DOI 10.1007/s10517-016-3365-7
141
Cell Technologies in Biology and Medicine, No. 1, May, 2016
Cell Technologies in Biology and Medicine Angiogenic Potential of Multipotent Stromal Cells from the Umbilical Cord: an In Vitro Study
I. V. Arutyunyan1,2, E. Yu. Kananykhina1,2, T. Kh. Fatkhudinov1,2,3, A. V. El’chaninov1,2,3, A. V. Makarov1,2,3, E. Sh. Raimova3, G. B. Bol’shakova2, and G. T. Sukhikh1 Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 3-12, January, 2016 Original article submitted July 1, 2015 The mechanisms of proangiogenic activity of multipotent stromal cells from human umbilical cord were analyzed in vitro. The absence of secreted forms of proangiogenic growth factor VEGF-A in the culture medium conditioned by umbilical cord-derived multipotent stromal cells was shown by ELISA. However, the possibility of paracrine stimulation of cell proliferation, mobility, and directed migration of endothelial EА.hy926 cells was demonstrated by using MTT test, Transwell system, and monolayer wound modeling. The capacity of multipotent stromal cells to acquire the phenotype of endothelium-like cells was analyzed using differentiation media of three types. It was found that VEGF-A is an essential but not sufficient inductor of differentiation of umbilical cord-derived multipotent stromal cells into CD31+ cells. Key Words: umbilical cord-derived multipotent stromal cells; endothelial cells; VEGF; migration; endothelial differentiation Therapeutic angiogenesis is a complex of methods aimed at stimulation of microcirculatory bed recovery in the ischemic tissue. It is accepted that this stimulation triggers two parallel processes: vasculogenesis and angiogenesis. Vasculogenesis normally occurring during the prenatal period and implies the formation of blood vessels from precursors of endothelial cells migrating to the focus of injury. Angiogenesis is a multistage process of vessel elongation, growth into damaged tissues, and maturation [5,12]. Thus, the most effective strategy is angiogenic therapy aimed at mobilization of endogenous progenitor cells and local 1 V. I. Kulakov Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of the Russian Federation; 2Research Institute of Human Morphology; 3N. I. Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation, Moscow, Russia. Address for correspondence: fatkhudinov@ gmail.com. T. Kh. Fatkhudinov
increase in the content of pro-angiogenic factors in the ischemic tissue. Cell therapy with multipotent stromal cells (MSC) is the most promising method of therapeutic angiogenesis due to simultaneous activation of various mechanisms (paracrine, replacement, trophic, and immunomodulatory) affecting all stages of vessel formation and maturation [9,26,36]. In most experimental studies in this field, the researchers used MSC isolated from the bone marrow (classical source of MSC) or adipose tissue; no significant differences between these cells in the realization of their proangiogenic potential were revealed [9,27]. Intensive studies with MSC from other sources, including umbilical cord-placental complex, are now in progress. Neonatal MSC are unique cells, because they represent an intermediate population (bridge) between embryonic and postnatal MSC [34]. MSC from the umbilical cord (UC) by their
0007-4888/16/16110141 © 2016 Springer Science+Business Media New York
142 biological properties differ from other types of MSC: they are characterized by the highest proliferative potential, plasticity, and immunomodulatory activity, high expression of genes involved in the development and function of the cardiovascular system, exhibit no tumorigenic properties, and are considered optimal resource for allogeneic transplantation [14,17,25,28]. In vivo studies demonstrated effectiveness of UC MSC transplantation into ischemic tissue [4,30], but the views on the mechanisms of angiogenic potential of these cells are rather fragmented. Discrepant data on the balance of pro- and anti-angiogenic factors secreted by UC MSC, in vitro interaction of MSC with endothelial cells (EC), and possible response to endothelial differentiation inductors were reported. We explored possible mechanisms of pro-angiogenic activity of human UC MSC: secretion of VEGFA, stimulation of proliferation, mobility, and targeted migration of EC, and their ability to acquire CD31+ phenotype under the action of various inductors.
