfunction (LV). Patients with severe ischemic heart failure have high morbidity and mortality, with heart transplantation being the only definitive therapy available.

Recently, different sources of stem cells have been tested in human patients who suffered an MI (myocardial infarction), including adult peripheral blood stem cells (APBSC) and bone marrow derived stem cells (BMDSC) (Losordo, DW, et. al., Intrarnyocardial Transplantation of autologous CD34 + stem cells for intractable angina: a phase I / IIa double-blind, randomized Controlled trial Circulation, 2007. 115 (25): pages 316572; Schachinger, V., et al., Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction.N Engl J Med, 2006. 355 (12): pages 1210-21). The improvement in the ejection fraction of the LV has been reported in most trials (Schachinger, V., et al., Intracoronary bone marrowderived progenitor cells in acute myocardial infarction. N Engl J Med, 2006. 355 (12): pages 1210-21, Passier, R., LW van Laake, and CL Mummery, Stem-cell-based therapy and lessons from the heart. Nature, 2008. 453 (7193): pages 322-9); however, the functional improvement is still modest (ejection fraction below 5%). Therefore, there is a need for alternative approaches to (i) increase the therapeutic effect of stem cells and to (ii) treat older patients who have adult stem cells with impaired biological activity (eg, diabetic patients, etc.). ) (Passier, R., LW van Laake, and CL Mummery, Stem-cell-based therapy and lessons from the heart. Nature, 2008. 453 (7193): page 322-9).

CD34 + cells isolated from human umbilical cord blood can be a promising cell therapy for heart regeneration. These stem cells can be used autologously, they can differentiate into vascular cells in vitro or in vivo (Le Ricousse-Roussanne, S., et al., Ex vivo differentiated endothelial and smooth muscle cells from human cordblood progenitors home to the angiogenic tumor vasculature Cardiovasc Res, 2004. 62 (1): pages 176-84) and increase neovascularization in animal models of myocardial ischemia (Ma, N., et al., Human cord blood cells induces angiogenesis following myocardial infarction in NOD / scid- mice Cardiovasc Res, 2005. 66 (1): pages 45-54; Hirata, Y., et al., Human umbilical cord blood cells improve cardiac function after myocardial infarction. Biochem Biophys Res Commun, 2005. 327 (2): pages 609-14). CD34 + cells isolated from human umbilical cord blood have several advantages compared to APBSC and BMDSC, including a higher proliferation rate, relatively low risk of unwanted cell production in vivo (as opposed to the risks described for BMDSC), and an adequate cell therapy for patients who suffered an MI and have human peripheral blood CD34 + cells with impaired function [5, 10]. For clinical efficacy, it is imperative that the stem cells or their progeny survive and graft into the host tissue. Unfortunately, many cells die a few days after grafting (Ma, N., et al., Human cord blood cells induces angiogenesis following myocardial infarction in NOD / scid-mice. Cardiovasc Res, 2005. 66 (1): page 45-54 ; Hirata, Y., et al., Human umbilical cord blood cells improve cardiac function after myocardial infarction. Biochem Biophys Res Commun, 2005. 327 (2): pages 609-14; Henning, RJ, et al., Human umbilical cord blood progenitor cells are attracted to infarcted myocardium and significantly reduce myocardial infarction size.Cell Transplant, 2006. 15 (7): pages 647-58).

General description

One aspect of the present disclosure describes a method to enhance the survival of hematopoietic stem cells, preferably CD34 + derived from human umbilical cord or peripheral blood, under hypoxic and serum-deprived conditions by culturing the cells in medium containing lysophosphatidic acid, preferably further comprising a gel, namely a biomimetic gel.

This combination surprisingly improved survival in hypoxia and serum deprivation conditions and preserves heart tissue after myocardial infarction. This mixture, especially CD34 + derived from human umbilical cord blood treated with lysophosphatidic acid in fibrin, showed better results.

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In one embodiment of the disclosed method, the biomimetic gel may be at least one of the following: fibrin, hyaluronic acid, alginate, agarose, collagen, PEG derivatives and mixtures thereof. Preferably, fibrin gel, more preferably, the fibrin gel comprises a fibrinogen at a final concentration of 1-100 mg / mL and thrombin at a final concentration of 1-500 U / mL. Even more preferably, the fibrin gel comprises a fibrinogen at a final concentration of 10-30 mg / mL and thrombin at a final concentration of 2-50 U / mL.

In one embodiment of the disclosed method, the concentration of lysophosphatidium varies between 1 and 1,000 pM, preferably 100 mM.

One aspect of the present disclosure describes a composition comprising: hematopoietic stem cells, preferably CD34 + derived from human umbilical cord or peripheral blood; with 100 pM lysophosphatidic acid, preferably further comprising a gel, specifically a biomimetic gel.

This combination surprisingly improved survival in hypoxia and serum deprivation conditions and preserves heart tissue after myocardial infarction. This mixture, especially CD34 + derived from human umbilical cord blood treated with lysophosphatidic acid in fibrin, showed better results.

