MAPCs have lymphvasculogenic and lymphangiogenic potential

When exposed to VEGF-A, mouse (m)MAPCs can be specified to arterial and human (h)MAPCs differentiate into arterial and venous endothelial cells36,37. Here, we investigated whether mouse and human MAPCs can differentiate down the lymphatic endothelial lineage under similar conditions. First, we confirmed that MAPCs gain general endothelial cell marker expression upon VEGF-A exposure (Supplementary Fig. S1a,b). In support of their lymphvasculogenic potential, Prospero homeobox 1 (Prox1), the lymphatic master switch, was significantly induced in MAPCs. This likely triggered expression of additional lymphatic genes (i.e., Pdpn and Itg9a), known to be upregulated by forced Prox1 expression (Supplementary Fig. S1c,d)40. A fraction (21 ± 6%) of VEGF-A-exposed MAPCs also expressed Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE1; shown for mMAPCs; Supplementary Fig. S1e). Notably, lymphatic marker gene induction in hMAPCs was not improved by lymphangiogenic growth factor VEGF-C (shown for LYVE1 in Supplementary Fig. S1f; PROX1 fold-induction versus day 0 was also comparable upon exposure to VEGF-A, VEGF-C or a combination: 26 ± 10, 26 ± 14 and 26 ± 11, respectively; n = 4 independent differentiations).

In ischaemic limbs, MAPCs had a limited direct contribution to blood vascular endothelium. Hence, their effect was mainly due to a side-supply of angiogenic growth factors37,38,41. We reasoned that MAPCs could have an equally important trophic effect on lymphangiogenesis. Accordingly, 72 hour mMAPC- or hMAPC-conditioned media significantly stimulated lymphatic endothelial cell sprouting, proliferation and migration (Fig. 1a–m). To explore the factors potentially responsible for this lymphangiogenic effect, we first performed quantitative (q)RT-PCR for known lymphangiogenic growth factors and found that while mMAPCs and hMAPCs expressed VEGF-A and angiopoietin-2 (ANG-2) to a similar extent, VEGF-C expression was only prominent in hMAPCs (Supplementary Fig. S2a). Furthermore, a more unbiased screen using antibody arrays on the (non-)conditioned media revealed that while mMAPCs and hMAPCs had a 62% overlap in their cytokine/growth factor secretion profile, hMAPCs not only secreted larger amounts, but also a broader complement of these factors, including VEGF-C (Supplementary Fig. S2b–d and Table S1).

Figure 1 MAPCs have lymphangiogenic potential. (a–c) Images of human lymphatic endothelial cell (LEC) spheroids exposed to LEC media (a; ‘L’) or conditioned media from mMAPCs (‘mCM’; b), and corresponding quantification (c; n = 4; *P = 0.029 versus ‘L’ by Mann-Whitney-U test). (d–m) Images of LECs stained with proliferation marker Ki67 (in green in bottom half; top half shows corresponding field of view (FOV) stained with Hoechst in blue in the presence of non-conditioned mMAPC media (NCM; f), mMAPC-CM (g), hMAPC-NCM (h) or hMAPC-CM (i) or LECs migrated across the membrane of a transwell (revealed by Wright-Giemsa staining; j–m) and the corresponding quantifications (d: proliferation, expressed as % of Ki67+ cells, n = 4; e: migration, expressed as number of cells per FOV, n = 4; *P = 0.029 versus corresponding NCM condition by Mann-Whitney-U test). Full size image