MATERIALS AND METHODS Isolation and properties of cell cultures. The primary MSC culture was isolated from Wharton’s jelly of human UC (n=5). MSC were verified according to ISCT criteria [15]: adhesion to untreated plastic, specific profile of surface antigens, and in vitro osteogenic, chondrogenic, and adipogenic differentiation. Immunophenotype was analyzed using Stemflow hMSC Analysis Kits on a FACSCalibur cytofluorometer (BD) using Cell Quest software. Targeted differentiation was induced in commercially available differentiation media: StemPro Adipogenesis Differentiation Kit, StemPro Osteogenesis Differentiation Kit, and StemPro Chondrogenesis Differentiation Kit (Gibco). Passage 3-4 cells were used in the study. EA.hy926 cell line retaining of the main morphological, phenotypical, and functional properties of vascular endothelial cells [8] were used as EC. To verify EA.hy926 cell line, the expression of endothelial marker CD31 was confirmed by immunocytochemical staining with ab24590 antibodies (Abcam). DMEM/F-12 (PanEco) supplemented with 10% fetal calf serum (GE) was used as the growth medium for both cell types. Measurement of VEGF-A-121 and VEGF-A-165 in the conditioned medium. EC and UC MSC (n=3) were transferred to a 96-well plate in concentration of 104 cells in 100 μl medium per well. The medium was changed in 24 h. Conditioned medium was sampled in 1, 3, 5, and 7 days. The content of secretory VEGFA121 and VEGF-A-165 was measured by ELISA using VEGF-IFA-Best kit (cat# 8784, Vector-Best) according to manufacturer’s recommendations. Opti-
Cell Technologies in Biology and Medicine, No. 1, May, 2016
cal density was measured at λ=450 nm (standardized at λ=590 nm) on a Multiskan GO spectrophotometer (Thermo Fisher Scientific). The data were analyzed using online application (http://elisaanalysis.com/app) and presented as mean±standard deviation. Preparation of the conditioned medium. After formation of a confluent monolayer, fresh portion of the growth medium was added to the plates with UC MSC or EC cultures. In 3 days, the medium was collected, centrifuged at 2800g, the supernatant was collected and filtered through 0.22-μ filters. Evaluation of the effect of MSC-conditioned medium on proliferation of EA.hy926 cells. EC and UC MSC (n=3) were transferred to a 96-well plate (3×103 cells in 100 μl medium per well). After the cells adhered to plastic, the medium was replaced with UC MSC-conditioned medium (n=3) or fresh growth medium (control). Proliferation was assessed by using MTT test. To this end, MTT (Sigma-Aldrich) was added to the culture medium in a final concentration of 1 mg/ml. After 3-h incubation at 37oC, the medium was removed, 100 μl DMSO (Sigma-Aldrich) was added to the wells, and the plates were shaken on a shaker for 15 min at 150 rpm; optical density was measured on a Multiskan GO spectrophotometer at λ=570 nm (standardized at λ=630 nm). The results are presented as mean±standard deviation. Targeted migration of EА.hy926 cells along a gradient of factors secreted by UC MSC. Cell migration was studied using Transwell system. UC MSC (105 cells in 600 μl medium; n=3) were transferred to a 24-well plate. Control wells contained 600 μl medium without cells. In 24 h (after cell adhesion), inserts with membranes (SPL Lifesciences, cat# 35224; pore size 8 μ) were placed into the wells. To the upper chamber, 105 EC in 250 μl medium were added. In 1, 2, or 3 days, the inserts were removed, the cells on the upper surface of the membrane were removed and EC on the lower surface were fixed with 4% paraformaldehyde (Serva) and stained with DAPI (Sigma-Aldrich). Migrated cells were counted at ×100 in 8 fields of view on each membrane under a fluorescent microscope Leica DM 4000B using LAS AF 3.1.0 build 8587 software (Leica Microsystems). The results are presented as mean±standard deviation. Evaluation of the effect of MSC-conditioned medium on mobility of EA.hy926 cells on the monolayer wound model. EC were transferred to a 96well plate in a concentration of 3×104 cells in 100 μl medium per well. After the cells adhered to plastic, monolayer confluence was evaluated and the medium was changed. Monolayer wound was modeled with a WoundMaker tool (Essen BioScience) designed for making 96 standard wounds without damage to cells. The wells were washed to remove detached cells and