In one embodiment of the disclosed composition, the biomimetic gel may be at least one of the following: fibrin, hyaluronic acid, alginate, agarose, collagen, PEG derivatives and mixtures thereof. Preferably, fibrin gel, more preferably, the fibrin gel comprises a fibrinogen at a final concentration of 1-100 mg / mL and thrombin at a final concentration of 1-500 U / mL. Even more preferably, the fibrin gel comprises a fibrinogen at a final concentration of 10-30 mg / mL and thrombin at a final concentration of 2-50 U / mL.

An embodiment of the disclosed composition may comprise 1 x 105-1 x 106 CD34 + cells; and 100 pM lysophosphatidic acid; and 100-200 mL of biomimetic gel.

In another aspect, the disclosed composition can be used in medicine or cosmetic application, specifically a pharmaceutical, medical or cosmetic composition, specifically with CD34 + cells and previous components of LPA are in a therapeutically effective amount and may further comprise adequate amounts of excipient. In particular, in the treatment of cardiac tissue and / or heart disease, and / or in the treatment of wound healing, namely, healing of diabetic wounds.

In one embodiment, the disclosure composition may be an injectable formulation.

In the present disclosure it is shown that hematopoietic stem cells, specifically CD34 + cells, treated with lysophosphatidic acid (LPA) and cultured under hypoxia and deprived serum conditions double their survival with respect to untreated cells. Surprisingly, cells proliferate and secrete high levels of cytokines such as IL-4, IL-8 and TNF-a compared to controls. Finally, cells treated with LPA, but not untreated cells, preserve cardiac function after myocardial infarction.

Description of the figures

The following figures provide preferred embodiments to illustrate the description and should not be seen as limiting the scope of the invention.

Figure 1: LPA chemical structure. Survival, apoptosis and necrosis of CD34 + cells cultured in hypoxia for 24 h in serum-free medium with or without drugs. The results are the average ± SEM (n = 2-13).

Figure 2: LPA improves the survival of CD34 + cells in cells grown under conditions of hypoxia and serum deprivation. (A) Survival, apoptosis and necrosis of CD34 + cells grown in serum-free medium for 24 hours in normoxia, hypoxia and hypoxia with treatment with LPA. (B) Effect of the concentration of LPA on the survival of CD34 + cells grown in serum-free medium for 24 h in hypoxia or normoxia. (C) Effect of LPA on the survival of cells grown in hypoxia for 1 (H1) or 3 days (H3), in hypoxia for 1 day and 3 days in normoxia (H1 + N3), or normoxia for 1 (N1) or 4 days (N4). In all graphs, cells were cultured with or without LPA (100 pM). The results are the average ± SEM (n = 4-50). In all figures, * denotes statistical significance: * P <0.05, ** P <0.01, *** P <0.001.

Figure 3: LPA affects cell proliferation and has a minimal impact on cell differentiation under conditions of hypoxia and serum deprivation. (A) Total number of cells after 1 day in hypoxia and 3 or 6 days in normoxia. (B) Cell differentiation measured by flow cytometry (n = 1). The cells were grown in hypoxia for 1 day or not for 6 days in normoxia. In all graphs, cells were cultured with or without LPA (100 pM).

Figure 4: LPA affects cell survival in CD34 + cells encapsulated in fibrin gel and cultured in hypoxia and deprived serum conditions. (A.1) The cells were grown in serum free medium with or without LPA (100 pM) and in hypoxia for 1 day. (A.2) The cells were grown in serum free medium with or without LPA (100 pM) and in hypoxia for 3 days. In all graphs, the results are the average ± SEM (n = 3-6). In all figures, * denotes 5 statistical significance: * P <0.05, ** P <0.01, *** P <0.001.

Figure 5: LPA induces the survival of CD34 + cells primarily through the peroxisome proliferator-activated receptor (PPAR). The cells were grown in serum free medium with LPA. Cells were previously treated with Rho kinase inhibitor (Y-2762, 50 pM), mitogen-activated protein kinase inhibitor (MAPK) (PD98059, 60 pM), specific inhibitor of LPA1 and LPA3 (Ki16425, 10 pM) or inhibitor of the 10 gamma receptor activated by the peroxisome proliferator (PPARg) (GW9662, 50 pM) for 1 hour before hypoxia and cellular medium containing LPA (100 pM) for 24 h. Cells without any pretreatment and cultured in serum-free medium with or without LPA, in hypoxia for 24 h, were used as positive and negative controls, respectively. The results are the average ± SEM (n = 3-6). * Denotes statistical significance: * P <0.05, ** P <0.01, *** P <0.001.

