Identification of highly clonogenic and vasculogenic precursor cells, distinct from peripheral blood or cord blood-derived ECFCs, from human BMA

Candidate VPCs with high clonogenic capacity were identified in the Endothelial Growth Medium culture of MNCs from human BMA. These BM-derived VPCs (BM-VPCs) generated a vascular network on Matrigel but did not express conventional surface markers of EPC-like or activated ECs, such as CD31 (PECAM-1), CD133 (prominin-1), CD144 (VE-cadherin), and CD309 (KDR/Flk-1/VEGFR2), reported in PB and CB (Additional file 1: Figure S4). These BM-VPCs were also distinct from MSC-like cells in terms of vasculogenic capacity but could cooperate with each other for complex vascular network formation on Matrigel.

To develop BM-VPCs for use in autologous vascular cell therapy, they were further characterized and their expansion efficiency was optimized (Fig. 1A–C). BM-VPCs were best cultured in the fetal bovine serum (FBS)-depleted EGM2 supplemented with 2% hPL (EGMPL) (Additional file 1: Figure S4). In the culture of BM-MNCs from 32 donors, the initial spindle-shaped colonies became apparent by day 4 and could be expanded over a three-week culture period, with a PDT of ~ 20 h (passage 3) (Fig. 1A). This cell expansion capacity yielded ~ 20 billion cells, starting with 10 million BM-MNCs (Fig. 1B, C, and Additional file 1: Table S1-3). The clonogenic capacity and cellular characteristics of BM-VPCs were compared with those of PB-ECFCs and CB-ECFCs, derived from EGM-2 cultures of PB and CB, respectively. These cells exhibited cobblestone morphology, similar to that of activated ECs, such as HUVECs, and formed initial colonies on days 7–10 (Fig. 1A). Because the PDT of these cells was much longer (by ~ 5.5- and 1.7-fold (passage 1–3 average), for PB-ECFCs and CB-ECFCs, respectively) than that of BM-VPCs, they cannot meet the cell expansion efficiency required for vascular therapeutics (Fig. 1B, C, Additional file 1: Table S2).

Fig. 1

Identification of VPCs, distinct from PB and CB-derived ECFCs, from BM. A Morphology of BM-VPCs, PB-ECFCs, CB-ECFCs in passages 0 and 3. Colonies were formed from mononuclear cells derived from the BM, PB, and CB. Scale bar = 500 μm. B, C Cell yield and PDT of CB-ECFCs, PB-ECFCs, and BM-VPCs (cell yield data of BM-VPCs presented as mean ± SD, n = 39). D Images showing the results of in vitro Matrigel tube formation assay for BM-VPCs, HUVECs, CB-ECFCs, and PB-ECFCs in the EGM-2 medium containing VEGF, EGF, FGF, IGF, and other growth factors, and in the MEMα + 0.2%hPL (lacking angiogenic growth factors). After overnight incubation, the morphology of tubes was observed using a phase contrast microscope. Scale bar = 500 μm. E UEA-1 binding ability of cells at passage 5 tested using flow cytometry. F Expression of markers of EPCs and MSCs in HUVECs, CB-ECFCs, PB-ECFCs, BM-VPCs, and BM-MSCs, analyzed using flow cytometry at passage 5

To determine vasculogenic capacity, we examined in vitro vessel formation on Matrigel (Fig. 1D). PB-ECFCs, CB-ECFCs, and HUVECs, used as a positive control for activated ECs, formed a good vascular network in EGM-2 medium supplemented with a variety of angiogenic factors, such as basic-fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and R3-insulin-like growth factor (IGF). However, HUVECs failed to form a vascular network and PB-ECFCs and CB-ECFCs generated rather disrupted vascular structures in the MEMα + 0.2%hPL medium deficient in angiogenic factors, highlighting the essential requirement of angiogenic factors. However, BM-VPCs formed huge and thick meshes in the EGM-2 medium and several small vascular meshes in the MEMα + 0.2%hPL medium (Fig. 1D, Additional file 1: Figure S5), which indicated its vasculogenic capacity even under angiogenic factor-deficient condition.

