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Table of Contents
REVIEW ARTICLE
Year : 2020  |  Volume : 3  |  Issue : 1  |  Page : 21-31

Cell therapy of critical limb ischemia: A review of preclinical and clinical research in China


Center of Laboratory Medicine, Union Hospital of Fujian Medical University, Fuzhou, Fujian, China

Date of Submission05-Jan-2020
Date of Decision05-Feb-2020
Date of Acceptance10-Feb-2020
Date of Web Publication30-Mar-2020

Correspondence Address:
Dr. Lianming Liao
Center of Laboratory Medicine, Union Hospital of Fujian Medical University, No. 29, Xinquan Road, Fuzhou, Fujian 350001
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/VIT.VIT_7_20

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  Abstract 


Critical limb ischemia (CLI) is the advanced stage of peripheral artery diseases and is characterized by limb pain. Current available treatments for CLI include pharmacological agents, bypass surgery, and endovascular therapy. When these conventional therapies fail, some patients may eventually suffer from amputation because of long-term blood flow insufficiency to the affected extremity. Novel therapeutic approaches are emergently needed. At present, proangiogenic gene/protein therapies and stem cell-based proangiogenic therapies have been proposed, and the promising results have been reported. Gene-modified stem cells and pretreated stem cells have been evaluated in the animal models. Proper bioscaffolds are also used to increase the survival and engraftment of delivered stem cells. Here, we address the current situation of stem cell research for CLI in China.

Keywords: Bioscaffold, critical limb ischemia, endothelial progenitor cell, gene therapy, mesenchymal stem cell


How to cite this article:
Liao L. Cell therapy of critical limb ischemia: A review of preclinical and clinical research in China. Vasc Invest Ther 2020;3:21-31

How to cite this URL:
Liao L. Cell therapy of critical limb ischemia: A review of preclinical and clinical research in China. Vasc Invest Ther [serial online] 2020 [cited 2020 Aug 4];3:21-31. Available from: http://www.vitonline.org/text.asp?2020/3/1/21/281598




  Introduction Top


Critical limb ischemia (CLI) is the advanced stage of peripheral artery diseases and is characterized by blood flow insufficiency to the affected extremity due to various reasons. Chronic blood flow insufficiency may cause by limb pain, nonhealing ulcers, and tissue necrosis with gangrene. CLI mortality rate in revascularizable candidates may exceed 50% at 5 years. However, in nonrevascularizable (known as no-option CLI [NO-CLI] patients), the 1-year mortality rate ranges from 10% to 40%.[1] Current treatments for CLI include pharmacological agents, endovascular therapy, and surgical bypass, which are very beneficial for many CLI patients. However, some patients will later suffer from sepsis/limb gangrene, uncontrollable infection, and eventually limb amputation. Therefore, novel therapies for CLI are urgently required to reduce the limb amputation rates and improve the quality of life in CLI patients.

In the past decade, proangiogenic growth factor therapy and stem cell-based therapy have been proposed for ischemic diseases. Various preclinical studies illustrated that the administration of stem cells can augment perfusion and formation of new blood vessels. These findings were quickly translated into many clinical pilot studies and the preliminary results are very promising, as cell transplantation could alleviate limb pain, increase patients' quality of life, and even reduce the amputation rate. A variety of cells, including bone marrow mononuclear cells (BMMNCs), peripheral blood mononuclear cells (PBMNCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and hematopoietic stem cells have been investigated to improve CLI symptoms. These cells were sometimes modified with exogenous factors, pretreated with molecules, and used with bioscaffolds to enhance survival and proangiogenic function after transplantation.

As China has a big population of diabetic patients, CLI is a great social, economic, and health burden in China. Many researchers in China have been engaged in the stem cell research for CLI. Most of the studies were funded by the government, indicating that Chinese government has been investing heavily in the every aspect of stem cells and considering stem cell industry as a new engine of economy.[2] Here, we review the preclinical and clinical studies by Chinese researchers in the past two decades that address different types of stem cells and their beneficial potentials in promoting blood vessel formation for CLI.


  Peripheral Blood and Bone Marrow Mononuclear Cells Top


PBMNCs and BMMNCs are the mixture of different types of cells, including EPCs, monocytes, macrophages, MSCs, and pericytes. Both PBMNCs and BMMNCs have proangiogenic features and have been used for CLI. Meng et al. reported that when BMMNCs were stimulatedin vitro with granulocyte colony-stimulating factor (G-CSF) before transplantation, their proangiogenic potential can be augmented in a rabbit diabetic lower limb ischemic model.[3]