15 Figure 6: CD34 + cells treated with LPA preserve cardiac function after myocardial infarction. Myocardial infarctions were induced by permanent ligation of the left anterior descending coronary artery (LAD). (A) Cardiac fractional shortening in animals with (simulated cells, gel + CD34 + cells, CD34 + cells treated with LPA + gel) or without LAD ligation (normal). In CD34 cells treated with LPA, cells were treated with 100 pM LPA, 1 h before transplantation. Cells (1 x 10 6 cells) were administered in a fibrin gel precursor solution (100 mL) in the infarcted heart of naked rats. (B) Cardiac fractional shortening in week 3 normalized in week 1. In all the graphs, the results are averages ± SEM (n = 6-10). * Denotes statistical significance: * P <0.05, ** P <0.01, *** P <0.001

Detailed description

In the present disclosure it is shown that hematopoietic stem cells, specifically CD34 + 25 cells treated with lysophosphatidic acid (LPA) grown under hypoxia and deprived serum conditions, surprisingly double their survival with respect to untreated cells. Preferably, cells proliferate and secrete high levels of cytokines such as IL-4, IL-8 and TNF-a compared to controls. The results show that the survival of CD34 + cells is mainly mediated by the receptor activated by the peroxisome proliferator. Finally, the present disclosure shows that cells treated with LPA, but not untreated cells preserve heart function after myocardial infarction.

The present invention allows the surprising increase in the survival of hematopoietic stem cells, specifically CD34 + cells, (in number and magnitude) cell proliferation and the therapeutic effect on tissue regeneration and maintain the effect of lysophosphatidic acid.

This effect was unexpectedly observed under conditions of hypoxia and serum deprivation, and it was also surprisingly observed that hematopoietic stem cells, in particular CD34 + cells express RLPA (LPA receptors) and the effects listed above are achieved by treating CD34 + with acid lysophosphatid (LPA).

Realizations

Isolation of CD34 + cells from UCB. In one embodiment, all human umbilical cord (UCB) blood samples were collected from donors, who signed an informed consent form, in accordance with Portuguese legislation. The collection was approved by the ethics committee of Maternidade Daniel de Matos. The samples were stored in sterile bags containing 35 mL of citrate phosphate dextrose anticoagulant solution. CD34 + cells were isolated from mononuclear cells, obtained from UCB samples after Ficoll density gradient separation (preferably, Histopaque-1077 Hybri Max, preferably, 45 Sigma-Aldrich, St. Louis, USA). CD34 + cells were positively selected (2 times) using the mini-MACS immunomagnetic separation system (preferably, Miltenyi Biotec, Bergisch Gladbach, Germany,
http://www.miltenyibiotec.com), according to the manufacturer's recommendations. CD34 + cells were used immediately for cell encapsulation studies or in vivo experiments without further treatment. The isolated cells had a purity greater than 95% for the CD34 antigen as confirmed by FACS. These cells are 50 CD34 + CD45 + CD31 + KDR-vWF-CD14 (Pedroso, DC, et al., Improved survival, vascular differentiation and wound healing potential of stem cells co-cultured with endothelial cells. PLoS One, 2011. 6 ( 1): page E16114).

Cellular treatment In one embodiment, UC34 CD34 + cells (1x106 cells / mL) were incubated in X-Vivo medium (Lonza) in a hypoxia chamber for 24 h (0.5% O2 and 5% CO2), in the presence or absence of pharmacological drugs, for further evaluation of cell survival / apoptosis and necrosis 55 by FACS analysis of the expression of annexin V and PI, respectively. Preferably, the cells were treated.

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previously with the respective drugs for 1 hour before hypoxia, and the treatment was maintained during hypoxia.

Immunostaining In one embodiment, the cells were fixed with 4% paraformaldehyde (v / v) (preferably, EMS, Hatfield, USA) for 15-20 minutes at room temperature. After blocking for 30 minutes with 1% (w / v) bovine serum albumin solution (BSA) (preferably, Sigma-Aldrich), the cells were stained for 1 h with PPARg and CD34 anti-human monoclonal antibodies. In each immunofluorescence experiment, a matching isotype IgG control was used. The binding of primary antibodies to specific cells was detected with a Cy3 conjugate of anti-mouse IgG (preferably, Sigma-Aldrich). The cell nucleus was stained with 4 ', 6-diamidino-2-phenylindole (preferably, DAPI, Sigma-Aldrich). After indirect labeling, the cells were preferably examined with a Zeiss fluorescence microscope.

Flow cytometry. In one embodiment, a multicolored analysis was performed for stem cell and progenitor phenotyping on a FACS Calibur cytometer (preferably, Becton Dickinson). Cells were stained for 1 h APC or anti-human CD45 with Pacific blue (preferably, e-Bioscience), anti-mouse CD45 with FITC (preferably, -eBioscience), anti-human CD33 with PeCy7 (preferably, BD Bioscience), anti-human CD11b with PE (preferably , BD eBioscience), anti-human CD19 with FITC (preferably, BD Bioscience), anti-human CD3 with PeCy7 (preferably, BD Bioscience), anti-human Glycoforin A with PE (preferably, BD Bioscience), anti-human CD41 with FITC (preferably, e-Bioscience) , and anti-human CD56 with PeCy7 (preferably, BD Bioscience), was washed with staining medium and analyzed.