Next, we examined the fluorescein-labeled UEA1-binding ability of the cells, a criterion for mature ECs, using flow cytometry. HUVECs, CB-ECFCs, and PB-ECFCs were shifted to UEA-1high whereas BM-VPCs were shifted to UEA-1low (Fig. 1E). CD45 and CD133 were not expressed in any of the tested cells. CD34, an hematopoietic stem cell (HSC) lineage marker, was not expressed in BM-VPCs and BM-MSCs but partially expressed in HUVECs, CB-ECFCs, and PB-ECFCs. vWF, an EC marker, was expressed in all cells, including BM-MSCs. EC markers, such as CD31, CD309, and CD144, were not expressed in BM-VPCs and BM-MSCs but expressed in over > 90% of HUVECs, CB-ECFCs, and PB-ECFCs, strongly suggesting that PB-ECFCs and CB-ECFCs may be similar to activated ECs. The characteristic MSC markers, such as CD29, CD44, CD73, and CD105, were expressed in all cells but CD90 was expressed only in BM-VPCs and BM-MSCs (Fig. 1F). Overall, in terms of the expression of surface markers and cell morphology, BM-VPCs did not exhibit the cellular phenotype of ECs or ECFCs but were autonomously vasculogenic in vitro.

BM-VPCs can recruit BM-MSCs to form a stable vascular network even in the presence of TNF-α

BM-VPCs exhibited a phenotype most similar to that of MSCs rather than ECs but retained a strong vasculogenic capacity even under angiogenic factor-deficient conditions. Previously, BM-MSCs were reported to differentiate into vascular smooth muscle cells [24] and pericytes in a reconstruction of blood–brain barrier on a chip [25]. We, therefore, compared the vasculogenic capacities of BM-VPCs, BM-MSCs, and their combination using the Matrigel tube formation assay in MEMα + 0.2%hPL wherein BM-VPCs and BM-MSCs were used at different ratios (Fig. 2A). BM-VPCs-only (1:0) formed a good vascular network, whereas BM-MSC-only (0:1) did not. However, a 2:1 combination of BM-VPCs and BM-MSCs recruited most BM-MSCs to the vascular network similar to vascular pericytes. In contrast, a 1:2 combination of BM-VPCs and BM-MSCs failed to form a vascular network, and a 1:1 combination partially disrupted the vascular network. Quantitatively, the number of isolated segments, an indicator of immature vessels, was lower in the combination of BM-VPCs and BM-MSCs (2:1) than in BM-VPCs-only (Fig. 2B), suggesting the possible role of BM-MSCs as vascular pericytes in endothelial tube maturation.

Fig. 2

Vasculogenic capacity of BM-VPCs and their cooperation with BM-MSCs and HUVECs in Matrigel tube formation. A Comparison of the in vitro tube-forming ability of various combinations of BM-VPCs and BM-MSCs. Scale bar = 500 μm. B Quantitative analysis of in vitro tube formation. Different parameters were measured using an angiogenesis analyzer in the Image J software. Values are mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, compared with 1:0, one-way ANOVA test followed by Turkey’s multiple comparison test, n = 4). C Images showing the results of Matrigel tube formation assay conducted using PKH green-labeled BM-VPCs and PKH red-labeled BM-MSCs. Pericyte-like localization of BM-MSCs in tubular networks formed by BM-VPCs is indicated with yellow arrowheads. Junctional points of the network tightened by BM-MSCs are indicated with white dotted lines. White scale bar = 200 μm, Yellow scale bar = 100 μm. D Images showing the results of in vitro tube formation assay conducted under conditions mimicking an inflammatory environment by adding 500 pg/mL TNF-α in MEMα + 0.2%hPL. Scale bar = 500 μm. E Quantitative analysis of panel D. Different parameters were measured using an angiogenesis analyzer in the Image J software. Values are mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA test followed by Turkey’s multiple comparison test, n = 5). F Images showing the results of Matrigel tube formation assay conducted using PKH green-labeled HUVECs and PKH red-labeled BM-VPCs and BM-MSCs. HUVECs only group included 6,000 cells/well and combination groups included 3,000 (HUVECs) + 3,000 (BM-VPCs or BM-MSCs) cells /well (1:1). Scale bar = 500 μm. G Flow cytometry analysis of CD31 expression of PKH-green labeled BM-VPCs and unlabeled BM-MSCs before and after the tube formation in MEMα + 0.2%hPL with or without 500 pg/mL TNF-α supplementation for 27 h. H Flow cytometry analysis of CD31 expression of PKH-green labeled BM-VPCs after the hybrid tube formation with unlabeled HUVECs in MEMα + 0.2% hPL media or EGM-2 media for 27 h

