Activated mesenchymal stem cells for the treatment of limb ischemia

Abstract

The present invention provides mesenchymal stem cells treated with analgin for treating limb ischemia in human patients. The present invention provides a pharmaceutical product, which is used for angiogenesis growth to replace occluded blood vessels to avoid limb amputation. By activating mesenchymal stem cells (MSCs), the secretion of angiogenesis growth factors VEGFA, HGF, bFGF, TEK is stimulated and the level of pro-inflammatory cytokines IF6, CXCF8, CCF2, IL1-RN is reduced. According to the treatment results, MSCs produce proteins and signaling molecules for the growth of new blood vessels, thereby accelerating the growth of new arteries.

Description

Technical Field

[0001] The progress and success of cell therapy rely on technologies that enable us to manipulate stem cells by stimulating proliferation, differentiation and integration into regenerative tissues and organs. Separated stem cells can be manipulated at different stages of cell therapy procedures, including cell isolation, cell propagation, transplantation and post-transplantation pretreatment (conditioning). The present invention relates to the use of clinically approved anti-inflammatory drugs to regulate the activity of mesenchymal stem cells isolated from adipose tissue. The secretion regulation of different growth factors including angiogenic factors and cytokines and the regulation of metabolic activity can be used to develop efficient MSC-based drug products for the treatment of various health conditions. Background Art

[0002] Mesenchymal stem cells (MSCs) are characterized by their ability to differentiate into a variety of cell lineages in vitro and to have immunomodulatory functions in regenerative processes (Augello & De Bari, 2010), (Shi et al., 2010). Unlike drug therapies that deliver specific active components at specific doses, MSCs exert therapeutic effects by secreting various bioactive compounds in response to external stimuli (Ma et al., 2014). Soluble factors produced by MSCs participate in anti-inflammatory and neovascularization processes, and have profound effects on tissue damage and regeneration. Diseases that can be treated with MSCs are, for example, immune and non-immune disorders such as myocardial infarction, diabetes, graft-versus-host disease, and liver cirrhosis (Wei et al., 2013) (Shi et al., 2010). Understanding the immunomodulatory properties of MSCs and how the inflammatory microenvironment affects their function is of great importance for developing better strategies to improve the therapeutic efficiency of MSCs with the goal of creating local and / or systemic conditions to stimulate healing and tissue regeneration.

[0003] Peripheral arterial disease is a condition characterized by limited oxygen and glucose supply due to vascular dysfunction. Peripheral vascular disease usually affects arteries and can cause critical limb ischemia (CLI) in the most advanced stage. To date, the most common treatment options include drug therapy and surgery (Norgren et al., 2007), (Hamdy et al., 2013), (Cullet al., 2014). Nonsteroidal anti-inflammatory drugs and synthetic glucocorticoid injections are widely used to reduce pain and inflammation in CLI patients. However, in many cases, the result of disease progression is gangrene and amputation. The development of new advanced therapies can improve clinical outcomes and improve the living standards of patients. The clinical potential of MSCs for the treatment of ischemic conditions has been described in several animal models and early human clinical trials (Liew & O'Brien, 2012). The efficiency and safety of administering MSCs to treat acute ischemic diseases show great potential. The therapeutic efficiency of MSCs depends on its ability to provide immunomodulatory and angiogenic factors to suppress inflammation and stimulate angiogenesis. Unactivated MSCs express low levels of immunosuppressive factors, but local conditions at the injection site affect their functionality. Different strategies have been developed to improve the therapeutic effects of MSCs. Stimulation of MSCs by IFNγ or TNFα has been used to induce the secretion of immunomodulatory factors (Crop et al., 2010). In addition, overexpression of CXCR4 in MSCs leads to more efficient homing of MSCs to ischemic tissues compared to unmodified cells (Cheng et al., 2008). Therefore, the immunosuppressive and angiogenic effects of MSCs can be stimulated by changing the conditions of the affected / lesional tissue or pretreatment of MSCs before transplantation. Since patients with limb ischemia are treated with different anti-inflammatory drugs, it is very important to understand the consequences of these drugs on the anti-inflammatory and angiogenic functions of MSCs. Most NSAIDs function by blocking prostaglandin synthesis via inhibition of cyclooxygenase. It is known that prostaglandin PGE2 is one of the important compounds secreted by MSCs, which is responsible for the regulation of inflammation. However, the role of NSAIDs in the complex of metabolic response and the secretion of anti-inflammatory and angiogenic factors in MSC anti-inflammatory therapy is still unclear.