Preparation of fibrin gels. In one embodiment, fibrin gels were formed by crosslinking fibrinogen in the presence of thrombin (both from Sigma-Aldrich). The fibrinogen solution was prepared by dissolving human fibrinogen in Tris-regulated saline (TBS) (preferably, Sigma-Aldrich), pH 7.4 (20 mg / mL), and then sterilized by filtering through a syringe filter. 0.22 pm (Acrodisc, Pall, NY, USA). Fresh thrombin solutions were prepared by dissolving human thrombin in TBS at pH 7.4 at a concentration of 50 U / mL. Fibrin gels (200 pL, unless otherwise indicated) were prepared by mixing three different components: fibrinogen (10 mg / mL), CaCl2 (preferably, Merck, NJ, USA) (2.5 pM) and thrombin (0.2 U / mL). This solution was allowed to gel at 37 ° C and 100% relative humidity.

Degradation of fibrin gels. In one embodiment, the gel precursor solution was prepared by mixing Alexa Fluor® 488 human fibrinogen conjugate (preferably, Invitrogen) (0.156 mg) with unlabeled fibrinogen (9,844 mg) in 1 mL of TBS. The degradation rate of fibrin gels with or without cells over time was indirectly estimated by decreasing their fluorescence. Its fluorescence was measured immediately at zero time and at the desired time points. Complete degradation of the gels was induced by incubation with 200 pL of a solution of human plasmin (preferably, Sigma-Aldrich) in TBS (0.006 U per gel) overnight at 37 ° C. After centrifugation, the fluorescence of the supernatant fractions was measured at 520 nm in a SPECTRAmax Gemini EM fluorescence microplate reader (preferably, Molecular Devices, Sunnyvale, CA, USA,
www.moleculardevices.com).

Cytokine secretion analysis. In one embodiment, cell culture supernatants were evaluated to determine the presence and concentrations of cytokines using a Bio-Plex Pro Human Cytokine 17-Plex panel assay (preferably, Bio-Rad, Hercules, CA, USA) , according to the manufacturer's instructions, in a Bio-Plex 200 System (Bio-Rad,
www.bio-rad.com). Panel 17-Plex of human Group I consisted of the following analytes: interleukin-1p (IL-1P), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8; IL-10, IL-12 (p70), IL-13, IL-17, granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor (GM-CSF), interferon-g (IFN -g), monocyte chemotactic protein (monocyte chemotactic activating factor [MCP-1 (MCAF)], macrophage inflammatory p protein (MIP-1p) and tumor necrosis factor a (TNF-a). Samples of the medium were collected supernatant, centrifuged to remove precipitates and freeze A standard range of 0.2 to 3,200 pg / mL was used Samples and controls were run in triplicate, standards and duplicate blanks.

Analysis of quantitative reverse transcription polymerase chain reaction (qRT-PCR). In one embodiment, the total RNA in the cells was extracted using the RNeasy Mini Kit (preferably, Qiagen, Valencia, USA), according to the manufacturer's instructions. The cells were initially centrifuged and homogenized in Trizol. In all cases, cDNA was prepared from 1 pg of total RNA using Taqman reverse transcription reagents (preferably, Applied Biosystems, Foster City, USA). Quantitative PCR (qPCR) was performed using Power SYBR Green PCR Master Mix (preferably Applied Biosystems) and detection was performed in a 7500 real-time rapid PCR system (preferably Applied Biosystems,
www.appliedbiosystems.com). The quantification of the target genes was performed with respect to the reference (human or mouse, depending on the type of cells under analysis) GAPDH gene: relative expression = 2 [- (Ctsample- CtGADPH)]. The minimum average values of the cycle threshold (Ct) were calculated from four independent reactions. The primer sequences are published as supporting information (Table S1).

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Animal model of myocardial infarction. In one embodiment, hairless rats were anesthetized with ketamine (75 mg / kg, IP) and dexmedetomidine (0.375 mg / kg, IP). They were anesthetized with 2-3% isofluorane in equilibrium with oxygen. The abdomen and anterior chest were rubbed with betadine and rinsed with 70% alcohol (with several cycles of betadine rub followed by alcohol rinse and application of betadine solution). The heart was approached by a transverse abdominal incision (diaphragmatic incision) with the animal's back extended gently on a soft towel or laterally through the intercostal space 4-5. The instruments were sterilized by autoclave. A small diaphragmatic incision was made to create a pericardial window. A 5 mm incision was made in the pericardium with an 11-0 scalpel. Myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery with a 6-0 proline suture, 2-3 mm below the origin of the artery. Paleness and abnormal movement of the regional wall of the left ventricle confirmed occlusion. The pericardium was left open or removed to prevent plugging. The pleural space was evacuated with a sterile 18 gauge needle and a 3 mL syringe after the closure of the pleural cavity. The abdominal wall and subcutaneous tissue were closed with Vicryl 4-0 followed by subcuticular closure with Vicryl 4-0. The animal was extubated and then allowed to recover. Each animal was maintained during surgical procedures and recovery under heating pads until he was awake and able to walk. Two days after the recovery of this procedure, the animals underwent echocardiographic evaluation under anesthesia with ketamine / midazolam. Animals that met the echocardiographic inclusion criteria (fractional shortening below 50%) were stratified into one of 4 groups. The rats were then subjected to a second thoracotomy followed by direct injection of 100 pL of therapeutic agent using a Hamilton syringe (preferably Hamilton Company) and a 30 gauge needle. The next day, the animals were monitored by echocardiography. Two weeks after implantation, the surviving rats were analyzed again by echocardiography. In week 3, the animals were sacrificed for euthanasia. The heart and various organs were collected, fixed in methyl Carnoy solution and processed for histological analysis.