I. V. Arutyunyan, E. Yu. Kananykhina, et al.
143
Fig. 1. Dynamics of VEGF-A-121 and VEGF-A-165 accumulation in the medium conditioned by EA.hy926 EC and UC MSC.
Fig. 2. Effect of UC MSC-conditioned medium on proliferative activity of EC EA.hy926 EC.
100 μl UC MSC-conditioned medium (n=4) or fresh medium (control) was added. EC migration was monitored over 36 h under an IncuCyte Zoom Time-Lapse microscope (Essen BioScience). Confluence of the wounded area was evaluated using an automated image acquisition and analysis system (Essen BioScience). Endothelial differentiation of UC MSC. UC MSC were cultured until confluence and then differentiation medium was added. Three differentiation media were used. Medium 1 consisted of growth medium and EC-conditioned medium (1:1); medium 2 contained additionally VEGF-A-165 (cat# 583702, BioLegend) to a final concentration of 50 ng/ml; medium 3 contained only growth medium and 50 ng/ ml VEGF-A-165. The content of fetal calf serum in the control and differentiation media was reduced to 5%. The medium was changed 2 times a week. In 3 weeks, the cells were fixed in 4% paraformaldehyde (Serva) and stained for endothelial marker CD31 (ab24590 antibodies, Abcam) using an imaging system HRP/DAB (ABC) Detection IHC Kit (ab64259 antibodies, Abcam).
secretion of VEGF-A by UC MSC [16]. It is widely agreed that almost complete absence of VEGF-A synthesis is an essential difference of UC MSC secretome; in contrast, secretion of this growth factor by bone marrow or adipose tissue MSC is higher by 103 and 102 times, respectively [6,23]. This unusual feature, the absence of VEGF-A (the key factor of angiogenesis in pre- and postnatal per iods [13]) in the secretome of UC MSC received little attention. We believe that this feature can be related to a specific structure of the cell source: unlike most tissues of the body, Wharton’s jelly contains no blood capillaries. It was demonstrated that active process of hematopoiesis and formation of blood capillaries in Wharton’s jelly occur during the 6th week of embryo nic development, but later (embryonic weeks 7-9), hematopoiesis cease and capillaries are lysed [3]. We can assume that loss of capillaries is preceded by cessation of VEGF-A synthesis in stromal cells of Wharton’s jelly in situ during this period. This assumption is confirmed by the fact that vegfr2-knockout mice lacking principal effector receptor of VEGF-A die on day 8 of fetal development due to vasculogenesis defect [31]. At the same time, the absence of blood capillaries did not impair the main protective function of Wharton’s jelly. VEGF-A did not appear in the secretome of UC MSC after transfer of Wharton’s jelly cells to in vitro conditions despite the presence of a detectable level of vegf gene transcription [23]. Thus, the proangiogenic potential of UC MSC is realized via a VEGF-A-independent pathway. Why it might be important? The fact is that the results of clinical trials using VEGF-A-121 or VEGF-A-165 (exogenous proteins or gene constructs) are contradictory and do not always meet expectations of researchers [33,37]. Analysis of published reports showed that the very first stage of VEGF-A machinery regulation occurs at the level of protein binding to receptors VEGFR1 and VEGFR2. VEGFR2 triggers intracellu-
RESULTS Isolation and properties of cell cultures. All 5 cultures of umbilical cord MSC demonstrated clonogenic growth on untreated plastic and typical MSC immunophenotype, were capable of targeted differentiation into mesodermal lineage cells in vitro, and did not express CD31. All EC expressed endothelial marker CD31 (data not shown). VEGF-A-121 and VEGF-A-165 concentrations in the conditioned medium. In wells with EC, the concentration of soluble VEGF-A gradually increased over 7 days, while in wells with MSC, the content of this factor did not change and remained at the level of growth medium (Fig. 1). These findings did no confirm