MAPCs support lymphatic capillary growth in healing wounds

Wound healing, a physiological repair process, requires blood and lymphatic vessel growth2,4. Since MAPCs showed the capacity in vitro to give rise and offer trophic support to blood vascular37,38 and lymphatic endothelial cells (this study), we tested their potential to ameliorate wound healing. Transplantation of mMAPCs from mice ubiquitously expressing enhanced (e)GFP significantly accelerated wound closure and resulted in smaller scars (Fig. 2a–c + Supplementary Fig. S3a–c) compared to phosphate-buffered saline (PBS)-injection. While all mMAPC-injected wounds were completely re-epithelialised, 60% of PBS-treated wounds were only partially covered with neo-epidermis at 10 days. In vivo fluorescence imaging revealed that 4 days after injection, eGFP+ mMAPCs were in close vicinity to blood vessels growing towards the wound bed (Supplementary Fig. S3d,e). In accordance, mMAPC transplantation also boosted growth of CD31+ vessels in the wound centre by 2-fold, in limited part (2.8 ± 0.4% of engrafted cells) by direct contribution to CD31+ cells (Fig. 2d–f + Supplementary Fig. S3f). mMAPCs only occasionally contributed to differentiated lymphatic endothelial cells but significantly increased LYVE1+ or podoplanin+ lymphatic capillary growth by 3-fold (Fig. 2g–i + Supplementary Fig. S3g–j). The vast majority of LYVE1+ cells were lymphatic endothelial cells and not macrophage intermediates – previously suggested to contribute to lymphatic vessels in transplanted kidneys26 – since they did not express panleukocytic marker CD45 (Supplementary Fig. S3k).

Figure 2 MAPCs stimulate blood vessel and lymphatic capillary growth in wounds. (a) Wound width in mice treated with PBS or mMAPCs (n = 5; *P < 0.05 versus PBS by repeated measures ANOVA with Fisher post-hoc test). (b,c) Representative pictures of cross-sections of 10 day (d)-old wounds from mice treated with PBS (b) or mMAPCs (c) stained with haematoxylin and eosin (H&E). Note the significantly smaller wound gap (the edges of which are indicated by arrowheads) in mMAPC-treated mice. (d–f) CD31-stained cross-sections of 10d-old wounds treated with PBS (d) or mMAPCs (e), and corresponding quantification (f; n = 5; *P = 0.008 versus PBS by unpaired two-tailed Student’s t-test). (g–i) LYVE1-stained (in red) cross-sections of 10d-old wounds treated with PBS (h) or mMAPCs (i), and corresponding quantification (g; n = 4–5; *P = 0.032 versus PBS by unpaired two-tailed Student’s t-test). (j,k) Cross-sections of wounds treated with PBS (j) or hMAPCs (k) 5d earlier, stained for pancytokeratin (PCK; arrowheads indicate wound borders, horizontal lines indicate distance covered by the epidermis). (l–n) CD31-stained cross-sections of wounds treated with PBS (l) or hMAPCs (m) 10d earlier, and corresponding quantification (n = 6–8; *P < 0.0001 versus PBS by unpaired two-tailed Student’s t-test). (o–q) LYVE1-stained (in red) cross-sections of 10d-old wounds after treatment with PBS (p) or hMAPCs (q), and corresponding quantification (o; n = 6–8; *P = 0.0007 versus PBS by unpaired two-tailed Student’s t-test). Haematoxylin or DAPI were used to reveal nuclei in b-e, j-m and h, i, p, q, respectively. Full size image