To visualize the interaction and localization of BM-MSCs in the vascular network of BM-VPCs, PKH green-labeled BM-VPCs and PKH red-labeled BM-MSCs were seeded on Matrigel (Fig. 2C). BM-MSCs is mostly localized to the junction of meshes formed by BM-VPCs, possibly stabilizing the junctional complex, but some were located on the abluminal surface of the BM-VPCs-vascular tubes, similar to vascular pericytes. Accordingly, BM-MSCs are apparently recruited to the network of BM-VPCs as vascular pericyte-like cells to tighten the vascular junction and stabilize the vascular tube by encircling the luminal side of the tube.

In the treatment of ischemic vascular diseases, most therapeutic cells may encounter inflammation at ischemic loci. To simulate the inflammatory environment, we supplemented TNF-α in the Matrigel tube assay (Fig. 2D). BM-VPCs alone formed a disrupted vascular network but a 2:1 combination of BM-VPCs and BM-MSCs maintained stable vascular networks even under TNF-α-supplemented conditions. Tubular structure was quantified (Fig. 2E). Because cell numbers and proliferation may affect the vascular network formation, the effect of TNF-α on cell viability and proliferation was tested (Additional file 1: Figure S6). TNF-α increased the viability of BM-VPCs in a dose-dependent manner, suggesting that TNF-α-mediated cytotoxicity of BM-VPCs is not involved in the disruption of vascular network even though TNF-α decreased the viability of BM-MSCs at high concentrations. Thus, BM-VPCs may survive well and retain their vasculogenic capacity even under inflammatory conditions only if BM-MSCs are co-transplanted, suggesting BM-MSCs’ role on vascular stabilization like pericytes under inflammatory condition.

BM-VPCs and HUVECs can cooperate to form a large vascular network

Upon transplantation, BM-VPCs may recruit endogenous ECs to cooperate in vascular repair or may be invited to arteriogenesis through bridging collaterals. To test this possibility, PKH green-labeled HUVECs were seeded at the lower cell density, which is insufficient for massive network formation, along with PKH red-labeled BM-VPCs or BM-MSCs for Matrigel tube assay (Fig. 2F). When seeded together at a 1:1 ratio, BM-VPCs and HUVECs formed segments of large vessel-like structures composed of both cells, but HUVECs and BM-MSCs could not. BM-MSCs themselves did not retain their vasculogenic capacity. Thus, BM-VPCs may collaborate with endogenous ECs present in the injury area to regenerate new vessels.

BM-VPC stimulates CD31 expression during tube formation when supplemented with TNF-α and cocultured with BM-MSC or HUVEC

In order to explore whether BM-VPCs can induce mature endothelial markers such as CD31, VEGFR2, and VE-cadherin during tube formation under inflammatory microenvironment, CD31 expression of BM-VPCs and BM-MSCs was analyzed by flow cytometry before and after the tube formation in the presence of TNF-α. PKH-green-labeled BM-VPCs in the tube was dissociated and separated by flow cytometry gating. The cell ratio of 2:1 in a combination of BM-VPCs and BM-MSCs was fairly well maintained even after the tube formation for 27 h (Fig. 2G). Only BM-VPCs increased CD31 expressing cells from 13.68% before the tube formation to 34.07% after tube formation in MEM-α + 0.2%hPL media and furthermore to 46.74% after tube formation in the presence of TNF-α, which was not accompanied in BM-MSCs. Also, the hybrid tubes with PKH-green labeled BM-VPCs and unlabeled-HUVECs at a ratio of 1:1 as shown in Fig. 2F were allowed to form in either MEM-α + 0.2%hPL (proangiogenic factor-deficient) or EGM-2 medium (proangiogenic factors sufficient) (Fig. 2H). CD31-expressing cells in BM-VPCs was increased from 13.68 to 57.74% in MEMa + 0.2%hPL media after the tube formation and furthermore to 70.34% in EGM-2 media. However, induction of CD309(VEGFR2) and CD144(VE-cadherin) was not accompanied under the same condition (Additional file 1: Figure S7). Even though the best condition for endothelial differentiation of BM-VPCs in vitro was not identified, BM-VPC’s plasticity toward endothelial cells, which is distinct from that of BM-MSC, was noted in the presence of TNF-a, BM-MSC, and HUVEC.