144 lar cascades providing survival, proliferation and migration of endothelial cells, mobilization of progenitor cells, formation and maturation of new blood vessels, while VEGFR1 is the main suppressor of VEGF-A [1,37]. VEGF-A (exogenous under in vitro conditions or endogenous in experiments with in vivo ischemia modeling) shifts the VEGFR1/VEGFR2 balance towards VEGFR1, thereby significantly reducing the effectiveness of angiogenic therapy [19,20]. Moreover, it was hypothesized that the absolute content of VEGFR1 and VEGFR2 and their proportion varies in human population, which explains individual differences in the response to VEGF-A therapy [19]. Under these conditions, cell therapy with UC MSC employing VEGF-A-independent mechanism of angiogenesis stimulation can be more efficient. Effect of MSC-conditioned medium on proliferation of EC EA.hy926. MTT assay showed that UC MSC-conditioned medium stimulated proliferation of EC (Fig. 2). Similar results were reported previously for other EC (HUVEC) [12] The data on the effect of MSC on EC proliferation are contradictory due to diversity of sources of both types of cells [9]. Thus, bone marrow MSC (including cells cultured under hypoxic conditions) had no effect on the growth EA.hy926 cells [10]. The absence in VEGF-A in UC MSC-conditioned medium suggests that EA.hy926 cells responded to some other inductor. It was previously reported that other factor of this cytokine family, VEGF-B, modulate proliferation of EA.hy926 cells [38], but we found no reports on VEGF-B synthesis by different types of MSC. Targeted migration of EА.hy926 EC along a gradient of factors secreted by UC MSC. UC MSC released factors that act are chemoattractants for EC (Fig. 3). Similar results were reported previously for other cell types: HUVEC [12,32], HMEC1 (microvascular EC), and N2a (neuroblastoma cells); the effect of UC MSC was more potent than that of bone marrow MSC [18]. Thus, UC MSC secrete factors that can attract endothelial and progenitor cells and stimulate their mobility. It is known that one of those factors, IL-8 can stimulate cytoskeleton rearrangement and targeted migration of EA.hy926 cells via activation of p38 MAPK [24]. It was also demonstrated that the rate of EC migration depended on the presence of HGF and MCP-1 (monocyte chemoattractant protein-1) in the medium conditioned by UC MSC. These findings are consistent with the results of other studies demonstrating more intensive secretion of IL-8, HGF, and MCP1 by UC MSC in comparison with bone marrow or adipose tissue MSC [6,16]. Evaluation of the effect of MSC-conditioned medium on mobility of EA.hy926 EC using the
Cell Technologies in Biology and Medicine, No. 1, May, 2016
monolayer wound model. Monolayer wound is one of the most convenient and simple models for in vitro studies of regeneration processes that essentially characterizes both proliferation and targeted migration of cells [29]. Proliferation and enhanced mobility of EC are the key mechanisms of angiogenesis [5,21]. The medium conditioned by UC MSC increased mobility of EC filling the monolayer wound: in test wells, confluence of the wounded area in 20 h was 91.79±4.56% (vs. 56.46±5.95% in the control wells). The dynamics of wound healing is shown in Figure 4. In a similar experiment, bone marrow MSC also stimulated mobility of EA.hy926 cells [9]. Endothelial differentiation of UC MSC. The data on possible differentiation of MSC into EC are contradictory, because the researchers use different inductors (the basic inductor is VEGF-A-165 in a concentration of 50 ng/ml), duration of differentiation (from 2 to 28 days), estimated markers (most common CD31, vWF, VE-cadherin, and VEGFR2), and final results [35]. It is currently accepted that in vitro study cannot confirm MSC differentiation into true endothelial cells; for MSC expressing specific markers, the term endothelium-like cells was proposed [9]. We compared three variants of differentiation media that contained VEGF-A-165 alone, or EC-conditioned medium alone, or their combination as the inductor. In all cases, fetal calf serum concentration was reduced to 5% to prevent excessive cell growth during long-term induction (3 weeks). UC MSC cultured in the control medium (without inductors) did not change their shape, typical