LPA induces the survival of CD34 + cells under conditions of hypoxia and serum deprivation

In a preferred embodiment, to identify molecules that promote the survival of CD34 + cells under conditions of hypoxia and serum deprivation, an assay was developed using CD34 + cells derived from human umbilical cord blood (2x105 cells per well of a 96-well plate ) suspended in X-Vivo medium (used in clinical trials (Schachinger, V., et al., Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med, 2006. 355 (12): pages 1210-21 )) and incubated in a hypoxia chamber with 0.5% O2, at 37 ° C, for 24 h. Some of the selected medications have been approved by the FDA for the treatment of cardiovascular diseases (for example, Nebivolol Lombardo, RM, et al., Effects of nebivolol versus carvedilol on left ventricular function in patients with chronic heart failure and reduced left systolic ventricular J Cardiovasc Drugs, 2006. 6 (4): pages 259-63; IbersatanMassie, BM, et al., Irbesartan in patients with heart failure and preserved ejection fraction.N Engl J Med, 2008. 359 (23): pages 2456-67), others are being evaluated in preclinical / clinical trials to improve heart function in patients / models with heart failure (for example, INO-1001 to novel poly (ADP-ribose) polymerase (PARP) inhibitor improves cardiac and pulmonary function after crystalloid cardioplegia and extracorporal circulation Shock, 2004. 21 (5): pages 426-32); erythropoietin (Belonje, AM, et al., Effects of erythropoietin after an acute myocardial infarction: rationale and study design of a prospective, randomized, clinical trial (HEBE III). Am Heart J, 2008. 155 (5): pages 817- 22); melatonin (Chen, Z., et al., Protective effect of melatonin on myocardial infarction. Am J Physiol Heart Circ Physiol, 2003. 284 (5): pages H1618-24); VX-702 (Ma, XL, et al., Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation, 1999. 99 (13): pages 1685-91)), others they are natural substances found in the human body (LPA). Cell viability was assessed by flow cytometry using annexin V / propidium iodide (PI) staining. Annexin V is a phospholipid binding protein with specificity for phosphatidyl serine, one of the first markers of cell transition to an apoptotic state. This phospholipid translocates from the inner leaflet to the outer leaflet of the plasma membrane (Koopman, G., et al., Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood, 1994. 84 (5): pages 1415-20). PI enters necrotic cells and binds to double-stranded nucleic acids, but is excluded from cells with normal integrity [20]. According to Figure 1, untreated CD34 + cells show very poor survival, with only ~ 31% of viable cells, ~ 31% of apoptotic cells (Annexin V + / PI-) with the majority of cells being ~ 39% in the necrotic stage (PI +). Of the ten drugs analyzed, LPA, prostaglandin E2 (PGE2) and erythropoietin significantly improved cell survival (P <0.001). LPA (10 pM) was the drug with the greatest effect in favor of survival (~ 69% of viable cells) and, therefore, was studied further (Figures 1 and 2A). Surprisingly, LPA improves cell survival by reversing the necrosis and apoptosis of CD34 + cells especially in hypoxic and serum-deprived conditions.

The effect in favor of LPA survival was concentration dependent (from 1-100 pM), with the survival of already statistically significant CD34 + cells (P <0.001, n = 6) compared to the control (untreated cells) at 1 pM of LPA (Fig. 2B). Importantly, the percentage of viable cells in CD34 + treated with 1 pM of lPa and cultured under hypoxia conditions is similar to untreated cells grown under normoxia conditions. The effect in favor of LPA survival decreases depending on the

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hypoxia time (Figure 2C). The percentage of viable cells in CD34 + cells treated with LPA decreased from 78% on day 1 to 40% on day 3. Overall, the results obtained surprisingly indicate that LPA is a molecule in favor of the survival of CD34 + cells and their effect is dependent on time and concentration.