It is noteworthy that Zhou et al. found that G-CSF-mobilized PBMNCs from diabetic patients had impaired proangiogenic capability.[4] Nevertheless, Huang et al.[5] reported that autologous transplantation of G-CSF-mobilized PBMNCs is still effective for CLI of diabetic patients. In their study, 28 diabetic patients with CLI were enrolled and randomized to either the transplant group or the control group. Patients in the transplant group received subcutaneous injections of recombinant human G-CSF (600 μg/day) for 5 days to mobilize stem/progenitor cells, and PBMNCs were harvested and transplanted by multiple intramuscular injections into the ischemic limbs. At the end of 3-month follow-up, patients in the transplant group had significantly less limb pain and higher blood perfusion of lower limbs and higher mean ankle–brachial pressure index. A total of 14 limb ulcers (77.8%) of the transplant group completely healed, whereas only 38.9% of limb ulcers (7 of 18) healed in the control group. No lower limb amputation occurred in the transplant group, in contrast to five in the control group. No adverse effects were observed. They further reported that PBMNCs achieved therapeutic neovascularization via the supply of abundant angioblasts and angiogenic factors.[6]

Sun et al. treated 87 CLI patients with PBMNCs. Three patients died and one patient was lost during the follow-up period. The 5-year amputation-free rate was 72.2%. They found that fibrinogen >4 g/L and fasting blood glucose >6 mmol/L were the risk factors for poor angiogenesis.[7] Pan et al.[8] enrolled 103 patients with NO-CLI and treated them with PBMNCs. At 6 months posttransplantation, there were 58 responders who achieved complete remission of CLI and 45 nonresponders. They found that age ≥50 years, blood fibrinogen >4 g/L, and arterial occlusion above the knee/elbow were the independent risk factors of poor angiogenesis. They further compared the efficacy of G-CSF-mobilized PBMNCs and BMMNCs for the treatment of lower limb arteriosclerosis obliterans (LASO).[9] They randomized 150 LASO patients to either the PBMNC group (n = 76) or the BMMNC group (n = 74). At 12 weeks after transplantation, significant improvement of pain was observed in both groups. PBMNC was superior to BMMNC in terms of ankle–brachial index (ABI), skin temperature, and rest pain. However, there was no significant difference between the two groups for pain-free walking distance, transcutaneous tissue oxygen tension (TcPO2), ulcer healing rate, and limb amputation rate. Their comparison research indicates that PBMNC is superior to BMMNC in the treatment of LASO.

Wan et al. treated severe CLI caused by thromboangiitis obliterans (TAO) with autologous PBMNCs.[10] A total of 64 patients with TAO (80 affected limbs) received PBMNCs. Amputation was performed in five patients (with 5 affected limbs) 4 weeks after transplantation. For the remaining 59 patients (75 affected limbs), pain and cold sensation of the affected limbs were improved with varying extent 3 months after transplantation. Claudication distance, ABI, TcPO2, and skin temperature were also improved. Most importantly, arteriography performed at 6 months after transplantation found formation of new collateral vessels in the affected limbs.

In an attempt to further improve the efficacy of autologous BMMNC transplantation for CLI, Gu et al. treated the patients with G-CSF before bone marrow aspiration in an attempt to increase the proangiogenic potential of BMMNCs.[11] They randomized 44 patients into two groups. Patients in the G-CSF group received subcutaneous injections of G-CSF (300 μg) before bone marrow aspiration (once a day for 2 days). In the control group, the patients did not receive G-CSF injection before bone marrow aspiration. Patients in both groups had 200 mL of bone marrow aspirated from the posterior superior iliac crest under local anesthesia, which was immediately proceeded for mononuclear cell isolation. At least 108 BMMNCs were infused into the ischemic leg muscles below the lowest patent artery. After 3 months, patients in the G-CSF group reported greater relief of symptoms. After 6 months, patients in the G-CSF group showed greater improvement in TcPO2, ABI, and angiographic score compared to the patients in the control group. They concluded that G-CSF-mobilized BMMNC should be considered for use in patients who are candidates for angiogenic therapy. Recently, they reported the 10-year results of autologous BMMNCs for TAO.[12] Patients were treated with either aspirin (100 mg/day) alone (n = 19) or aspirin and BMMNC injection (n = 40), according to patient preference between January 2005 and July 2006. The 10-year amputation-free rate was 85.3% (29/34) in the BMMNC group, higher than that of the aspirin group (40%, 6/15, P = 0.0019). Combination of BMMNC and aspirin was also superior to aspirin alone in terms of ulcer area, toe–brachial index, TcPO2, and pain score. In a randomized, placebo-controlled trials, Li et al. allocated 58 patients to receive placebo (0.9% NaCl) or BMMNCs (1 × 107).[13] At 6 months, BMMNCs significantly improved rest pain, ABI, skin ulcer, pain-free walking, and ulcer healing compared to placebo.