waveform arrangement typical of MSC monolayer, retained contact inhibition, and did not acquire CD31+ phenotype (Fig. 5). MSC cultured in EC-conditioned medium did not change their shape, but cell density in the monolayer was higher and solitary islets consisting of CD31+ cells appeared (Fig. 5). MSC cultured in the presence of VEGF-A-165 and EC-conditioned medium demonstrated most drastic changes. Instead of typical “waves”, a new pattern resembling coarse network appeared. We observed the appearance of tubular structures (3-5 per 35-mm Petri dish) consisting of dozens of narrow elongated CD31+ cells with elongated nucleus (Fig. 5). The use of VEGF-A-165 as the only inductor led to the loss of contact inhibition, MSC grew in layers, their morphology was similar to the above-described, but the cells did not form tubular structures and did not express CD31 (Fig. 5). Thus, UC MSC can acquire CD31+ phenotype in vitro, VEGF-A-165 being a necessary but not sufficient differentiation inducer. Our findings differ from the results of other studies that revealed no expression
I. V. Arutyunyan, E. Yu. Kananykhina, et al.
145
Fig. 3. Targeted migration of EА.hy926 EC along a gradient of factors secreted by UC MSC. a) lower surface of the membrane, nuclei of migrated cells are stained with DAPI; b) quantitative values of migration.
146
Cell Technologies in Biology and Medicine, No. 1, May, 2016
Fig. 4. Effect of UC MSC-conditioned or control medium on the rate of wound filing in the monolayer of EA.hy926 EC. a) Time-Lapse microscopy; b) quantitative evaluation of the dynamics of monolayer confluence in the wounded area.
I. V. Arutyunyan, E. Yu. Kananykhina, et al.
147
Fig. 5. Endothelial differentiation of UC MSC in vitro. Immunocytochemical staining for endothelial cell marker CD31; cell nuclei are poststained with hematoxylin. Media used: growth medium (a); growth medium+EA.hy926 EC-conditioned medium (b); growth medium+EA. hy926 EC-conditioned medium+VEGF-A-165 (c), growth medium+VEGF-A-165 (d).
148 of endothelial markers in UC MSC cultured for 14 days more in complex differentiation media containing hEGF, VEGF, hFGF-B, IGF-1, hydrocortisone, and other inducers [12]. In another study [11], UC MSC cultured for 12 days in a medium supplemented with VEGF, EGF, and hydrocortisone started uniformly expressing endothelial markers (vWF, VE-cadherin, and VEGFR2), but their morphology and organization remained unchanged. The possibility of endothelial differentiation of MSC is often questioned [9]. It should be noted that VEGF-A levels in ischemic tissue in vivo is ~1000-fold lower than in induction media supplemented with this factor (up to 50 ng/ml) [22] and is close to VEGF-A concentration used in EC-conditioned medium used in our experiments. However, we have previously demonstrated that UC MSC can acquire CD31+ phenotype without addition of VEGF-A during long-term co-culturing with EC in the basement membrane matrix [2]. Thus, the role VEGF-A in endothelial differentiation of MSC can be less important than was previously assumed. This assumption is confirmed by the fact that MSC do not express surface VEGF receptors and its effects are mediated by PDGF-receptor [7]. Thus, UC MSC via a paracrine mechanism stimulate proliferation, mobility, and targeted migration EA.hy926 line EC. UC MSC can acquire the phenotype of endothelium-like cells in vitro, VEGF-A-165 being a necessary but not sufficient differentiation inducer. The proangiogenic potential of UC MSC is realized via a mechanism that significantly differs from that for MSC from other sources, first of all, by activation of the VEGF-A-independent pathway. The part of the study including real-time monitoring of cell migration to the monolayer wound area and data processing was performed on an interactive IncuCyte Zoom (Essen BioScience) microscope provided by Quadros-Bio company. The authors are grateful to L. A. Strukova, the leading product expert of QuadrosBio Company for her help in the experiment. The study was supported by Ministry of Education and Science of the Russian Federation (RFMEFI61314X0008).