Next, hMAPCs applied onto circular wounds accelerated wound closure (Supplementary Fig. S4a–c). Live imaging and cross-sections through the wound area showed their homogenous engraftment in the wound bed, with only occasional in situ differentiation to (lymphatic) endothelial cells, as shown by the intercalation of human-specific CD31+ cells in CD31+ host vessels and by the co-localisation of the hMAPC-derived vimentin signal and LYVE1, the latter both staining lymphatic endothelial cells from human donor and mouse recipient origin (Supplementary Fig. S4d–g). hMAPCs accelerated epithelial coverage (% coverage at 5 days: 46 ± 5 in hMAPC-treated versus 7 ± 2 in vehicle-treated wounds; n = 6, P < 0.0001 by unpaired two-tailed Student’s t-test; Fig. 2j,k), likely by increasing keratinocyte numbers in the advancing epithelial tongues (number of keratinocytes/mm at 5 days: 1,160 ± 87 in hMAPC-treated versus 440 ± 30 in PBS-treated wounds; n = 6, P < 0.0001 by unpaired two-tailed Student’s t-test) and increased granulation tissue formation by two-fold (Supplementary Fig. S4h–j). All wounds were completely re-epithelialised in hMAPC-treated mice versus only 46% of PBS-treated mice and hMAPC-treated wounds showed improved collagen remodelling at 10 days (determined by the % organised red-birefringent collagen; Supplementary Fig. S4k–m). hMAPC transplantation improved wound vascularisation by about 2-fold at 10 days (determined by the % CD31+ area in the entire wound; Fig. 2l–n). hMAPCs significantly boosted lymphangiogenesis as evidenced by the 3-fold increased LYVE1+ fractional area and the 2-fold increase in podoplanin+ vessel density at 10 days (Fig. 2o–q + Supplementary Fig. S4n–p). Double immunofluorescence staining for Prox1 and smooth muscle α-actin (αSMA) revealed that the vast majority (97 ± 2%) of lymphatic vessels in granulation tissue at 10 days were capillaries devoid of αSMA coverage.

MAPCs support lymphatic capillary and pre-collector restoration in elevated skin flaps

To test and compare the potential of mMAPCs and hMAPCs to functionally restore lymph flow through repair of a discontinued draining lymphatic system of the skin, we disrupted lymph drainage to the axillary lymph nodes by making a full-thickness skin incision in the abdomen (Fig. 3a)42. This intervention abrogated lymph drainage in the majority (7 out of 10) of PBS-treated animals shown by the lack of fluorescent dye crossing the wound border 2 weeks following skin incision (Fig. 3b; Table 1). MAPC transplantation almost completely (in 5 out of 6 and 6 out of 6 cases for mMAPC- or hMAPC-treated mice, respectively) restored drainage across this border (Fig. 3c,d; Table 1). While drainage to axillary lymph nodes was only obtained in 1 out of 10 PBS-injected mice, 3 out of 6 mMAPC-injected and 6 out of 6 hMAPC-injected mice showed lymph node drainage after 2 weeks. In a second set of mice injected with PBS or mMAPCs, fluorescent dye crossed the wound border in 5 out of 5 mMAPC-treated mice and lymph node drainage was restored in 4 out of 5, while there was no restoration of drainage across the wound border and into the axillary lymph nodes in any of the PBS-injected mice 4 weeks after skin incision (Table 1). Histological analysis of the skin wound area around the transplantation sites revealed that, in addition to a 1.8-fold expansion of CD31+ blood vessels (Supplementary Fig. S5a–d), MAPC-injected mice had a ~two-three-fold increase in fms-like tyrosine kinase (Flt)4+ (VEGFR3+) and LYVE1+ fractional area in the wound borders (Fig. 4a–d + Supplementary Fig. S5e–h, respectively) 2 weeks after skin incision. The average number of functional (dextran-filled) lymphatic vessels per cross-section around the incision at 2 weeks was significantly increased by MAPC injection (Fig. 4e–h). Notably, some mMAPCs persisted until 2–4 weeks and lodged in the vicinity of draining lymphatic vessels (Fig. 4i–k). Compared to the wound healing models, deep sparsely αSMA-coated Prox1+ pre-collector vessels were more frequently observed here (a representative example is shown in Fig. 4l), yet the majority (67 ± 5%) of skin lymphatics was still devoid of αSMA coating. Nevertheless, in addition to expanding the LYVE1+ capillary network, hMAPC transplantation increased the number of draining pre-collectors by 3-fold after 2 weeks (Table 1).