  Mesenchymal Stem Cells Top


MSCs are multipotent stem cells that can differentiate into different cell types including fat, cartilage, muscle, and bone. MSCs are described as potent biofactories, which home to the site of ischemia and improve the processes of angiogenesis and arteriogenesis by secreting a wide range of angiogenic and immunomodulatory factors. Studies in rodent ischemic limb models showed that MSCs promoted angiogenesis by producing angiogenic factors in ischemic tissues. MSCs were first isolated from bone marrow and later from many other tissues and organs. Historically, MSCs have been shown to exhibit low immunogenic potential because of their limited expression of major histocompatibility complex (MHC) I molecules, the lack of MHC II expression, and costimulatory molecules. Indeed, by labeling MSCs with aminopropyltriethoxysilane-modified magnetic iron oxide nanoparticles and monitored with a 3 Tesla magnetic resonance imaging, Li et al. showed that MSC remained in the murine ischemic lower-limb for at least 3 weeks.[14]

In addition, MSCs can be induced from pluripotent stem cells. Lian et al. induced induced pluripotent stem cells (iPSCs) into functional MSCs with a protocol that was feasible for clinical application because they circumvented the use of animal products, transfection of genetic material, or mouse feeder cells[15] They even found that iPSC-MSCs were superior to bone marrow mesenchymal stem cells (BMMSCs) in attenuating ischemia and promoting vascular and muscle regeneration in mice hindlimb ischemic model.

In the study by Wei et al., they established a large-scale protocol for the generation of MSCs from human embryonic stem cells (hESCs). Bone marrow stromal cells (BMSCs) derived from hESC had hematopoietic-supporting effectsin vitro and could notably relieve symptoms of hindlimb ischemia.[16]

Proangiogenic potential of BMMSCs was first shown in animal heart infarct model. The proangiogenic potential of MSCs can be further enhanced through long-term expression of proangiogenic factors. As neural guidance cues, netrin-1 is the most extensively studied gene in the field of angiogenesis. In the hindlimb ischemic rats, Li et al. found that when 1 μg netrin-1 and MSCs were injected into the muscle of the ischemic limb, greater angiographic score, capillary density, and function of the ischemic limb were achieved compared with MSCs alone.[17] Chen et al. reported similar results in a rat chronic limb ischemia model.[18] Ke et al. transfected BMMSCs with netrin-1 expressing recombinant adenovirus and showed BMMSCs over-expressing netrin-1 formed significantly more microvessel-like tubes in vitro on Matrigel compared with the untreated BMMSCs.[19] In the rat ischemic hind limb model, rats treated with netrin-1-overexpressing BMMSCs had greater blood perfusion scores and vessel densities, as well as better function of the ischemic limbs than rats treated with the control BMMSCs.[19] Zhang et al. reported that PEP-1-CAT transduction decreased MSC apoptosis rate while downregulating malondialdehyde content and blocking lactate dehydrogenase release, leading to greater superoxide dismutase activity. Importantly, PEP-1-CAT-overexpressing MSCs significantly enhanced ischemia-induced angiogenesis by upregulating vascular endothelial growth factor (VEGF) expression[20] and prolonging MSC survival in lower limb ischemia rat model. The proangiogenic potential of BMMSCs could also be enhanced by expressing human basic fibroblast growth factor (hbFGF).[21]

More simply, Zhang et al. found that supplementation with physiologic amounts of zinc prevented MSC apoptosis, enhanced cell viabilities, increased VEGF production, and upregulated Akt activation. Zinc-treated MSCs transplanted into ischemic hind limbs resulted in significant improvements in limb blood perfusion.[22] Zhu et al. and Zhang et al. reported that local administration of MSC with low-dose simvastatin could act in a synergistic way to induce functional neovascularization in a mouse model of hindlimb ischemia.[23],[24]

Wang et al. enhanced the proangiogenic function of MSCs by another way. They seeded BMMSCs onto collagen scaffold (CS) to construct BMSCs-CS. The ischemic hindlimbs of rabbit models were implanted with autologous BMSCs-CS or autologous BMMSCs. By measuring oxygen saturation parameters and capillary and mature vessel areas, they showed that the CS cellular delivery system could improve ischemic hindlimb perfusion and angiogenesis of BMMSCs.[25]

To improve cells retention in ischemic tissues, Wang et al. constructed a thermoresponsive and reversible hydrogel based on methylcellulose salt system.[26] Stem cells were then encapsulated in the hydrogel. The thermogel was biocompatible and cytoprotective. Transplantation of hydrogel and stem cells effectively inhibited the fibrosis and muscular atrophy of lower limb ischemia, accelerated the recovery of lower limb blood flow, and promoted angiogenesis, indicating that the reversible thermogel was a novel and potential approach for the targeted delivery of therapeutic cells in CLI patients.