REFERENCES 1. I. V. Arutyunyan, E. Y. Kananykhina, and A. V. Makarov, Role of VEGF-A165 receptors in angiogenesis. Geny Kletki, 8, No. 1, 12-18 (2013). 2. I. V. Arutyunyan, T. H. Fatkhudinov , A. V. El’chaninov, A. V. Makarov, E. Yu. Kananykhina, N. Yu. Usman, E. Sh. Raimova, D. V. Goldshtein, and G. B. Bol’shakova, Effect of Endothelial Cells on Angiogenic Properties of Multipotent Stromal Cells from the Umbilical Cord during Angiogenesis Modeling in the Basement Membrane Matrix. Bull. Exp. Biol. Med., 160, No. 4, 575-582 (2016).
Cell Technologies in Biology and Medicine, No. 1, May, 2016
3. N. N. Dorofienko, Formation of umbilical cord at early stages of gestation. Byull. Fiz. Patol. Dukh., No. 41, 38-41 (2011). 4. A. V. Makarov, I. V. Arutyunyan, T. Kh. Fatkhudinov, A. V. Elchaninov, E. Yu. Kananykhina, P. A. Volodina, O. A. Vasyukova, A. A. Kim, E. E. Kiriyenko, and G. B. Bolshakova, Effect of multipotent stromal cells of the umbilical cord on the ischemic muscle tissue in rat limb. Klin. Eksp. Morfologiya, No. 4, 45-52(2013). 5. E. V. Parfenova and V. A. Tkachuk, Therapeutic angiogenesis: achievements, problems, prospects. Kardiovestnik, 2, No. 2, 5-14 (2007). 6. P. R. Amable, M. V. Teixeira, R. B. Carias, J. M. Granjeiro, and R. Borojevic, Protein synthesis and secretion in human mesenchymal cells derived from bone marrow, adipose tissue and Wharton’s jelly. Stem Cell Res. Ther., 5, No. 2, 53, doi: 10.1186/scrt442 (2014). 7. S. G. Ball, C. A. Shuttleworth, and C. M. Kielty, Vascular endothelial growth factor can signal through platelet-derived growth factor receptors. J. Cell Biol., 177, No. 3, 489-500 (2007). 8. D. Bouïs, G. A. Hospers, C. Meijer, G. Molema, and N. H. Mulder, Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research. Angiogenesis, 4, No. 2, 91-102 (2001). 9. A. Bronckaers, P. Hilkens, W. Martens, P. Gervois, J. Ratajczak, T. Struys, and I. Lambrichts, Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate angiogenesis. Pharmacol. Ther., 143, No. 2, 181-196 (2014). 10. A. Burlacu, G. Grigorescu, A. M. Rosca, M. B. Preda, and M. Simionescu, Factors secreted by mesenchymal stem cells and endothelial progenitor cells have complementary effects on angiogenesis in vitro. Stem Cells Dev., 22, No. 4, 643-653 (2013). 11. M. Y. Chen, P. C. Lie, Z. L. Li, and X. Wei, Endothelial differentiation of Wharton’s jelly-derived mesenchymal stem cells in comparison with bone marrow-derived mesenchymal stem cells. Exp. Hematol., 37, No. 5, 629-640 (2009). 12. M. Choi, H. S. Lee, P. Naidansaren, H. K. Kim, E. O, J. H. Cha, H. Y. Ahn, P. I. Yang, J. C. Shin, and Y. A. Joe, Proangiogenic features of Wharton’s jelly-derived mesenchymal stromal/stem cells and their ability to form functional vessels. Int. J. Biochem. Cell Biol., 45, No. 3, 560-570 (2013). 13 A. S. Chung and N. Ferrara, Developmental and pathological angiogenesis. Annu. Rev. Cell Dev. Biol., 27, 563-584 (2011). 