Figure 3 MAPCs restore lymph drainage across a severed lymphatic network. (a) Image displaying the skin flap model. R1/R2 indicate areas from which images in panel b-d are shown. Arrows/‘X’ indicate injection spots of fluorescently-labelled dextran for lymphangiography or MAPCs/PBS, respectively, and arrowheads show the area through which blood supply to the skin flap is preserved. (b–d) Merged pictures of bright field/fluorescence images 15 minutes after injection of dextran (FITC (green)-labelled in b,d or Rhodamin-B-(red)-labelled in c) of regions R1 (left panels; and enlarged image of the corresponding inset (i) in the middle panels) and R2 (right panels) of mice injected 2 weeks (w) earlier with PBS (b), mMAPCs (c) or hMAPCs (d). Arrowheads indicate filled afferent lymphatic vessels. LN: lymph node. Dashed lines delineate border of the opened skin in R1 or the flap border in R2. Full size image

Table 1 Lymphatic function/anatomy in skin flap and lymph node transplantation models. Full size table

Figure 4 MAPCs restore lymphatic capillaries and pre-collectors. (a–d) Flt4-stained wound cross-sections from PBS (a), mMAPC (‘mM’; b) or hMAPC-treated (‘hM’; c) mice, and corresponding quantification (d; n = 6; P = 0.0074 by Kruskal-Wallis test; *P < 0.05 versus PBS by Dunn’s post-hoc test). (e–h) Wound cross-sections from PBS (e), mMAPC (f) or hMAPC-treated (g) mice revealing functional (dextran (red or green)-perfused) lymphatics in cell-treated mice, and corresponding quantification (h; n = 5–10; P < 0.0001 by Kruskal-Wallis test; *P < 0.05 versus PBS by Dunn’s post-hoc test). Inset (i1) in e shows corresponding Prox1-stained (in red) region. Note diffuse fluorescence signal in e representing FITC-dextran that failed to be drained. (i) Merged bright field/fluorescence image of the wound transplanted with eGFP+ mMAPCs (in green; indicated by arrowheads) 2 w earlier. (j) Merged green/red fluorescence images of the wound transplanted with eGFP+ mMAPCs (circled by dashed line) 4 w earlier. Note Rhodamin-dextran-filled lymphatic vessels (red; indicated by arrowheads) in the vicinity of transplanted cells. (k) Cross-section through the wound, revealing transplanted eGFP+ mMAPCs (in green) adjacent to functional (red Rhodamin-dextran-filled) lymphatics (asterisks). (l) Merged picture of green (FITC-labelled dextran), red (Prox1) and far-red (αSMA) fluorescence images of a wound transplanted with hMAPCs 2 w earlier, revealing a functional sparsely αSMA-coated (indicated by arrowheads) Prox1+ lymphatic pre-collector and two functional Prox1+/αSMA− lymphatic capillaries (circled by white dashed lines). Haematoxylin or DAPI were used to reveal nuclei in a–c and e–g, k, l, respectively. Full size image

hMAPCs reconnect transplanted lymph nodes to the host lymphatic network

Thus far, we showed that MAPC transplantation increased lymphangiogenesis and reinstated lymphatic drainage mainly by boosting restoration of small caliber lymphatic vessels. However, the underlying problem of secondary lymphedema most often relates to damaged lymph nodes and large lymphatic collectors to which the lymphatic capillaries and pre-collectors normally connect. Hence, an appropriate remedy must equally imply restoration of lymphatic collectors. We applied a stringent model in which axillary lymph nodes and their surrounding lymphatic (collector) network were surgically ablated, such that drainage of a lymph node transplanted in this area becomes critically dependent on restoration of lymphatic collectors and their reconnection to the host lymphatic network9. To test the potential of hMAPCs, we applied them in Matrigel around a transplanted lymph node derived from mice ubiquitously expressing DsRed or eGFP in the right axillary cavity (Fig. 5a). Transplantation of the lymph node alone (and covering it with Matrigel containing PBS) failed to resolve inflammation-induced edema in the right upper limb, evident from interstitial fluid accumulation measured by magnetic resonance imaging (MRI) 4 and 16 weeks after surgery upon challenge of the paw with mustard oil – an inflammatory agent (Fig. 5b,c + Supplementary Fig. S6a). At 16 weeks, fluid accumulation was significantly less prominent upon application of hMAPCs around the transplanted lymph node, suggesting functional restoration of lymph drainage from the front paw to the axillary region (Fig. 5b,d + Supplementary Fig. S6b). Indeed, lymphangiography revealed that lymph fluid drainage was significantly improved in hMAPC-treated mice and that the injected fluorescent dye reached and filled the transplanted lymph node in ~35% and 50–60% of hMAPC-treated mice, 8 and 16 weeks post-transplantation, respectively, a result that was reproduced with two hMAPC populations and not at all in PBS-treated mice (Fig. 5e–g + Supplementary Fig. S6c; Table 1). This suggested that hMAPC transplantation functionally reconnected the transplanted lymph node to the host lymphatic network. Notably, while all lymph nodes implanted along with hMAPCs persisted, half of them could not be traced in PBS-injected mice at 16 weeks, suggesting a positive effect of hMAPC transplantation on lymph node survival (Table 1). Moreover, unlike in hMAPC-treated mice, the mean size of the engrafted lymph nodes was decreased in PBS-treated mice (Table 1).