Sublethal hypoxic preconditioning of MSC induced glycogen storage and stimulated glycogen catabolism and cellular ATP production, thereby preserving cell viability in long-term ischemia. In the mouse limb ischemia model, Zhu et al. showed that hypoxic preconditioning MSCs' retention time in the ischemic thigh muscles was significantly increased, associated with improved limb salvage, perfusion recovery, and angiogenesis.[27] Similar results were observed by Liu et al.[28] They showed that hypoxic preconditioning enhanced cell autophagy mediated by elevated expression of hypoxia inducible factor-1α (HIF-1α) in the BMMSCs. The adenosine monophosphate-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway was also activated during hypoxia. Hypoxia pretreatment significantly enhanced survival of BMMSCs and promoted angiogenesis in the lower limb of ischemic diabetic rats. The expression levels of VEGF-1α, matrix metalloproteinase (MMP)-9, and VEGF receptor were increased, and the expression of pAKT was upregulated in the ischemic muscle. In addition, Liu et al. reported that BMMSCs pretreated with hypoxic preconditioning promoted the proliferation and inhibited the apoptosis of endothelial cells (ECs).[29]

The beneficial effects of BMMSC injection in CLI patients have been demonstrated in many clinical studies. In a small double-blind randomized trial, Lu et al. reported that BMMSCs were more effective than BMMNCs in increasing lower limb perfusion and promoting foot ulcer healing in diabetic patients.[30] However, because BMMSCs represent about 0.001%–0.01% of the total population of bone marrow-nucleated cells and a large number of BMMSCs are required for CLI treatment, cultured-expanded BMMSCs, or MSCs from other sources are now used to circumvent these limitations.

Adipose tissues are not a mere mechanical buffer, energy reservoirs, or thermal insulators. There are adult stem cells known as adipose-derived stem cells (ADSCs) in the adipose tissues. ADSCs and BMMSCs have many features in common. They share similar surface markers, can differentiate into lineage cells forming fat, bone, and cartilage tissues, and possess immunosuppressive and immunomodulatory properties. ADSCs can also be used to induce angiogenesis and neovascularization. Mechanistic studies have demonstrated that their proangiogenic effects are attributed to the paracrine secretary features that are similar to BMMSCs. However, ADSCs have several advantages over BMMSCs: a less invasive surgical procedure for cell harvesting, a higher yield, and a higher cell division capacity. Qin et al. reported that white adipose tissue was a very rich source of MSCs that could promote limb ischemia recovery in mice.[31] Lu et al. found ADSCs, umbilical cord-derived MSCs (UCMSCs), and endometrium MSCs (EMSCs) had similar immunophenotype and differentiation potentials.[32] However, ADSCs formed more organized capillary-like network when cultured on matrigel. When implanted into nude mice with hindlimb ischemia, ADSCs were significantly superior to UCMSCs and EMSCs in promoting functional vessel formation, perfusion recovery, and limb salvage. Furthermore, ADSC-conditioned medium (CM) contained more proangiogenic factors (such as VEGF-A, platelet-derived growth factor BB, and bFGF) and less inhibitory factor (such as thrombospondin-1), when compared with UCMSC-CM and EMSC-CM.[32]

To further enhanced the therapeutic effect of ADSCs, periostin, an extracellular matrix protein that exhibits a critical role in wound repair as well as promotes cell adhesion, survival, and angiogenesis, was transfected into ADSCs (P-ADSCs).[33] In the ischemic hindlimb model, the laser Doppler perfusion index was significantly higher in the P-ADSC group compared with that in the ADSC group after 4 weeks. Microvessel densities were significantly improved in the P-ADSC group. Periostin activated integrinβ1/FAK/PI3K/Akt/endothelial nitric oxide synthase (eNOS) signal pathway of ADSCs and increased secretion of growth factors.

In the study by Piao et al., Sendai viral vector (SeV) harboring human angiopoietin-1 gene (SeVhAng-1) was transfected into the MSCsin vitro and injected into the ischemic limb of rats.[34] Both MSCs and SeVhAng-1-modified MSCs improved blood flow recovery and increased capillary density of the ischemic limbs. However, in contrast to MSCs, SeVhAng-1-modified MSCs performed better in improving blood flow recovery and survival of MSCs. More simply, Fan et al. showed that selectively targeting mTOR complex-1 using low-dose rapamycin treatment could inhibit production of proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor-α by ADSCs and promote cell viability and antiapoptotic/proangiogenic efficacy in vivo.[35]

Liang demonstrated that apelin promoted functional survival of ADMSCs in ischemic hindlimbs and provoked a synergetic effect with ADMSCs to restore hindlimb blood perfusion and limb functions. Mechanistically, apelin increased the viability of AD-MSCs via promoting protective autophagy during hypoxia, which was accompanied by the activation of AMPK and inhibition of mTOR. On the contrary, apelin suppressed autophagic cell death during reoxygenation, which was accompanied with the activation of Akt and inhibition of Beclin 1.[36]

Li et al. developed injectable three-dimensional (3D) microscale cellular niches (microniches) based on biodegradable gelatin microcryogels (GMs).[37] The microniches were constituted byin vitro priming ADSCs seeded within GMs, resulting in tissue-like ensembles with enriched extracellular matrices and enhanced cell–cell interactions. The primed 3D microniches facilitatedin vivo cell retention, survival, and ultimate therapeutic functions in mouse CLI models compared with free cell-based therapy. In particular, 3D microniche-based therapy with 105 ADSCs realized better ischemic limb salvage than treatment with 106 free-injected ADSCs, the minimum dosage with therapeutic effects for treating CLI in the literature.