LPA induces cell proliferation without the expansion of early multipotent progenitor cells

LPA is highly mitogenic for quiescent cells [21]. The mitogenic action of LPA involves the activation of a pertussis toxin insensitive G protein with subsequent mobilization of Ca2 + and stimulation of protein kinase C, release of arachidonic acid in a GTP-dependent manner and activation of a pertussis toxin-sensitive Gi protein which mediates the inhibition of adenylate cyclase (van Corven, EJ, et al., Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins. Cell, 1989. 59 (1): pages 45-54). To determine whether LPA can induce proliferation of CD34 + cells, a suspension of untreated or LPA-treated cells (2x105 cells in 200 pl of X-vivo medium) was exposed to hypoxia for 24 h and then cultured under normoxia conditions during 6 additional days Cells treated with LPA increased their number approximately 3 times during the 7-day period while untreated cells decreased to half of their initial number (Fig. 3A).

To examine the effect of LPA on the self-renewal / differentiation of CD34 + cells, CD34 + cells not treated and treated with LPA were grown in X-live medium under hypoxic conditions for 1 day or not for 6 days in normoxia and finally characterized by FACS. After 1 day of hypoxia, both untreated CD34 + cells and those treated with LPA began to differentiate into mast cells (between 14 and 17%) and neutrophils (between 5 and 8%) (Figure 3B). Only 78% and 72% of CD34 + cells not treated or treated with LPA express the CD34 marker. CD34 expression is higher in cells that have been exposed to hypoxia than in normoxia conditions since only 6% of cells grown under normoxia conditions for 24 hours express the CD34 marker. Cells grown for 1 day in hypoxia and then 6 days in normoxia are further differentiated into several cell lineages that include dendritic cells (CD), basophils, monocytes, neutrophils and mast cells. Similar differentiation profiles were observed for untreated and LPA treated cells.

LPA induces the survival of CD34 + cells in cells encapsulated in fibrin gels and are grown under conditions of hypoxia and serum deprivation.

Injectable structures are very promising vehicles for administering stem cells for regenerative medicine as they provide favorable structural support for cell survival and proliferation (Pedroso, DC, et al., Improved survival, vascular differentiation and wound healing potential of stem cells co - cultured with endothelial cells. PLoS One, 2011. 6 (1): page e16114; Kraehenbuehl, TP, R. Langer, and LS Ferreira, Three-dimensional biomaterials for the study of human pluripotent stem cells. Nat Methods, 2011. 8 (9): pages 7316; Nakamuta, JS, et al., Cell therapy attenuates cardiac dysfunction post myocardial infarction: effect of timing, routes of injection and a fibrin scaffold. PLoS One, 2009. 4 (6): page e6005). Several studies have shown that transplanted cells with structures in the cardiac context improved cell survival, induced angiogenesis and preserved cardiac function after infarction (Nakamuta, JS, et al., Cell therapy attenuates cardiac dysfunction post myocardial infarction: effect of timing, routes of injection and a fibrin scaffold PLoS One, 2009. 4 (6): page e6005; Christman, KL, et al., Fibrin glue alone and skeletal myoblasts in a fibrin scaffold preserve cardiac function after myocardial infarction. , 2004. 10 (3-4): pages 403-9; Kraehenbuehl, TP, et al., Human embryonic stem cell-derived microvascular grafts for cardiac tissue preservation after myocardial infarction. Biomaterials, 2011. 32 (4): pages 1102 -9; Kutschka, I., et al., Collagen matrices enhance survival of transplanted cardiomyoblasts and contribute to functional improvement of ischemic rat hearts. Circulation, 2006. 114 (1 Suppl): pages 1167-73). Recently, it was shown that CD34 + cells have a higher initial adhesion to fibrin gels than to polystyrene, collagen and fibronectin plates (Pedroso, DC, et al., Improved survival, vascular differentiation and wound healing potential of stem cells cocultured with endothelial cells. PLoS One, 2011. 6 (1): page e16114). In addition, it is shown that fibrin gels support the survival of CD34 + cells for at least 10 days and the gels resist degradation of metalloprotease enzymes (Pedroso, DC, et al., Improved survival, vascular differentiation and wound healing potential of stem cells cocultured with endothelial cells PLoS One, 2011. 6 (1): page e16114). In a preferred embodiment, to examine whether fibrin gels support the survival of CD34 + cells under conditions of hypoxia and serum deprivation, cells untreated or treated with LPA (2x105) were encapsulated in fibrin gel (200 pL) and incubated in a 0.5% O2 hypoxia chamber, at 37 ° C, for 1 and 3 days. Cell viability was assessed by flow cytometry using annexin V / propidium iodide (PI) staining. Untreated CD34 + cells have poor survival in fibrin gels (23.2 ± 2.8% of viable cells, n = 3, on day 1, 12.6 ± 1.2% of viable cells, n = 5, on day 3) that show that the matrix alone has none in favor of survival (Fig. 4). In contrast, CD34 + cells treated with LPA encapsulated in fibrin gels have high survival, comparable to the values observed in cells treated with LPA not encapsulated in fibrin gels (day 1: 69.0 ± 0.7% vs. 76 , 7 ± 1.5% for encapsulated and non-encapsulated; respectively, day 3: 51.1 ± 0.8% versus 37.2 ± 6.6% for encapsulated and non-encapsulated, respectively).