BM-VPCs highly express CD141, thrombomodulin, as a unique marker distinct from BM-MSCs

To distinguish between BM-VPCs and BM-MSCs, we screened 376 surface markers (Additional file 1: Figure S8). The expression of seven markers, namely CD141, CD157, CD197, CD282, CLEC4D, ROR1, and TGF-βRII, differed by more than 50% between the two cells (Fig. 3A). The expression of CD141 and CD282, which are expressed in ECs and are involved in vasculogenesis [26,27,28], was verified in passage 3 BM-VPCs and BM-MSCs derived from to 15–20 donors using flow cytometry (Fig. 3B). CD141 (thrombomodulin) was expressed in 73–98% of the BM-VPCs population but was barely expressed in BM-MSCs. CD282 was expressed in 2–100% of the BM-VPCs population, with large variations between donors, but was most not expressed in BM-MSCs. Thus, CD141 may be the most suitable marker for distinguishing BM-VPCs from BM-MSCs.

Fig. 3

CD141 is a critical marker that distinguishes BM-VPCs from BM-MSCs. A List of markers with an expression difference of > 50% between BM-VPCs and BM-MSCs screened among 376 surface markers. B Differential expression of CD141 and CD282 in 15–20 donors assessed using flow cytometry analysis (***p < 0.001, Student's t-test, two-tailed). C–F BM-MNCs were sorted using magnetic bead-conjugated CD141 antibody. The selected MNCs were plated in EGMPL. C Colonies that appeared from CD141+ sorted BM-MNCs on days 5, 7, and 9. Scale bar = 500 μm. D Marker expression profile of ex vivo-cultured cells from CD141+ sorted colonies at passages 1 to 4 analyzed using flow cytometry. E, F CD141+ sorted BM-MNCs were cultured in EGMPL at passage 0; from passage 1 to 3  and then, the cells were divided and cultured in EGMPL or StemMACS. E Morphology of replated CD141+ sorted cells in EGMPL and StemMACS at passages 1 to 3, and their tube-forming ability in MEMα + 0.2%hPL at passage 2. Scale bar = 500 μm. F Marker expression profile of CD141+ sorted cells cultured in StemMACS

CD141+BM-VPCs preexist in the bone marrow and can preferentially be expanded under specific culture conditions

To check whether CD141-expressing BM-VPCs preexist in the BM or are induced in specific ex vivo culture, CD141-positive cells were sorted from whole BM-MNCs using magnetic cell separation and cultured in the EGMPL medium. Colonies emerged from CD141-sorted cells and expanded with PDT similar to that of unsorted BM-MNC cultures, up to passage 4 (Fig. 3C, Additional file 1: Table S4). In addition, their surface marker expression pattern—positive for CD141, CD29, CD44, CD73, CD90, CD105, and c-MET, and negative for CD31, CD34, CD45, and CD309—was identical to that of unsorted BM-MNC culture in the EGMPL medium (Fig. 3D). Thus, CD141+BM-VPCs preexisted in the human BM and could be expanded under specific growth conditions provided by EGMPL used in this study. Unsorted BM-MNCs cultured in StemMACS lacked vasculogenic capacity (Fig. 2A) and CD141 expression (Fig. 3B, Additional file 1: Figure S8, S9). Similarly, the CD141-sorted cells at P0 in the EGMPL could not maintain their CD141 expression or vasculogenic capacity when the culture medium was switched to StemMACS from passage 1 (Fig. 3E, F, Additional file 1: Figure S10). Maintenance of the phenotype and expansion of preexisting CD141-expressing vasculogenic clones may require specific culture conditions, such as those provided by EGMPL.

CD141+BM-VPCs possess multipotent differentiation capacity similarly to BM-MSCs

Expression of CD141 and other markers in BM-VPCs and BM-MSCs was confirmed using western blot analysis and immunofluorescence staining (Fig. 4A, B). CD141 was expressed in BM-VPCs but not in BM-MSCs. Alpha-smooth muscle actin (α-SMA), a marker for MSCs, pericytes, and myofibroblasts [29], was expressed in BM-MSCs but mostly not in BM-VPCs. Transgelin (TAGLN), a marker for smooth muscle cells [30] was expressed in both the cell types even though its expression was lower in BM-VPCs.