Wang et al. constructed a bioactive hydrogel by mixing chitosan and hyaluronic acid and immobilized the hydrogel with C domain peptide of insulin-like growth factor 1 (IGF-1C).[38] IGF-1C-modified hydrogel could increasein vitro viability of ADSCs. Cotransplantation of hydrogel and ADSCs into ischemic hind limbs of mice promoted blood perfusion and muscle regeneration. Improved ADSC retention, angiopoietin-1 secretion, and neovascularization, as well as reduced inflammatory cell infiltration, were observed with hydrogel and ADSCs.

Interestingly, Song et al. found that ultrasound-induced microbubble destruction was able to promote targeted delivery of ADSCs to improve hindlimb ischemia of diabetic mice by upregulating the expression of proinflammatory cytokines VEGF, P-selectin, intercellular adhesion molecule-1, and stromal cell-derived factor-1 (SDF-1).[39]

The therapeutic effects of diabetic ADSCs (D-ADSCs) are impaired by diabetes partially through intracellular reactive oxygen species (ROS) accumulation. To overcome this limitation, Peng et al explored whether overexpression of methylglyoxal-metabolizing enzyme glyoxalase-1 (GLO1), which mediates ROS metabolism in D-ADSCs, could restore their proangiogenic function in a treptozotocin-induced diabetic mice model of CLI.[40] GLO1 overexpression in D-ADSCs from type 2 diabetic mice (BKS. Cg-m +/+Leprdb) (G-D-ADSCs) significantly decreased intracellular ROS accumulation. Notably, their proangiogenic capacity was restored to the same level of the nondiabetic ADSCs under high-glucose conditions. G-D-ADSC transplantation induced improved reperfusion and an increased limb salvage rate compared to D-ADSCs in the diabetic CLI mice. Higher expression of VEGF-A and SDF-1α and lower expression of HIF -1α were detected in the ischemic muscles of the G-D-ADSC group than that of the D-ADSC group. To achieve the same aim, Lian et al treated ADSCs isolated from diabetic mice with mitoTEMPO, a mitochondrial ROS scavenger.[41] mitoTEMPO pretreatment increased the proliferation, differentiation and the migration and proangiogenic capacities of D-ADSCs to levels similar to those of ADSCs from normal mice. mitoTEMPO pretreatment enhanced the mitochondrial antioxidant capacity of D-ADSCs. In addition, mitoTEMPO pretreatment improved the survival and function of D-ADSCs in diabetic mice with CLI.

Umbilical cord is another rich resource for MSCs. Yin et al. investigated whether UCMSCs can relieve hindlimb ischemia in a tree shrew model.[42] UCMSCs were isolated from the umbilical cord of tree shrews. Angiographic results showed that UCMSC injection significantly promoted angiogenesis. Moreover, the ABI, TcPO2, blood perfusion, and capillary/muscle fiber ratio were all markedly increased by UCMSCs.

The role of VEGF in stimulating angiogenesis in hindlimb ischemia in animals are well documented.[43],[44],[45] Li et al. used VEGF-overexpressing UCMSCs to treat lower limb ischemia in type 2 diabetic rats.[44] VEGF overexpression increased human umbilical cord (hUC)-MSC proliferation and VEGF secretion. VEGF-UCMSC transplantation increased VEGF expression in the skeletal muscle tissues of rats. Importantly, the vascular proliferation and blood perfusion of limbs injected with VEGF-UCMSCs were improved, accompanied with increased expression of ERK, AKT, MMP-2, and MMP-9. Their study suggests that VEGF-overexpressing UCMSCs are more effective in stimulating angiogenesis than UCMSCs and may be a new choice to improve the lower limb vascular lesions of diabetics. Wang et al. compared gene expression between BMMSCs and UCMSCs and found that the expression of angiogenesis-related factors was upregulated in UCMSCs.In vitro assays indicated that UCMSCs were superior to BMMSCs in promoting human umbilical vein EC proliferation, migration, and tube formation. In the mouse model of hindlimb ischemia, UCMSCs had stronger proangiogenic ability than BMMSCs.[46]

By overexpressing miR-21 in human UCMSC, Zhou et al. identified miR-21 induced UCMSCs proliferation, migration, and angiogenesis in vitro.[47] Transplantation of UCMSCs overexpressing miR-21 increased neovascularization of the muscles of CLI nude mice. Furthermore, carboxyl terminus of Hsc70-interacting protein (CHIP) was found to be the target gene for miR-21-mediated activation of HIF-1α in the UCMSCs. Li et al. showed overexpression of erythropoietin in UCMSCs also augmented the proangiogenic effects of UCMSCs.[48]

Similar to umbilical cord, placenta is also rich in MSCs. Xie et al. isolated human placenta mesenchymal stem cells (PMSCs) and evaluated their proangiogenic effects in mouse hindlimb ischemia model.[49] Intramuscular injection of PMSCs significantly enhanced microvessel density and blood perfusion in ischemic hindlimbs as compared. Du et al. isolated VCAM-1+ MSCs from the placenta chorionic villi using flow cytometry.[50] These cells expressed many angiogenic genes, including hepatocyte growth factor (HGF), ANG, IL-8, IL-6, VEGF-A, transforming growth factor-β, bFGF, angiogenin, angiopoietin-2, CSF2, CSF3, MCP-3, CTACK, and OPG. They showed remarkable vasculo-angiogenic abilitiesin vitro and enhanced hindlimb blood perfusion of ischemic BALB/c nude mice.