[0004] The effects of NSAIDs (acetaminophen, analgin, ketoprofen and diclofenac) and the glucocorticoid prednisolone, which are used in clinical practice to treat ischemic diseases, on the cell cycle, metabolic activity, and on angiogenesis and inflammatory cytokine expression by AdMSCs at therapeutic doses have been analyzed and will be described below. Summary of the invention

[0005] The object of the present invention is to provide a pharmaceutical product for the angiogenic growth to replace occluded blood vessels to avoid limb amputation.

[0006] The inventors have developed a protocol to stimulate mesenchymal stem cells to secrete regulators that affect the regeneration of ischemic limbs and improve neovascularization and arteriogenesis. MSCs were treated with analgin, whose active ingredient is analgin, which alters the cell cycle, stimulates the synthesis of angiogenic trophic factors VEGF, HGF, TEK and bFGF, and reduces the expression of inflammatory cytokines and chemokines such as IL6, IL1RN, CCL2, IL8 / CXCL8.

[0007] Using a rat model of limb ischemia, it was demonstrated that treatment of MSCs with analgin prior to transplantation stimulated neoangiogenesis and arteriogenesis in the operated limb.

[0008] According to the following research examples, AdMSC is a promising and promising cell source for cell therapy to treat critical limb ischemia (CLI). Although the preliminary study of applying MSC to treat CLI patients is promising, the potential of MSC is not very effective, and there are many problems about their feasibility use in the clinic. Recently, it has been shown that MSC pretreatment and preactivation significantly improve their immune and therapeutic potential. The present invention proposes a new strategy for treating CLI by preactivating AdMSC via NSAID analgin. Analgin affects the proliferation and metabolic activity of AdMSC initiation, changes the cell cycle, dynamically regulates the expression profile of inflammatory cytokines and chemokines, and induces the expression of angiogenesis markers, which is important in the case of disease treatment. Preclinical experiments on rats have shown that AdMSC activated by analgin are more effective for their therapeutic application in treating limb ischemia through their accelerated and reliable new arterial generation and new angiogenesis.

[0009] In the present invention, mesenchymal stem cells (MSCs) are activated to stimulate the secretion of angiogenic growth factors VEGFA, HGF, bFGF, and TEK and reduce the levels of pro-inflammatory cytokines IL6, CXCL8, CCL2, and IL1-RN.

[0010] The present invention provides mesenchymal stem cells treated with analgin for treating limb ischemia in human patients. The pharmaceutical product of the invention comprises mesenchymal stem cells treated with analgin. The product is in an injectable form and preferably comprises 1 million cells / kg patient. The cells are administered in an amount of 750,000-1.5 million / kg patient body weight. The cells treated with analgin are trace concentrations.

[0011] The present invention also discloses a method for producing a product comprising mesenchymal stem cells treated with analgin. The method comprises the following steps:

[0012] Collecting fat tissue

[0013] Isolation of mesenchymal stem cells from adipose tissue

[0014] MSC replication

[0015] Influencing MSCs with active ingredients

[0016] According to the study, treatment of MSCs with Analgin provides advantages and assumptions for the effectiveness of the product.