Fig. 4

Multipotency of BM-VPCs and angiogenic marker expression. A Comparison of the expression of CD141, α-SMA, and TAGLN in BM-VPCs and BM-MSCs using western blot analysis. GAPDH was used as a loading control. B Immunofluorescence images showing expression of CD141 and α-SMA in BM-VPCs and BM-MSCs. Green = CD141; Red = α-SMA; Blue = DAPI. Scale bar = 100 μm. C Images of Oil red O-, Alizarin red S-, and Alcian blue-stained differentiation-induced BM-VPCs and BM-MSCs. yellow scale bar = 50 μm, white scale bar = 200 μm, black scale bar = 300 μm. D Comparison of c-MET expression in BM-VPCs and BM-MSCs using western blot analysis. α-tubulin was used as a loading control. E HGF concentration at passages 1 to 3 in cell culture supernatants measured using ELISA (***p < 0.001, one way ANOVA test followed by Turkey’s multiple comparison test, n = 4). F HGF secretion by BM-VPCs and BM-MSCs under growth factor-defective culture condition (MEMα + 0.2%hPL), assessed using ELISA. Values are mean ± SD (***p < 0.001, Student's t-test, two-tailed, n = 3). G Images showing in vitro Matrigel tube-forming ability of BM-VPCs with or without PHA-665752 200 nM, a c-MET inhibitor. Scale bar = 500 μm. H Quantitative analysis of in vitro tube formation. Different parameters were measured using an angiogenesis analyzer in the Image J software. Values are mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, student’s t-test, n = 5). Full-length blots are presented in Figure S17

According to the definition of MSCs proposed by International Society for Cell and Gene Therapy (ISCT), MSCs adhere to plastic surfaces, are CD105+, CD73+, CD90+, CD34−, CD45−, CD14−, CD11b−, CD79−, CD19−, and HLA-DR−, and exhibit multipotent differentiation potential for adipocytes, osteocytes, and chondrocytes [31]. Because BM-VPCs matched several MSC criteria (Fig. 1F, Additional file 1: Figure S8, S11), we explored their multipotent differentiation capacity (Fig. 4C). Similar to BM-MSCs, BM-VPCs differentiated into adipocytes, osteocytes, and chondrocytes under appropriate conditions. Therefore, BM-VPCs are CD141-expressing immature cells that retain multipotency, but preferentially reveal their vasculogenic capacity.

HGF/c-MET signaling may play a role as an autocrine vasculogenic factor, especially in BM-VPCs

HGF is an angiogenic factor that stimulates EC motility and growth [32]. To investigate the potential involvement of HGF and c-MET in the autocrine vasculogenic capacity of BM-VPCs (Fig. 1D), the expression levels of HGF and c-MET in BM-VPCs were assessed (Fig. 4D, E). The expression of c-MET was much higher in BM-VPCs than in BM-MSC, and that of HGF in the culture supernatant was also higher in BM-VPCs, being 30–45 ng/ml in all passages of BM-VPCs, but was negligible in BM-MSCs. Furthermore, to explore autocrine vasculogenic contribution of BM-VPCs-secreted HGF, HGF levels were examined in the BM-VPCs culture in angiogenic factor-deficient MEMα + 0.2%hPL medium (Fig. 4F). BM-VPCs secreted HGF at ~ 10 ng/ml, which was approximately nine-fold higher than that secreted by BM-MSCs. Pretreatment with PHA-665752, a c-MET inhibitor, reduced vascular network formation in BM-VPCs (Fig. 4G, H). Thus, BM-VPCs highly express HGF and c-MET compared to BM-MSCs, which may help their vasculogenic capacity using HGF as an autocrine factor. Collectively, through extensive phenotypic characterization, BM-VPCs were identified as CD141-positive vasculogenic multipotent stem cells.

Transplantation of a combination of CD141+ VPCs and BM-MSCs is more effective than transplanting them individually

To evaluate the efficacy on vascular repair, a nonclinical hindlimb ischemia model was established in nude mice using different surgical techniques (Additional file 1: Figure S12). Severe hind limb ischemia, which substantially reduced the blood flow up to 20% of that in the contralateral normal side, was chosen to assess the extent of restoration achievable with cell therapy. The severity of limb damage was standardized by scoring gross views of the mouse limbs, feet, and toes and classified into seven stages—grade 0 for limb salvage and grade 6 for limb loss (Additional file 1: Figure S3).