Small molecular hydrogels have shown attractive abilities to enhance the therapeutic effects of human MSCs via promoting their proliferation or maintaining their differentiation potential. Huang et al. designed and synthesized a new bioactive and biocompatible hydrogel, Nap-GFFYK-Thiol, to enhance the retention and engraftment of human PMSCs in a mouse ischemic hind limb model.[51]In vitro results demonstrated that the Nap-GFFYK-Thiol hydrogel increased cell viability. Moreover, it enhanced the proangiogenic and antiapoptotic effects of PMSCs. In vivo, Nap-GFFYK-Thiol hydrogel improved the PMSC retention in the murine ischemic hind limbs as visualized by bioluminescence imaging. Furthermore, cotransplantation of PMSCs with hydrogel improved blood perfusion and limb salvage. The Nap-GFFYK-Thiol hydrogel fabricated using disulfide bonds as cleavable linkers serves as a niche for PMSCs.

In a pilot study, Wang et al. treated four diabetic patients with CLI with PMSCs. Cells were intramuscularly injected for 3 times at an interval of 4 weeks. There were no adverse events during the 24-week follow-up period. The clinical ischemic features of patients, including resting pain, limb coldness, and pain-free walking distance, were improved 24 weeks after PMSC therapy. The findings of magnetic resonance angiography showed increased collateral vessel formation in one patient.[52]


  Endothelial Progenitor Cells Top


First identified by Asahara et al., EPCs are characterized by expression of CD133, CD34, c-kit, VEGFR2, VE-cadherin, and SCA-1. These cells improve blood supply to the ischemic tissues by secreting angiogenic factors (VEGF-A, VEGF-B, SDF-1, and IGF-1) and forming new blood vessels. Liu et al. used EPC to enhance the neovascularization effect of transmuscle laser revascularization in ischemic hindlimbs of nude rats.[53] EPCs also activate the resident ECs by secreting HFG, VEGF, and exosomes and releasing microvesicles from the cell membrane to transmit mRNA to ECs.

The number of circulating EPCs in the CLI patients was reported be lower compared to the healthy controls.[54] Systemic administration of EPCs into the animal hindlimb ischemia models led to the incorporation of EPCs into the microvascular endothelium, promotion of blood flow, and increased capillary density in the ischemic hindlimb.[55] Consistently, the efficacy of intramuscular injection of EPCs for CLI patients was demonstrated in several clinical trials. Interestingly, Duan et al. proposed that hUC blood has two types of EPCs with different angiogenic potential.[56] They named them circulating angiogenic cells (CACs) and high proliferative potential EPCs (HPP-EPCs). Both types of cells possessed similar characteristics of EPCs. Unlike CACs, which expressed CD14 but not CD133, HPP-EPCs expressed CD133 but not CD14. HPP-EPCs display stronger proliferation and clonogenic potentialin vitro and show stronger ability to promote vascular growth in the hindlimb model of ischemia in mice (BALB/C-nu) in vivo.

To further improve the function of EPCs, Zhang et al. administered GDF11 with EPCs to promote vascularization and blood flow in diabetic rats with hindlimb ischemia.[57] Hu et al. reported that simvastatin could significantly reduce apoptosis of ischemic skeletal muscle cells in part through downregulation of Bax, upregulation of Bcl-2, and increasing the ratio of phospho-Akt to Akt in an athymic nude mouse model of hindlimb ischemia,[58] in addition to enhance the proangiogenic potential of EPCs.

Several research teams applied exogenous expression of proangiogenic genes to further enhance EPCs' therapeutic potentials. HIF-1 alpha is a key determinant of oxygen-dependent gene regulation in angiogenesis. Jiang et al. reported HIF-1 alpha overexpression could promote EPCs to secret VEGF and enhance EPC differentiation, proliferation, and migration. The expressions of EC markers CD31, Flk-1, and eNOS as well as VEGF and nitric oxide (NO) secretions were also increased. In the hind limb ischemia model, HIF-1 alpha-transfected EPCs exhibited a higher revascularization potential.[59],[60] When EPC mobilization was induced with SDF-1 alpha, Yu et al. demonstrated VEGF-overexpressing EPC-mediated vasculogenesis was further promoted in vivo.[61] A synergic proangiogenic effect of VEGF-A and HO-1 for rat hindlimb ischemia was also reported by Long et al.[62]