[0017] As a result of the treatment, MSCs produce proteins and signaling molecules for the growth of new blood vessels, thereby accelerating the growth of new arteries. The effects of different drugs on MSCs and the drug used in the present invention, analgin, on MSCs have been studied and will be described below. The products of the invention have been successfully tested on animals.

[0018] It is known that stem cells are involved in angiogenesis and arteriogenesis, and previous laboratory studies and animal experiments have aimed to find the most effective cell drug combination. Since time is of the essence for patients with severe leg ischemia, it is important to find a cell drug that works as quickly as possible. Although the product of this invention has only been tested on animals so far, it is believed to have the same effect on human patients based on the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following figures are shown:

[0020] Figure 1 . Assay of WST metabolic activity in MSCs treated with anti-inflammatory drugs (AIDs)

[0021] For control and AID-treated (acetaminophen, analgin, diclofenac, ketoprofen, prednisolone) AdMSCs, absorbance was measured at 440 nm after 2 hours of incubation with WST-1 reagent. AdMSC metabolic activity was measured on days 1, 2, 3, and 5 after treatment.

[0022] Figure 2 Cell cycle analysis of control and AID-treated AdMSCs AID-treated and control AdMSCs were stained with propidium iodide (PI) 24 h after treatment and analyzed by flow cytometry using BD Accuri C6.

[0023] Figure 3 Effect of AID on the expression of angiogenic markers in AdMSCs

[0024] The VEGFA, HGF, bFGF and TEK mRNA expression levels in AdMSCs treated with AID for 24 h were measured in triplicate by RT-qPCR and normalized with GAPDH mRNA expression levels. The data from AID-treated cells were calculated relative to control untreated AdMSCs, and the results were expressed in a logarithmic scale. Positive values ​​indicate that mRNA levels are increased, and negative values ​​indicate that mRNA levels are reduced, compared to control untreated cells.

[0025] Figure 4 .Relative mRNA expression of inflammatory genes in LPS-stimulated and AID-treated 24h is presented as a heat map. Expression levels were measured in triplicate by RT-qPCR and normalized with GAPDH mRNA expression levels. Data from LPS-stimulated AID-treated AdMSCs were calculated as relative fold differences compared to LPS-treated cells and converted to a logarithmic scale. Quantitative changes in gene expression are represented by color: red indicates upregulation, while blue indicates downregulation. Expression data were visualized using GENE-E software.

[0026] Figure 5 Changes in inflammatory factors in the culture medium. Secreted IL6, CXCL8, CCL2, and IL1RN levels in the conditioned medium from control, LPS-stimulated, and LPS-stimulated and acetaminophen or prednisolone-treated AdMSCs were measured by ELISA at 24, 48, and 72 h and expressed as normalized to the control 24 h probe.

[0027] Figure 6 .Heatmap of inflammatory gene expression profiles in LPS-stimulated and AID-treated AdMSCs. The mRNA expression levels of inflammatory genes in AdMSCs stimulated with LPS and treated with AID for 48 h (A) and 72 h (B) were calculated as fold differences compared to LPS-treated AdMSCs and presented as heatmaps. The expression levels of inflammatory gene mRNAs were measured in triplicate by RT-qPCR, normalized with GAPDH mRNA levels, and converted to a logarithmic scale. Positive values ​​(red) indicate higher expression, and negative values ​​(blue) indicate lower expression relative to control LPS-treated AdMSCs.

[0028] Figure 7 . Correlation analysis between angiographic and perfusion differences. (A) shows angiogenesis and perfusion differences, (B) demonstrates arteriogenesis and perfusion differences. Solid regression lines are fitted, while dashed lines represent 95% confidence intervals.

[0029] Figure 8Kaplan-Meier survival analysis of treatment groups. Dashed lines indicate 95% confidence intervals. BEAULI n=8, CYTORI n=13, MSC n=8, MSCD n=8 and SALINE n=16.