14. J. De Kock, M. Najar, J. Bolleyn, F. Al Battah, R. M. Rodrigues, K. Buyl, G. Raicevic, O. Govaere, S. Branson, K. Meganathan, J. A. Gaspar, T. Roskams, A. Sachinidis, L. Lagneaux, T. Vanhaecke, and V. Rogiers, Mesoderm-derived stem cells: the link between the transcriptome and their differentiation potential. Stem Cells Dev., 21, No. 18, 3309-3323 (2012). 15. M. Dominici, K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, F. Marini, D. Krause, R. Deans, A. Keating, Dj Prockop, and E. Horwitz, Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8, No. 4, 315-317 (2006). 16. S. S. Edwards, G. Zavala, C. P. Prieto, M. Elliott, S. Martínez, J. T. Egaña, M. R. Bono, and V. Palma, Functional analysis reveals angiogenic potential of human mesenchymal stem cells from Wharton’s jelly in dermal regeneration. Angiogenesis, 17, No. 4, 851-866 (2014).
I. V. Arutyunyan, E. Yu. Kananykhina, et al.
17. K. Gauthaman, C. Y. Fong, C. A. Suganya, A. Subramanian, A. Biswas, M. Choolani, and A. Bongso, Extra-embryonic human Wharton’s jelly stem cells do not induce tumorigenesis, unlike human embryonic stem cells. Reprod. Biomed. Online, 24, No. 2, 235-246 (2012). 18. J. Y. Hsieh, H. W. Wang, S. J. Chang, K. H. Liao, I. H. Lee, W. S. Lin, C. H. Wu, W. Y. Lin, and S.M. Cheng, Mesenchymal stem cells from human umbilical cord express preferentially secreted factors related to neuroprotection, neurogenesis, and angiogenesis. PLoS One, 8, No. 8, doi: 10.1371/journal. pone.0072604 (2013). 19. P. I. Imoukhuede, A. O. Dokun, B. H. Annex, and A. S. Popel, Endothelial cell-by-cell profiling reveals the temporal dynamics of VEGFR1 and VEGFR2 membrane localization after murine hindlimb ischemia. Am. J. Physiol. Heart Circ. Physiol., 304, No. 8, H1085-H1093 (2013). 20. P. I. Imoukhuede and A. S. Popel, Quantification and cell-tocell variation of vascular endothelial growth factor receptors. Exp. Cell Res., 317, No. 7, 955-965 (2011). 21. J. M. Isner, P. Vale, J. Symes, D. W. Losordo, and T. Asahara, Angiogenesis and cardiovascular disease. Dialog. Cardiovasc. Med., 6, No. 3, 145-170 (2001). 22. Q. Jiang, S. Ding, J. Wu, X. Liu, and Z. Wu, Norepinephrine stimulates mobilization of endothelial progenitor cells after limb ischemia. PLoS One, 9, No. 7, doi: 10.1371/journal. pone.00101774 (2014). 23. P. Kuchroo, V. Dave, A. Vijayan, C. Viswanathan, and D. Ghosh, Paracrine factors secreted by umbilical cord-derived mesenchymal stem cells induce angiogenesis in vitro by a VEGF-independent pathway. Stem Cells Dev., 24, No. 4, 437450 (2015). 24. Y. Lai, X. H. Liu, Y. Zeng, Y. Zhang, Y. Shen, and Y. Liu, Interleukin-8 induces the endothelial cell migration through the Rac 1/RhoA-p38MAPK pathway. Eur. Rev. Med. Pharmacol. Sci., 16, No. 5, 630-638 (2012). 