Figure 5 hMAPCs support functional reconnection of transplanted lymph nodes. (a) Merged bright field/fluorescence image of right axillary region 16 weeks (w) post-transplantation of an eGFP+ lymph node (LN; green; arrowhead) and treatment with Matrigel containing hMAPCs (dashed and full white lines indicate Matrigel-covered area and open skin border, respectively). (b) Edema extent in right upper limb (shown as rigth/left ratio in arbitrary units) 4 w or 16 w after LN transplantation and treatment with Matrigel containing PBS or hMAPCs. n = 4–9; *P = 0.011 versus 4 w by unpaired two-tailed Student’s t-test. (c,d) T 2 -weighted MR images of antebrachial regions 16 w after LN transplantation and treatment with Matrigel containing PBS (c) or hMAPCs (d). Hyperintense areas (arrows) indicate fluid accumulation. L: left; R: right. (e,f) Merged bright field/fluorescence image of right axillary region 16 w post-transplantation of an eGFP+ LN (green; arrowhead) and treatment with Matrigel containing PBS (e) or hMAPCs (f). Insets (i1,2; red channel only) zoom in on boxed areas in e,f. Note significantly improved drainage of Rhodamin-labelled (red) lectin in hMAPC-treated mice (arrow and white lines indicate lymphangiography injection spot and open skin border, respectively). (g) Merged bright field/fluorescence image zooming in on an eGFP+ (in green) LN transplanted in a mouse treated with Matrigel containing hMAPCs 16 w earlier, revealing uptake of red Rhodamin-labelled lectin. Arrowheads indicate connecting lymph vessel. Full size image

Inspection of the skin area leading up to the transplanted lymph node revealed a two-fold more elaborate blood vascular network in hMAPC-treated mice (Fig. 6a–c) with significantly more blood vessels in the immediate surrounding of the lymph nodes, compared to PBS-injected mice (Fig. 6d + Supplementary Fig. S6d,e). Some hMAPCs persisted until 16 weeks and were found in the vicinity of the transplanted lymph node (Supplementary Fig. S6f). All transplanted lymph nodes in hMAPC-treated mice showed signs of (outward) branching of their internal (lymph)vascular network from 4 weeks onwards, while this was never observed in PBS-treated mice (Fig. 6e–g + Supplementary Fig. S6g,h; Table 1). At 8 weeks, hMAPC transplantation resulted in a significant 4-fold expansion of LYVE1+ lymphatic capillaries in the area surrounding the lymph node as compared to PBS-treatment (Fig. 6h–j). Finally, to test whether the beneficial effect of hMAPCs was related to functional reconnection of lymphatic collector vessels, we performed αSMA/Prox1 immunofluorescence stainings on cross-sections taken from the area around the transplanted lymph nodes and found lymph-filled Prox1+αSMA+ collectors (Fig. 6k–m). Collector identity was confirmed by negative staining for LYVE1 (Fig. 6n).