[0030] Fig. 9 . One-way analysis of variance (ANOVA) of perfusion differences between treatment groups. (A) shows data before surgery, (B) demonstrates data immediately after surgery, (C) represents data 3 days after surgery, (D) represents data 7 days after surgery, and (E) represents data 14 days after surgery. BEAULI - group of animals operated and treated with lipoaspirate; CYTORI - group of animals treated with cells derived from the Cytori Cellution 800 / CRS system; MSC - laboratory isolated and expanded mesenchymal stem cells; MSCD - mesenchymal stem cells treated with 10 μM analgin; SALINE - control animals treated with 0.9% NaCl.

[0031] Fig.10 One-way ANOVA of angiogenesis and arteriogenesis between treatment groups. ANOVA of (A) angiogenesis and (B) arteriogenesis. BEAULI – group of animals operated and treated with lipoaspirate; CYTORI – group of animals treated with cells derived from the CytoriCellution 800 / CRS system; MSC – mesenchymal stem cells isolated and expanded in the laboratory; MSCD – mesenchymal stem cells primed with 10 μM analgin; SALINE – control animals treated with 0.9% NaCl.

[0032] Fig.11 .Angiograms from a series of MSCD-treated animals.

[0033] Fig.12 .Angiograms from a series of MSC-treated animals.

[0034] AID affects the metabolic activity and cell cycle progression of MSCs

[0035] Using AdMSC pools isolated from at least three donors, the effects of acetaminophen (4.4 mM), diclofenac (10 μM), analgin (10 μM), ketoprofen (50 μM) and prednisolone (0.1 μM) on metabolic activity were analyzed to reduce the impact of donor variation. Metabolic activity assay (WST-1) data showed that AID slightly altered the metabolic activity of AdMSC ( Figure 1). During the first 48 h, all analyzed AIDs stimulated the metabolic activity of AdMSCs. Analgin treatment significantly stimulated cell metabolism within the first 24 h, while all investigated drugs demonstrated a long-term inhibitory effect on the metabolic activity of AdMSCs ( Figure 1 ).

[0036] The effects of drugs on cell cycle changes measured by flow cytometry are shown in Figure 2 Compared with untreated control cells (G0 / G1 phase - 82%), the percentage of cells in the G0 / G1 phase was not significantly affected after treatment with diclofenac (81%), ketoprofen (80%), and prednisolone (77%) ( Figure 2 The percentage of cells in the G0 / 1 phase was lower in cultures treated with analgin (65%) and acetaminophen (71%), whereas the percentage of cells in the G2 / M cell cycle was increased in these cultures ( Figure 2 ). The results obtained showed that analgin and acetaminophen were the only investigated substances that affected the cell cycle, since they accumulated AdMSCs in the G2 / M phase.

[0037] In conclusion, all investigated AIDs affected the metabolic activity of AdMSCs, whereas analgin and acetaminophen also slightly altered cell cycle progression.

[0038] AID affects the expression of angiogenic factors

[0039] Many soluble factors produced by MSCs are involved in the regulation of angiogenesis and neovascularization in vivo (Estrada et al., 2009). On the other hand, MSCs have been revealed to inhibit angiogenesis under certain conditions (Otsu et al., 2009). In addition, the trophic factors VEGF and bFGF have been shown to stimulate angiogenesis in ischemic treatment (Leung et al., 1989). The effect of AIDs on the expression of angiogenic factors in AdMSCs was analyzed. Different AIDs have different effects on the expression of angiogenic factors, stimulating or inhibiting their expression. Treatment of AdMSCs with analgin and ketoprofen resulted in upregulation of VEGFA, HGF, bFGF, and TEK mRNA expression ( Figure 3 ), diclofenac had no effect, and acetaminophen and prednisolone inhibited VEGFA and HGF expression. In addition, acetaminophen induced bFGF but strongly inhibited TEK expression, while prednisolone had the opposite effect ( Figure 3 ).