To promote homing of EPCs to ischemic tissues, Sun et al. expressed α1,3-fucosyltransferase VI (FucT VI) in the EPCs to enhance sLe (x) synthesis, which is crucial for E- and P-selectin-binding, and EPC adhesion to ECs. In a mouse model of hindlimb ischemia, in which EPCs were injected intravenously, FucT VI expression increased EPC homing, neovascularization, and blood flow in ischemic muscles.[63] On the other hand, Zhou et al. established an optimal hypoxia preconditioning, i.e., 1% O2 for 2 h, to promote EPC survival after transplantation.[64]

Li et al. constructed a polymeric drug depot-anchoring hydrogel scaffold with self-assembling peptide (RADA16) to achieved long-term delivery of VEGF.[65] VEGF was encapsulated in the hydrogel. EPCs cultured on the scaffold had greater cell proliferation and differentiation potential in vitro. Furthermore, this scaffold laden with EPCs promoted neovascularization in an animal model of hind limb ischemia.

Generation of large quantities of ECs is highly desirable for the treatment of ischemia diseases. To achieve this goal, Zhang et al. developed a simple, chemically defined culture system that could differentiate human pluripotent stem cells into ECs via a MESP1 mesoderm progenitor stage.[66] MESP1+ cells highly expressed sphingosine-1-phosphate (S1P) receptor, and the addition of S1P significantly increased the endothelial differentiation efficiency. Upon seeding in a novel 3D microniche and priming with VEGF and bFGF, MESP1+ cells underwent endothelial differentiation and proliferation. Transplanting a small number of culture-expanded endothelial-primed MESP1+ cells in the 3D microniches wer sufficient to mediate rapid repair of a mouse model of CLI.[66] In a rabbit ischemic model, Fan et al. showed similar effects of culture-expanded EPC in promoting neovascularization in ischemic hindlimbs.[67]

More directly, Lai et al. produced functional EC from human BMMNC (BM-EC), hESCs (hESC-EC, and human-induced pluripotent stem cells (hiPSC-EC). All cells expressed major angiogenic factors including epidermal growth factor, HGF, VEGF, placental growth factor, and SDF-1. Transplanting BM-EC, hESC-EC, or hiPSC-EC significantly attenuated severe hindlimb ischemia in mice via the enhancement of neovascularization.[68]


  Cd34+ and Cd133+ Cells Top


Human CD34+ cells and CD133+ cells are well known as hematopoietic stem cells used for stem cell transplants in patients who have bone marrow ablation by chemotherapy or radiation therapy. Hematopoietic stem cells can differentiate into EPCs in the setting of ischemia. CD34 is also a surface marker of EPCs and mature ECs. Hence, it is desirable to use CD34+ cells and CD133+ cells for ischemic conditions.[69],[70] Li et al. reported that G-CSF-mobilized PBMNCs deprived of CD34+ cells had weak therapeutic efficiency in response to ischemia-induced neovascularization.[70]

Angiitis-induced CLI (AICLI) patients constitute a remarkable proportion of NO-CLI patients. Dong et al. investigated the feasibility, safety, and efficacy of the intramuscular injection of CD34+ cells isolated from G-CSF-mobilized PBMNCs for the management of AICLI in 25 patients (25 lower extremities and three upper extremities). The patients were given low-dose (105/kg), medium-dose (5 × 105/kg), and high-dose (106/kg) CD34+ cells. During the follow-up time of 6–33 months, the overall outcomes showed that the Wong-Baker FACES pain rating scale score, the peak pain-free walking time, the ABI, and TcPO2 all improved. The Kaplan–Meier estimate of the rate of freedom from major amputation at 6 months was 84%. The comparison among the three groups revealed no significant difference. They further reported the 5-year follow-up.[71] Peak pain-free walking time and Wong-Baker FACES remained improved. The ulcer healing rate was 85.71%, and the recurrence rate was 11.11%. The quality of life of patients was significantly improved.

Lian et al. reported culture-expanded autologous CD34+ cells transplantation for NO-CLI patients. Human CD34+ cells were isolated from the peripheral blood and cultured for up to four passages.[72] Seven days after induction of limb ischemia, ischemic muscles of nude mice were injected with CD34+ cells. Two weeks later, CD34+ treatment significantly alleviated injury and increased vascular density. Furthermore, when patients with severe lower extremity arterial ischemia were injected with autologous CD34+ cells in the affected calf, foot, or toe, significant improvements were observed in peak pain-free walking time, ABI, and TcPO2. These findings demonstrate that culture-expanded CD34+ cells may provide a rich source of stem cells for CLI patients.

CD133+ is also a surface marker for EPCs and hematopoietic stem cells, and CD133+ cells are used for therapeutic vasculogenesis.[73],[74] Yang et al. isolated CD133+ cells from the cord blood of 52 neonates and cultured them in fibronectin-coated flask in M199 medium supplemented with 50 ng/ml VEGF, 20 ng/ml IL-3, and 50 ng/ml SCF.[73] One day after the unilateral ischemic limb surgery was performed in nude mice, 5,000,000 cells were transplanted into the nude mice via tail vein (EPC group). They found that transplanted EPCs were incorporated into the capillary networks in the ischemic limbs of nude mice. The ratio between the blood perfusion of the ischemic limb and nonischemic limbs was 19.1% before EPC treatment. Two weeks after transplantation, the ratio increased to 77.3%, with increased density of capillaries in the muscles of ischemic hind limbs.