The efficacy of transplantation of CD141+VPCs or BM-MSCs and their combination was compared using the CLI model by intramuscularly injecting 1.2 × 105 cells/50 μL of a 2:1 mixture of human BM-derived CD141+ VPCs and BM-MSCs at passage 3 or the same number of either cell type at five different sites along the artery-excised area (Fig. 5A, Additional file 1: Figure S2). As evident from gross views of the limb at 4 weeks, only the dual cell group could consistently rescue the limb or part of it, whereas the limb was lost within 7 days in the saline-injected group (Fig. 5B–D). Furthermore, transplantation of either CD141+ VPCs or BM-MSCs could not salvage the limb, and toe or foot was lost. The effect of cell transplantation was evident on day 1 and almost plateaued on day 7. A clear difference was noted between dual cell and singular transplantation as early as on day 2, which almost plateaued at day 3, resulting in limb salvage at day 7, which was sustained for up to 4 weeks. In contrast, the BM-MSC-only group showed a higher ischemic score than the CD141+VPC-only group over 4 weeks (Fig. 5C). At 4 weeks, the dual cell and CD141+VPC-only groups rescued the limb in ~ 90% and ~ 20%, but BM-MSC-only group could not (Fig. 5D). On day 7, the blood flow in the ischemic limb in the dual cell group, measured using a laser Doppler blood flow imager, was recovered to ~ 80% of that in the contralateral normal side and continuously increased to 100% at 4 weeks (Fig. 5E, F). In addition, the group that received singular transplantation showed lower recovery compared to the group that received the dual cell transplantation. The CD141+VPCs-only group showed better recovery than the BM-MSCs-only group, for which recovery was not sustained at later stages. According to the ischemic score and blood flow recovery, the combination of CD141+VPCs and BM-MSCs was the most effective, and the CD141+VPCs-only group was more effective than the BM-MSCs-only group.

Fig. 5

Limb salvage in hind limb ischemia requires a combination of CD141+ VPCs and BM-MSCs. A To compare the efficacy of dual stem cell therapy with singular cell type therapy, CD141+VPCs, BM-MSCs and their combination were administered in a CLI model. B Changes in the limb over a 4-week period. Dotted circle: CLI-induced site. C Ischemic necrosis grade over the 4-week period; 2-Way ANOVA test with Tukey multiple comparisons. D Distribution of grades for ischemic necrosis across all groups at 4 weeks post-surgery. E Images of laser Doppler measurements over 4 weeks was acquired and F blood flow was relatively quantified; 2-Way ANOVA test with Tukey multiple comparisons G, H Immunofluorescence images for human CD31 G and human a-SMA expression H at 4 weeks I, J Qualification of human CD31 (I) and human a-SMA (J); Kruskal–Wallis test with Tukey multiple comparisons. N = 8/group. Scale bar = 100 μm. p values < 0.05 were considered statistically significant. ***p < 0.001 dual cells vs. saline; ++p < 0.01, +++p < 0.001, dual cells vs. CD141+VPC; #p < 0.05, ##p < 0.01, ###p < 0.001 dual cells vs. BM-MSCs; $p < 0.05, $$p < 0.01, $$$p < 0.001 saline vs. CD141 + VPCs; &&p < 0.01, CD141 + VPCs vs. BM-MSCs

The transplanted cells may be involved in vessel regeneration at the ischemic site for blood flow recovery and limb salvage. Cross sections of dual cell-transplanted ischemic limb were stained with human CD31-specific antibodies for ECs and human α-SMA-specific antibodies for smooth muscle cells and pericytes (Fig. 5G–J, Additional file 1: Figure S13). Dual cell-transplanted mice exhibited large CD31+/ α-SMA+ vasculature. In contrast, CD141+VPCs-transplanted mice displayed only a few CD31+ but α-SMA− vessel, whereas BM-MSCs-transplanted mice showed a few α-SMA+ but CD31− vessels (Fig. 5I, J).

Dose response of dual cells on vascular repair

To determine the optimal cell number for vascular repair and blood flow recovery, we injected three different doses of 2:1 combination of CD141+ VPCs and BM-MSCs: low (1.2 × 104 cells/50 μL), mid (1.2 × 105 cells/50 μL), and high (1.2 × 106 cells/50 μL) to each mouse (Fig. 6). Based on the ischemic score and blood flow recovery over 4 weeks, the mid dose was the most and the low dose was the least effective; the high dose was effective in limb salvage but was apparently excessive (Fig. 6A–C).