[0040] Effect of AID on the expression of immune regulatory factors

[0041] A large number of inflammatory cytokines and chemokines secreted by MSCs are involved in immunomodulatory processes, thereby affecting immunocompetent cells. Quantitative differences in the levels of cytokines secreted by MSCs determine the local conditions of the microenvironment and induce anti-inflammatory responses. Identification of inflammatory biomarker profiles in response to MSC therapy combined with AID treatment could predict the outcome of such an intervention on the overall immune status. The effects of AID treatment on the inflammatory profile of AdMSCs were analyzed at the protein and gene expression levels.

[0042] Exposure of AdMSCs to acetaminophen, diclofenac, analgin, ketoprofen, and prednisolone in standard culture conditions had little effect on the synthesis and secretion of immunomodulatory factors (Table 1). Only acetaminophen resulted in a significant decrease in IL6, CXCL8 / IL8, and CCL2 levels in the culture medium compared to untreated cells.

[0043] To simulate inflammatory conditions, AdMSCs were exposed to lipopolysaccharide (LPS) before AID treatment. After LPS treatment, the levels of inflammatory cytokines IL6, CXCL8, CCL2, and IL1RN increased (Table 1). Then, LPS-treated AdMSCs were exposed to AID to study their effects on cytokines under inflammatory conditions.

[0044] Acetaminophen and prednisolone significantly reduced IL6 and CXCL8 levels in LPS-stimulated AdMSC cultures (Table 1). Analgin had an inhibitory effect on detectable CCL2 and IL1RN expression. In contrast, ketoprofen and diclofenac stimulated the expression of IL6 and CXCL8 in LPS-stimulated AdMSC cultures. Interestingly, exposure of AdMSC to ketoprofen and diclofenac did not result in an effect on CCL2 levels.

[0045] Table 1 AdMSCs secrete inflammatory factors in response to AID treatment The concentrations of secreted IL6, CXCL8, CCL2 and IL1RN proteins were measured by ELISA from culture medium of control, LPS-stimulated and AID-treated AdMSCs for 24 hours in pg / ml.

[0046]

[0047]

[0048] The effect of AID on the mRNA expression of inflammatory cytokines in AdMSCs was analyzed using RT-qPCR. The analysis results were visualized as a heat map using the GENE-E platform ( Figure 4Short-term treatment of AdMSCs with AIDs affected the expression of inflammatory cytokines. Exposure of AdMSCs to all studied AIDs for 24 h significantly reduced the levels of IL-1RN, IDO1, and chemokines CXCL9 and CXCL10, while inducing IL4 mRNA expression ( Figure 4 ). The expression of proinflammatory cytokines CCL2, CCL3, TNF, IL6, IL1A, and IL1B was inhibited by ketoprofen and analgin treatment. NSAID treatment had opposite effects on the expression of different TGFβ family members, while acetaminophen and diclofenac treatment inhibited TGFβ1 and TGFβ3, while stimulating TGFβ2 expression ( Figure 4 Together, these data suggest that various AIDs differentially alter AdMSC transcriptome profiles, stimulating or inhibiting the expression of specific inflammatory cytokines, even under short-term treatment.

[0049] To investigate whether AID treatment of LPS-induced AdMSCs presents prolonged changes in cytokine levels, the effects of acetaminophen and prednisolone on cytokine levels in the culture medium were analyzed. Even a single treatment of LPS-induced AdMSCs under serum-deficient conditions significantly affected their cytokine profile ( Figure 5 ). In the culture medium of control untreated AdMSCs, the levels of CCL2, IL6, and IL-1RN increased over the 3-day period. Activation of AdMSCs by LPS induced the expression of CCL2, CXCL8, IL6, and IL-1RN at each analyzed time point. Our results indicate that acetaminophen treatment reduced the levels of CCL2 in LPS-treated cultures compared to control cultures and had no significant effect on CXCL8, IL6, and IL-1RN expression during this time period ( Figure 5 In the case of CCL2 and IL6 expression, the effects of prednisolone treatment were opposite to those of acetaminophen, likely due to the effects of the various mechanisms underlying these drugs. Thus, prednisolone significantly stimulated CCL2 expression at all time points, while inhibiting IL-1RN and modestly inducing IL6 levels 72 h after treatment ( Figure 5 ).