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LPA induces survival of CD34 + cells primarily through the peroxisome proliferator activated receptor (PPAR)

The biological roles of LPA are diverse and include developmental, physiological and pathophysiological effects (Lin, ME, DR Herr and J. Chun, Lysophosphatidic acid (LPA) receptors: signaling properties and disease relevance. Prostaglandins Other Lipid Mediat, 2010. 91 ( 3-4): pages 130-8). To date, up to five LPA receptors (LPAR) have been identified: LPA1-LPAS (Choi, J.W., et al., LPA receptors: subtypes and biological actions. Annu Rev Pharmacol Toxicol, 2010. 50: pages 157-86). The receptors are G-protein coupled receptors (GPCR) and their presence can be found in multiple tissues. LPA1, LPA2 and LPA3 are widely expressed in most tissues (Ishii, I., et al., Functional comparisons of the lysophosphatidic acid receptors, LP (A1) / VZG-1 / EDG-2, LP (A2) / EDG-4, and LP (A3) / EDG-7 in neuronal cell lines using a retrovirus expression system, Mol Pharmacol, 2000. 58 (5): pages 895-902). LP4 is expressed in specific organs, such as the pancreas, ovaries and thymus (Lee, CW, et al., LPA (4) / GPR23 is a lysophosphatidic acid (LPA) receptor utilizing G (s) -, G (q) / G (i) -mediated calcium signaling and G (12/13) -mediated Rho activation. J Biol Chem, 2007. 282 (7): pages 4310-7). LPA5 is expressed at low levels in multiple tissues (Lee, CW, et al., GPR92 as a new G12 / 13- and Gq-coupled lysophosphatidic acid receptor that increases cAMP, LPA5. J Biol Chem, 2006. 281 (33): pages 23589-97). To identify the receptor that is mediating the effect in favor of the survival of LPA (100 pM), Ki16425 (10 pM), a specific antagonist of LPA1 and LPA3, and GW9662 (50 pM), a receptor antagonist and activated was used by the peroxisome proliferator (PPARy). Studies indicate that LPA has a high affinity for this last receptor (Mclntyre, TM, et al., Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPARgamma agonist. Proc Natl Acad Sci USA, 2003. 100 (1): pages 131-6; Gustin, C., M. Van Steenbrugge, and M. Raes, LPA modulates monocyte migration directly and via LPA-stimulated endothelial cells. Am J Physiol Cell Physiol, 2008. 295 (4) : pages C905-14). This approach was complemented by inhibiting the targets of the receptors later, including Rho kinase and mitogen-activated protein kinase (MAPK) using Y-2762 (50 pM) and PD98059 (60 pM) antagonists, respectively. It is known that all LPARs are coupled with and activate G proteins, which in turn activate MAPK (LPA1, LPA2, LPA3 and LPA4) and Rho kinase (LPA1, LPA2, LPA4 and LPA5) (Choi, JW, et al., LPA receptors: subtypes and biological actions, Annu Rev Pharmacol Toxicol, 2010. 50: pages 157-86). The cells were treated for 1 h in X-Vivo medium containing a specific antagonist followed by culture under hypoxia conditions for 24 h. Cellular survival was evaluated by FACS analysis after annexin V / PI staining. The results indicate that, under the conditions analyzed, PPAR and mainly mediates the effect in favor of LPA survival (Fig. 5B). The PPARy antagonist significantly decreased (P <0.001, n = 11) the number of viable cells induced by LPA from ~ 77% to ~ 53%; however, it did not completely block the effect in favor of LPA survival, since non-LPA cells have a 39% survival (P <0.01). The inhibition of LPA1 and LPA3 by Ki16425 had a relatively small effect on the survival of CD34 + cells (Fig. 5B). The number of viable cells decreased from 77% (+ LPA) to 67% (P <0.01, n = 19). Importantly, the inhibition of the MAPK signaling pathway suppressed the effect in favor of LPA survival at higher levels than the inhibition of the Rho kinase signaling pathway. Because the two signaling pathways are the later targets of different LPARs (see above), this could indicate different LPAR contributions in the survival of CD34 + cells. Taken together, the data shows that LPA can promote the survival of CD34 + cells in hypoxic and serum-deprived conditions primarily through the activation of PPARy.