Fig. 6

Optimization of dose for dual cell transplantation in CLI model. Dual cells were transplanted into CLI mice model at three different doses (High: 1.2 × 106; Mid: 1.2 × 105; Low: 1.2 × 104). A Assessment of ischemic necrosis grade over 4 weeks; 2-Way ANOVA test with Tukey multiple comparisons. B Distribution of grades for ischemic necrosis across all groups at 4 weeks post-surgery. C Images of laser Doppler measurements over 4 weeks together with relative quantification data; 2-Way ANOVA test with Tukey multiple comparisons. D Immunofluorescence images for human/mouse CD31 expression. WGA was used to label the muscle structure. E Immunofluorescence images for TAGLN. DAPI was used for counterstaining and WGA was used to label the muscle structure. F, G The quantification of human/mouse CD31 and TAGLN vessel number (Kruskal–Wallis test with Dunn’s multiple comparisons). N = 8/group. Scale bar: 100 μm. ##p < 0.01, ###p < 0.001 High dose vs. Saline; + p < 0.05, High dose vs. Low dose; *p < 0.05, ***p < 0.001 Mid dose vs. Saline; $p < 0.05, $$p < 0.01, $$$p < 0.001 Mid dose vs. Low dose

In immunofluorescence staining, large vessels encircled by a thick TAGLN+ smooth muscle layer were frequently detected at the injury site in the mid- and high-dose injection groups (Fig. 6D–G). Thus, 1.2 × 105 cells/50 μL was considered to be the optimal dose for this CLI model.

Dual cell transplantation led to the formation of arteries and arteriole-like vessels at the injury site on day 7, which increased in diameter at 4 and 12 weeks

Based on the ischemic necrosis score (Additional file 1: Figure S3), the therapeutic effect of dual cell transplantation became obvious on day 3, and blood flow recovery was apparent on day 7 (Figs. 5F, 6C), indicating rapid manifestation of beneficial effects. The effect of dual cell transplantation on vascular repair was explored in the early phase (Fig. 7A–G). The difference in ischemic scores between the dual cell and saline groups was evident on day 2, and limb necrosis was evident on day 3 in the saline group (Fig. 7A). In the dual cell transplantation group, blood flow recovery was obvious on day 3, reaching approximately 80% on day 7; however, blood flow insufficiency was obvious on day 3 in the saline group (Fig. 7B). Macroscopic examination of the injured limb on day 7 revealed restoration of a large vessel (white arrowhead) within the muscle, where the superficial femoral artery was removed (white dotted line), in the dual cell group. In contrast, in the saline group, the muscle displayed severe inflammation (black star) and did not show neovascularization (Fig. 7C).

Fig. 7

Dual cells induced the formation of arteriole-like vasculature at injury site within 7 days of transplantation. A Scoring of ischemic necrosis. **p < 0.01 and ***p < 0.001 vs. saline-treated group (Student's t-test, two-tailed). B Quantification of blood restoration. ***p < 0.001 vs. saline-treated group (Student's t-test, two-tailed). C Injury site at day 7 (White dotted line: excised femoral artery; white arrowhead: new vessels; yellow arrowhead: femoral nerve; black star: purulent; 1–3: ligated site). D, E Immunofluorescence images for human/mouse TAGLN; DAPI was used for counterstaining and WGA was used to label muscle structure. Scale bar = 100 μm. F, G Quantification of TAGLN+ vasculature. H Comparison of the vasculature in the ischemic zone at 4 weeks post-dual cell transplantation (white arrowhead: new vessels; yellow dotted lines: excision of the femoral artery; yellow arrowhead: femoral neuron). I, J Whole-mount images of CD31+ vessels at injury site and their quantitative analysis. Scale bar = 200 μm. K–N Immunofluorescence images for human/mouse TAGLN+ vessels and their density and diameter at 4 weeks post-transplantation. Scale bar = 100 μm. O–R Immunofluorescence images for human/mouse TAGLN+ vessels and their density and diameter at 12 weeks post-transplantation. Scale bar = 100 μm. N = 8/group. p < 0.05 indicates statistical significance. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. saline-treated group (Student's t-test, two-tailed). WGA: Wheat germ agglutinin

Arteries and arterioles, characterized by their large size and sufficient coverage of the vascular smooth muscle cell (VSMC) media layer, were detected at the injury site by immunofluorescence staining of TAGLN, a representative VSMC marker, and Alexa 488-labeled wheat germ agglutinin (WGA) for muscle and vessels (Fig. 7D, E). The dual cell group showed numerous TAGLN+ vessels on day 3, which became larger on day 7, whereas the saline group displayed fewer and smaller TAGLN+ vessels. Quantitative analysis of vessel density and diameter further confirmed that dual cell transplantation increased the vessel density and the number of vessels with diameters larger than 20 μm was approximately 2.7-fold more than in the saline group on day 7 (Fig. 7F, G). In mice, vessel with diameters > 20 μm are classified as arterioles [33].