[0050] The effects of repeated AID treatment (48 and 72 h) on the mRNA expression of cytokines in AdMSCs were analyzed using RT-qPCR. Figure 6In is represented as a heat map. The analysis results show different expression profiles of cytokine genes between two time points. After exposure to various drugs, complex expression patterns of pro-inflammatory factors and anti-inflammatory factors were observed. Although the expression of most pro-inflammatory cytokines such as IL1B, CXCL9 or CCL2 was downregulated after AID treatment, the levels of some factors (TNF or MIF) recovered during this time, indicating their dynamic regulation after drug treatment. However, stable induced expression of anti-inflammatory factors (IL4, TNFAIP6, IDO1) was not observed.

[0051] Together, these data suggest that the expression of inflammatory factors can be significantly altered by various AIDs in a time-dependent manner.

[0052] Establishment of an animal model of muscle ischemia using Cytori-derived ADRC as a reference system

[0053] The goal of preclinical studies is to evaluate the conditions and cells that best promote neoangiogenesis and neoarteriogenesis by comparing differently isolated and conditionally manipulated human AdMSCs. These types of preclinical studies imply the use of the hind limb ischemia model (HLIM), in which the revascularization of ischemic muscle is restored due to the regenerative potential of the administered drug (cells), rather than the animal's own regenerative capacity. This ischemic window should last at least 2-3 weeks. Further, in this model, the Hellingman model of HLIM, which is very suitable for in vivo regeneration testing of AdMSCs, was successfully developed.

[0054] Therapeutic suspension preparations

[0055] BEAULI: Obtaining lipoaspirate using body-jet (human medicine, routine clinical practice for the treatment of soft tissue defects). Obtaining lipoaspirate using water-jet-assisted liposuction. or The harvested fat is gently separated from the remaining fluid and used immediately for fat transfer. This technique has been adopted as the clinical standard for fat cell transfer.

[0056] CYTORI: Lipoaspirate obtained by water-jet-liposuction was processed with the Cytori Cellution 800 / CRS system (Cytori Therapeutics INC.). Cytori Cellution-derived regenerative cells are the gold standard in regenerative medicine for enriching fat grafts and for improving angiogenesis in the graft area. The Cytori Cellution 800 / CRS system uses lipoaspirate, digests it with collagenase, and washes and separates the regenerative cells by centrifugation. Prior to injection, the amount of nucleated cells was analyzed with a cell counter. The average dose of viable nucleated cells was calculated using a Nucleocounter NC100 (Chemometec). The average count of viable nucleated cells was 0.9x10 6 cells / ml.

[0057] MSCs: Human AdMSCs were obtained from freshly isolated subcutaneous adipose tissue and characterized as previously reported (Liet al., 2007). For each administration, AdMSC pools from at least 3 individuals and passaged 3 were cultured in low glucose Dulbecco's modified Eagle's medium (DMEM-LG) (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (PAA, Pasching, Austria) and 1% penicillin-streptomycin (PEST) (Life Technologies). To obtain approximately 80-90% confluence, cells were collected and 2×10 cells were used per animal. 5 cells.

[0058] MSCD: AdMSCs were cultured under standard culture conditions as described above until 80-90% confluence, 12 h before treatment, the medium was changed to DMEM-LG containing 1% FBS and 1% PEST, stimulated with LPS (0.1 ug / ml; Sigma-Aldrich, Steinheim, Germany) for 2 h, washed centrally with phosphate-buffered saline (PBS), and treated with analgin (10 μM) for 24 h. Cells were collected and 2×10 cells were used per animal. 5 cells.