Desferrioxamine (DFO), an iron chelator, mimics hypoxia by inhibiting HIF-1α degradation and upregulated angiogenic factors. Du et al. used DFO to promote migration and increase the tube formation and expression of angiogenic factors in CD34+ cells in vitro. In the ischemic model, DFO increased blood flow, the function of the ischemic hindlimbs, and VEGF levels.[75] The number of CD34+ cells migrating to the ischemic sites was also increased.


  Stem Cell-Derived Microvesicles Top


Microvesicles are generated by the outward budding and fission of membrane vesicles from the cell surface. Microvesicles are spontaneously released by many cell types, including stem cells. Microvesicles contain biologically active proteins and lipids, as well as mRNA or microRNA, indicating their potential role in the exchange of genetic material between cells. Stem cells are reported to contribute to the process of angiogenesis partially by releasing exosomes and microvesicles to transmit RNA and proteins to ECs, resulting in EC proliferation.

Ju et al. harvested exosomes secreted by cardiac MSCs (C-MSC-Exo), then injected C-MSC-Exo or phosphate buffered saline (PBS) intramuscularly into ischemic hindlimbs.[76] Laser Doppler imaging showed that ischemic limbs treated with C-MSC-Exo exhibited improved blood perfusion compared to ischemic limbs treated with PBS at 2 weeks and 1 month after induction of limb ischemia. They further identified that mmu-miR-7116-5p was the most abundant miRNA in C-MSC-Exo and may be responsible for C-MSC-Exo's proangiogenic effect.

Zhu et al. harvested extracellular vesicles (EVs) with a diameter of 30–150 nm secreted by human urine-derived stem cells (USCs) and evaluated the proangiogenic effects of USCs-EVs.[77] The USCs-EVs were positive for exosomal markers, such as CD9, CD63, and Tsg101. After intramuscularly injection of USCs-EVs into an ischemic mouse hindlimb, ischemic limb perfusion and function were markedly increased after USCs-EVs administration. Moreover, angiogenesis and muscle regeneration levels were significantly increased. They results revealed for the first time that USCs-EVs represent a novel therapy hindlimb ischemia.

Exosomes could also be secreted by human iPS-MSCs.[78] Hu et al. demonstrated that exosomes derived from iPS-MSCs (iMSCs-Exo) had a diameter of 57 ± 11 nm and expressed CD63, CD81, and CD9. They possessed the ability to attenuate limb ischemia and promote angiogenesis after injection into ischemic limbs of mice. Interestingly, Sun et al. reported that iPS-MSCs were insensitive to proinflammatory interferon-γ-induced HLA-II expression and had a stronger immune privilege after transplantation.[79]

Du et al. explored compounds that could enhance the proangiogenic potential of exosomes from PMSCs.[80] They found that exosomes released from human PMSCs after NO stimulation have greater angiogenic potentialin vitro and in the murine model of hind limb ischemia. Further analysis demonstrated that NO stimulation increased VEGF and miR-126 levels in exosomes.


  Conclusion Top


Stem cell research in China is attracting more and more attention from around the world. In fact, the beginnings of stem cell research in China may be traced back to 1963, 34 years before Dolly the sheep was cloned, when the late embryologist Tong et al. transferred DNA from a cell of a male Asian carp to an egg of a female Asian carp and produced the world's first cloned fish.[81] In the past two decades, stem cell research keeps to be a hot field in China, as evidenced by the expansion of publications by Chinese researchers and wide applications of stem cells in hospitals in China.

Stem cell therapies for CLI have shown very promising results both in the animal models and many pilot clinical studies. There are several important issues that challenge the application of stem cells for CLI treatment. Preclinical designs are heterogeneous in terms of cell dose and administration route, and there are few randomized clinical trials. Preclinical studies should be focused on stem cells that are more possible to be used for patients in the near future. For example, animal studies should address the optimal routes of stem cell administration (intravenous injection, intra-arterial injection, multiple injections to different ischemic sites, or other innovative deliver system). The safety of gene-modified stem cells needed to be evaluated in more preclinical studies. Approaches to promote survival of stem cells by providing a better microenvironment for the injected cells within the ischemic area after transplantation are also crucial. Chinese researchers have paid much attention on these issues.

In summary, stem cell-based therapies seem to be efficient for CLI. However, more clinical studies, including comparison research, are required to optimize these novel therapeutic strategies.

Financial support and sponsorship

The project was supported by the Natural Science Foundation of Fujian Province of China (No. 2018J01302) and the Joint Funds for the Innovation of Science and Technology, Fujian Province (No. 2017Y9001).

Conflicts of interest

There are no conflicts of interest.



 
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