At 4 weeks, a new large vessel restored at the injury site, where numerous arteries and arterioles bifurcated from the main artery, was clearly observed in the dual cell group (Fig. 7H). Severe inflammation and fibrosis were evident in the remaining tissues in the saline group. After tissue clearing, the 3-D architecture of CD31+ vessels was observed using whole-mount analysis (Fig. 7I, J). Normal limb showed a large CD31+ femoral artery with mean diameter of 321.7 ± 21.9 μm. In the dual cell group, abundant CD31+ vessels were detected, corresponding to approximately 90% of the vessel density in normal limb and mean diameter of 88.1 ± 5.5 μm in the large vessel. However, in the saline-treated group, thin CD31+ vessels were scarcely detected, and CD31+ vessel density was approximately 20% of that in the normal limb.

The characteristics of the restored vessels at 4 weeks were analyzed using immunofluorescence staining with TAGLN and WGA, followed by quantitative analysis of vessel density and size (Fig. 7K–N). The dual cell group displayed numerous vessels with a larger diameter and a thicker TAGLN+ VSMC media layer than the saline group. Quantification of TAGLN+ vasculature showed that the dual cell group exhibited a varied range of vessel diameters, including 28% of TANGL+ vessels with a diameter > 20 μm (Fig. 7N; < 20 μm: 72%; 20–50 μm: 25%; > 50 μm: 3%; average: 17.71 μm) but the saline group showed small vessels with a diameter mostly < 20 μm.

In addition, immunofluorescence staining at 12 weeks revealed numerous large-diameter vessels uniformly covered by the TAGLN+ thick VSMC media layer in the dual cell group (Fig. 7O). The vessel density in the dual cell group was more than that in the saline group and the mean diameter was ~ 37 μm, which was larger than that at 4 weeks. Vessels with diameter > 50 μm were ~ 13%, most clearly detected in the dual cell group (Fig. 7P–R). The effects of the dual cell transplantation on blood flow recovery and limb salvage were maintained for up to 24 weeks (Additional file 1: Figure S14).

Dual cells were engrafted in the restored large vessel as human CD31+ intima and human α-SMA+ VSMC layer at 4 and 12 weeks

The fate of transplanted human CD141+VPCs and BM-MSCs in restored vessels was confirmed using immunofluorescence staining with human CD31 and human α-SMA-specific antibodies and WGA staining for vascular and muscle histology (Fig. 8A). CD141+VPCs were anticipated to function as the vessel endothelium and BM-MSCs to cover vessels as the vascular smooth muscle of arteries or arterioles. The human CD31+ inner lining and human α-SMA+ media layer of vessels, both of which are stained with WGA, were detected in the muscle at the injury site in the dual cell group at 4 weeks (Fig. 8A). Double immunofluorescence staining with human/mouse reactive α-SMA and human-specific CD31 antibodies showed a human CD31+ inner lining of the large vessel, tightly encircled by a thick α-SMA-covered smooth muscle layer (Fig. 8B). Among the vessels at the injury site, the endothelial lining engrafted by transplanted human cells was distinguished from that of mouse vessels using double immunofluorescence staining with human CD31 and human/mouse-reactive CD31 antibodies (Fig. 8C). The human cell-engrafted vasculature was identified by human CD31+ and human/mouse CD31+ vessels (white arrowheads), which were mostly larger than human CD31− and human/mouse CD31+ vessels, presumably mouse vessels (yellow arrowheads). Human CD31+ vessels were frequently detected among large-diameter vessels, possibly contributed by human CD141+VPCs in the transplanted dual cells. At 12 weeks, human α-SMA+ large vessel, which was also stained with TAGLN+ media layer, was detected in serially sectioned muscle samples (white asterisk), indicating the long-term vascular integration of transplanted human cells, possibly BM-MSCs (Fig.