[0059] SALINE: A control series of rats were treated with saline (0.9% NaCl) injections.

[0060] Animal studies

[0061] For preclinical animal studies, four different treatments were tested—BEAULI, CYTORI, MSC, and MSCD. In each series, 11 animals were operated on—8 treated with different cells injected into the gastrocnemius muscle, and 3 control animals treated with saline. In addition, 6 animals were operated on for a separate control series.

[0062] All animal experiments were performed in accordance with European Directive 2010 / 63 / EU, local legislation for animal protection (LoKS) and approved by the Ethics Committee of the Estonian Ministry of Agriculture (now Ministry of Rural Affairs).

[0063] Female Sprague-Dawley (SD) rats were housed under standard animal facility conditions (2-4 animals per cage in a temperature (22±22° C.) and humidity (55±10%) controlled room with a 12h:12h light:dark cycle). Standard maintenance rodent diet and water were given freely to the animals.

[0064] In our series, adult rats (4 ± 1 months old (n = 50) and in one series 12 to 13 months old (n = 11)) were used. For details on the number and size of groups, see the section "Statistical Analysis".

[0065] Surgical method

[0066] The animals were operated on in the supine position under general anesthesia (a mixture of ketamine (100 mg / ml; Vetoquinol, France) and medetomidine (1 mg / ml; Syva, Spain) 75 mg / kg and 0.5 mg / kg, respectively, given intraperitoneally). An incision was made in the right limb. The external iliac artery, femoral artery, popliteal artery and all side branches were exposed. Tantalum microclips of different sizes were used to occlude the arteries and side branches. Tantalum microclips are good landmarks for angiographic studies. The iliac artery and femoral artery and vein were resected using electrocautery. In the gastrocnemius muscle of the animals, 0.4 ml of the cell suspension was injected in the treatment group or 0.9% saline in the control group. The skin was closed with interrupted sutures and secured with a skin stapler. Anesthesia was reversed by subcutaneous injection of atipamezole (5 mg / m; Syva, Spain) 1 mg / kg. Animals were weighed daily, monitored for signs of pain, and given analgesia during the recovery period (buprenorphine (0.3 mg / ml; Richterpharma AG, Austria) 0.01-0.03 mg / kg every 6-12 hours as needed during a minimum of the first three days, combined with ketoprofen (10 mg / ml; Merial, France) 5 mg / kg every 24 hours as needed during a minimum of five days. Euthanasia was administered when body weight loss exceeded 20% or signs of severe pain and distress or moribund conditions were observed.

[0067] Laser Doppler blood perfusion measurement

[0068] Animals were under general anesthesia (deep surgery was performed on the day of surgical intervention, and perfusion was measured only as half a dose). In the measurement area, hair was removed using an electric shaver and depilatory cream. The animals were placed on a heated, temperature-controlled surface for 5 minutes at a temperature of 37°C. During measurement, PeriCam PSI (Perimed AB) was used. The measuring distance was 15 cm. The measurement was performed on a heated and temperature-controlled surface. On both limbs, a region of interest (ROI) was selected. Measurements were performed before surgery, immediately after surgery, and 3, 7, and 14 days after surgery. Although standard conditions were different, because perfusion was different in different animals, the differences and comparative differences between the surgical limbs and the control limbs on the same animal were used for analysis.

[0069] Angiography

[0070] Angiography was performed on the 14th day after surgery. The animals were under general anesthesia, in the supine position, on a heated and temperature-controlled surface. A midline incision was made in the abdominal wall, wherein the abdominal aorta was exposed. A MicroSlide kit (Galt MedicalCorp) was used for aortic cannulation together with the contrast agent Omnipaque 300. Digital subtraction angiography was performed with a Ziehm Vision RFD, 20kW (Ziehm Imaging GmbH).

[0071] The angiographic results were analyzed by two vascular surgeons who were unaware of the study groups. In the analysis, the total number of detectable vessels was counted, as well as the number of coiled vessels to separate neoarteriogenesis from angiogenesis.