25. X. Li, J. Bai, X. Ji, R. Li, Y. Xuan, and Y. Wang, Comprehensive characterization of four different populations of human mesenchymal stem cells as regards their immune properties, proliferation and differentiation. Int. J. Mol. Med., 34, No. 3, 695-704 (2014). 26. A. Liew and T. O’Brien, Therapeutic potential for mesenchymal stem cell transplantation in critical limb ischemia. Stem Cell Res. Ther., 3, No. 4, 28. doi: 10.1186/scrt119 (2012). 27. R. Z. Lin, R. Moreno-Luna, B. Zhou, W. T. Pu, and J. M. Melero-Martin, Equal modulation of endothelial cell func-
149 tion by four distinct tissue-specific mesenchymal stem cells. Angiogenesis, 15, No. 3, 443-455 (2012). 28. F. Lv, M. Lu, K. M. Cheung, V. Y. Leung, and G. Zhou, Intrinsic properties of mesemchymal stem cells from human bone marrow, umbilical cord and umbilical cordblood comparing the different sources of MSC. Curr. Stem Cell Res. Ther., 7, No. 6, 389-399 (2012). 29. L. G. Rodriguez, X. Wu, and J. L. Guan, Wound-healing assay. Methods Mol. Biol., 294, 23-29 (2005). 30. D. Santos Nascimento, D. Mosqueira, L. M. Sousa, M. Teixeira, M. Filipe, T, Resende, A. F. Araújo, M. Valente, J. Almeida, J, Martins, J. M. Santos, R. N. Bárcia, P. Cruz, H. Cruz, and P. Pinto-do-Ó, Human umbilical cord tissue-derived mesenchymal stromal cells attenuate remodeling after myocardial infarction by proangiogenic, antiapoptotic, and endogenous cell-activation mechanisms. Stem Cell Res. Ther., 5, No. 1, 5. doi: 10.1186/scrt394 (2014). 31. F. Shalaby, J. Rossant, T. P. Yamaguchi, M. Gertsenstein, X. F. Wu, M. L. Breitman, and A. C. Schuh, Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature, 376, 62-66 (1995). 32. C. Shen, P. Lie, T. Miao, M. Yu, Q. Lu, T. Feng, J. Li, T. Zu, X. Liu, and H. Li, Conditioned medium from umbilical cord mesenchymal stem cells induces migration and angiogenesis. Mol. Med. Rep., 12, No. 1, 20-30 (2015). 33. M. Shimamura, H. Nakagami, Y. Taniyama, and R. Morishita, Gene therapy for peripheral arterial disease. Expert Opin. Biol. Ther., 14, No. 8, 1175-1184 (2014). 34. R. R. Taghizadeh, K. J. Cetrulo, and C. L. Cetrulo, Wharton’s Jelly stem cells: future clinical applications. Placenta, 32, Suppl. 4, S311-S315 (2011). 35. C. Vater, P. Kasten, and M. Stiehler, Culture media for the differentiation of mesenchymal stromal cells. Acta Biomater., 7, No. 2, 463-477 (2011). 36. J. Yan, G. Tie, T. Y. Xu, K. Cecchini, and L. M. Messina, Mesenchymal stem cells as a treatment for peripheral arterial disease: current status and potential impact of type II diabetes on their therapeutic efficacy. Stem Cell Rev., 9, No. 3, 360-372 (2013). 37. S. Ylä-Herttuala, T. T. Rissanen, I. Vajanto, and J. Hartikainen, Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine. J. Am. Coll. Cardiol., 49, No. 10, 1015-1026 (2007). 38. G. H. Zhang, R. Qin, S. H. Zhang, and H. Zhu, Effects of vascular endothelial growth factor B on proliferation and migration in EA.Hy926 cells. Mol. Biol. Rep., 41, No. 2, 779-785 (2014).