LPA modulates the release of cytokines from CD34 + cells

To determine the effect of LPA on the release of signaling cytokines and growth factors by CD34 + cells, a matrix of cytokine beads was used. The cells were incubated in serum-free medium X-Vivo for 24 h under conditions of normoxia or hypoxia, in the presence or absence of LPA (100 pM). Untreated CD34 + cells in normoxia express high levels (> 100 pg / mL) of IL-8 and MIP-1 b, medium levels (between 100 and 1 pg / mL) of IL-6, TNF-a and GM-CSF , and low levels (<1 pg / mL) of IL-1 p, IL-4 and IL-17 (Fig. 5C). CD34 + cells grown under hypoxia and serum-deprived conditions express significantly higher levels of IL-1 p (~ 8 times), IL-4 (~ 2 times), IL-6 (~ 1.2 times), IL- 8 (~ 10 times), IL-17 (~ 4 times) and GM-CSF (~ 2 times), which under normoxia conditions. Interestingly, CD34 + cells grown under hypoxia and serum deprivation conditions but in the presence of LPA increase the secretion of IL-4 (from ~ 0.9 to ~ 2.7 pg / mL), IL-8 (from ~ 10,000 to ~ 17,000 pg / mL) and TNF- a from ~ 3 to ~ 15 pg / mL) and decrease the secretion of IL-1 p (from ~ 9 to ~ 2 pg / mL), IL-6 (from ~ 4 to ~ 2 pg / mL) and gM -CSF (from ~ 4 to ~ 1 pg / mL) compared to cells grown under the same conditions but in the absence of LPA.

CD34 + cells treated with LPA retain cardiac function after myocardial infarction

It has been shown that transplantation of human CD34 + cells in the heart after infarction improves left ventricular ejection fraction and preserves heart tissue. To assess the therapeutic potential of CD34 + cells treated with LPA, the cells were administered (1x106 cells) treated with LPA (100 pM) in a fibrin gel precursor solution (100 pL) in the infarcted heart of hairless rats. Myocardial infarctions were induced by permanent ligation of the left anterior descending coronary artery (LAD). Hearts infarcted without any treatment (simulated) or treated with CD34 + cells suspended in a fibrin gel precursor solution were used as controls. After 2 weeks after implantation, the functional properties

of the heart were evaluated by echocardiography. The left ventricle of the control rat had a mean fractional shortening of 42.7 ± 1.6 (n = 10) and 41.6 ± 3.8 (n = 10) at 1 day and 2 weeks, respectively; rats treated with CD34 + cells encapsulated in a fibrin gel precursor solution had a mean fractional shortening of 36.7 ± 3.4 (n = 6) and 44.5 ± 3.0 (n = 6) at 1 day and 2 weeks, respectively; and finally rats treated with CD34 + cells treated with LPA and encapsulated in a fibrin gel precursor solution had a mean fractional shortening of 37.5 ± 1.2 (n = 6) and 47.0 ± 2.0 (n = 6) at 1 day and 2 weeks, respectively (Fig. 6A). Because the initial fractional shortening is different between the experimental groups, the difference in fractional shortening at 2 weeks and on day 1 was used to compare the therapeutic effectiveness of the treatments (Fig. 6B). The median of the differences in fractional shortening was 7.5 and 2.6 in the 10 cells treated with LPA + gel and the simulated groups, respectively, the differences being statistically significant (P <0.05). No statistical differences were observed between hearts treated with CD34 + cells and simulations (P> 0.05). Taken together, the administration of CD34 + cells treated with LPA in the infarcted heart improves cardiac fractional shortening, and the effect was superior to untreated CD34 + cells.

Claims (17)

5 10 fifteen twenty 25 30 1. Method to improve the survival of hematopoietic stem cells in hypoxic and serum-deprived conditions by culturing the cells in a medium containing lysophosphatidic acid. 2. Method according to claim 1, wherein the hematopoietic stem cells are CD34 + cells. 3. Method according to any of the preceding claims, wherein the concentration of lysophosphatidic acid varies between 1 and 1,000 pM. 4. Method according to claim 3, wherein the concentration of lysophosphatidic acid is 100 pM. 5. Composition comprising hematopoietic stem cells and lysophosphatidic acid in which the concentration of lysophosphatidic acid is 100 pM. 6. Composition according to claim 5, wherein the hematopoietic stem cells are CD34 + cells. 7. Composition according to any of claims 5-6, further comprising a gel, in particular a biomimetic gel. 8. Composition according to any of claims 5-7, comprising 1x105-1x106 CD34 + cells; and 100 pM lysophosphatidic acid; and 100-200 mL of biomimetic gel. 9. Composition according to any of claims 7-8, wherein said gel is at least one of the following: fibrin, hyaluronic acid, alginate, agarose, collagen or PEG derivatives. 10. Composition as described in any of claims 5-9, wherein said composition is a pharmaceutical, medical or cosmetic composition. 11. Composition according to claim 10, wherein the components are in a therapeutically effective amount. 12. Composition as described in any of claims 5-11 for use in medicine or cosmetics. 13. Composition according to claim 12 for use in cell therapy, namely cardiac tissue cell therapy. 14. Composition according to any of claims 12-13 for use in the treatment of heart disease or in the treatment of wound healing. 15. Composition for use according to claim 14, wherein said wound healing is healing of diabetic wounds. 16. Composition according to any of claims 5-11 or composition for use according to any of claims 12-15, further comprising suitable amounts of excipient. 17. Composition according to any of claims 5-11 or composition for use according to any of claims 12-16, wherein the composition is an injectable formulation.