[0072] Statistical analysis

[0073] Statistical analysis was performed by Microsoft Excel and JMP10.0 SAS statistical analysis software. Linear regression was used to analyze the relationship between angiogenesis or arteriogenesis and perfusion. Survival was analyzed using the Kaplan-Meier method, and one-way analysis of variance (ANOVA) was used to compare the mean differences in perfusion and the mean values ​​of new vessels between the different treatment groups. For mean comparisons, Dunnett's post hoc test was used to compare the treatment groups with the saline control group. If there was no change in the perfusion difference before and after surgery (less than 3 perfusion units), the ischemia modeling in the animal was considered unsuccessful, and all these cases (3 saline-treated control animals) were excluded from the statistical analysis. There were two series of CYTORI-treated animals - one of them used older rats, but there were no statistically significant differences in any measurement between older and younger CYTORI-treated rats, and these two series of 8 animals were considered as one treatment group (n=16).

[0074] result

[0075] Correlation analysis between laser Doppler blood perfusion measurements and angiographic results Figure 7 Shown in.

[0076] There was a statistically significant negative correlation between the difference in perfusion measured 7 days after surgery (perfusion of the control leg - perfusion of the operated leg) and both angiogenesis and arteriogenesis (angiography performed 14 days after surgery). R = 0.51; P value for angiogenesis was 0.0031 and R = 0.46; P value for arteriogenesis was 0.0095, respectively, n = 31 for both. As expected, the correlation analysis showed that if there was better perfusion in the operated leg, the number of new vessels was also greater.

[0077] Survival analysis was Figure 8 Shown in.

[0078] There were no statistically significant differences in survival rates between the different treatment groups.

[0079] Perfusion difference analysis Fig. 9 Shown in.

[0080] like Fig. 9 As shown, there were no statistically significant differences between the groups before, after, and 3 days after surgery. Compared with the saline-treated controls, there were statistically significant improvements in the MSCD group after 7 days and in the CYTORI group after 14 days, with P values ​​of 0.0008 and 0.0322, respectively. The analysis showed that on the seventh day after surgery, animals treated with analgin-induced MSCs (study group MSCD) recovered blood perfusion faster than other groups (P value 0.0008). The difference between the MSC and SALINE groups was not statistically significant, probably because the MSC group was small (only 3 of 8 animals survived to day 7).

[0081] Analysis of angiogenesis between different cell therapy groups Fig.10 Shown in.

[0082] Significantly better neovascularization was observed for CYTORI, MSC, and MSCD treated animals compared to saline control animals (P values ​​less than 0.0001 for all groups for both angiogenesis and arteriogenesis; and P value of 0.034 for arteriogenesis analysis in the MSC group).

[0083] Two vascular surgeons who were blinded to the treatment analyzed the results of the angiography. In the analysis, the total number of all detectable vessels and the amount of coiled vessels (all vessel segments) were counted to evaluate overall angiogenesis and neoarteriogenesis, respectively. It was clear from the angiographic images that effective neoarteriogenesis and neovascularization were observed in the operated limbs of the animals treated with MSC and MSCD.

Claims

1. Use of a product comprising mesenchymal stem cells treated with analgin in the preparation of a medicament for treating human limb ischemia.

2. A method for producing a product comprising mesenchymal stem cells treated with analgin, wherein: The method comprises the following steps: - Collection of adipose tissue - Isolation of mesenchymal stem cells from adipose tissue -Replication of MSCs - Influencing MSCs with an active ingredient, wherein the active ingredient is metamizole.

3. An in vitro method for stimulating the secretion of angiogenic growth factors VEGFA, HGF, bFGF and TEK and reducing the levels of pro-inflammatory cytokines IL6, CXCL8, CCL2 and IL1-RN by activating mesenchymal stem cells (MSCs) with metamizole.

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