TNFSF15 facilitates human umbilical cord blood haematopoietic stem cell expansion by activating Notch signal pathway

Abstract The lack of efficient ex vivo expansion methods restricts clinical use of haematopoietic stem cells (HSC) for the treatment of haematological malignancies and degenerative diseases. Umbilical cord blood (UCB) serves as an alternative haematopoietic stem cell source. However, currently what limits the use of UCB‐derived HSC is the very low numbers of haematopoietic stem and progenitor cells available for transplantation in a single umbilical cord blood unit. Here, we report that TNFSF15, a member of the tumour necrosis factor superfamily, promotes the expansion of human umbilical cord blood (UCB)‐derived HSC. TNFSF15‐treated UCB‐HSC is capable of bone marrow engraftment as demonstrated with NOD/SCID or NOD/Shi‐SCID/IL2Rgnull (NOG) mice in both primary and secondary transplantation. The frequency of repopulating cells occurring in the injected tibiae is markedly higher than that in vehicle‐treated group. Additionally, signal proteins of the Notch pathway are highly up‐regulated in TNFSF15‐treated UCB‐HSC. These findings indicate that TNFSF15 is useful for in vitro expansion of UCB‐HSC for clinical applications. Furthermore, TNFSF15 may be a hopeful selection for further UCB‐HSC application or study.

registries. 6 Umbilical cord blood (UCB) serves as an alternative haematopoietic stem cell source and the use of umbilical cord blood (UCB)-derived HSC is a highly demanded alternative approach because of reduced need for HLA matching, decreased risk of chronic graft-vs-host disease (GVHD) despite HLA disparity, relatively convenient acquisition of the cells and retained graft-vs-tumour effects. 7,8 However, currently what limits the use of UCB-derived HSC is the very low numbers of haematopoietic stem and progenitor cells available for transplantation in a single umbilical cord blood unit, hindering otherwise a great application of these cells in allogeneic transplantation, particularly in adult recipients. 9,10 Moreover, the low haematopoietic stem and progenitor cell dose given with UCB transplantation patients could have negative effects on outcomes. 11 The fundamental of overcoming the obstacle in UCB transplantation is increasing the number of HSC in a single UCB unit by ex vivo UCB expansion. Many studies have translated into significant clinical benefits on UCB-HSC expansion to enhance UCB-HSC dose over the last decades. However, currently available techniques are likely expanding more progenitors rather than true stem cells. 12 It is necessary to develop new strategies to expand UCB-derived HSC ex vivo.
TNFSF15 is a cytokine involved in maintaining vascular homeostasis. This cytokine uniquely exhibits a number of activities: it inhibits neovascularization and vascular hyperpermeability but promotes lymphangiogenesis. [13][14][15][16][17][18] TNFSF15 can also enhance the survival of bone marrow-derived Lin − Sca1 + endothelial progenitor cells in vitro and at the same time inhibit the differentiation of these cells into endothelial cells. 15 Lin − Sca1 + mononuclear cells were considered as EPCs, while HSC has the same biomarker. As a result, we speculated that TNFSF15 (VEGI) directly regulated and may inhibit the differentiation HSC. Then, we accidentally discovered TNFSF15 (VEGI) injection by intraperitoneal at 5 mg/kg significantly increased the percentage of Lin − -c-Kit + -Sca-1 + cells in the bone marrow which suggests that VEGI promotes HSC production. In this study, we investigated the effect of TNFSF15 on UCB-HSC expansion ex vivo. We found that TNFSF15 was able to stimulate the expansion of UCB-HSC ex vivo and promote the engraftment of HSC in immune-deficient mice by activating Notch signal pathway. All the results showed that TNFSF15 was a hopeful selection for further UCB-HSC application or study.

| Colony formation assay
Fresh CD34 + cells were suspended with in expansion medium and seeded into 96 well plate with 1 × 10 4 cells/200 µL each well. After treated with TNFSF15 (2 µg/mL, 7 days), 10 µL cell suspension (equivalent to 200 initial cells) was mixed with Methocult H4434 gently and evenly. Then the mixture was plated into 6-well plate and cultured at 37°C with 5% CO 2 for 10 days. The number of CFU-GM, CFU-E, BFU-E, CFU-G, CFU-M and CFU-GEMM was scored according to morphological features. The experiment was repeated for three times. Freshly isolated CD34 + cells from human umbilical cord blood were treated with TNFSF15 (2 µg/mL) or vehicle for 7 days in expansion medium, then rinsed with PBS, resuspended in 0.1 mL of HLTM containing 1 µmol/L hydrocortisone, and added to the M 2 -10B 4 cultures at different cell number concentrations with 12 replicates each concentration.

| Cobblestone area-forming cells (CAFC) assay
The CAFC cultures were maintained for 5 weeks at 37°C with 5% CO 2 for five weeks with changing half medium weekly. Cobblestone areas in the cultures were then scored. CAFC frequencies were calculated by using Poisson analysis based on the proportion of negative wells and maximum likelihood. Statistical analysis was performed by using the L-Calc software for limiting dilution assay.

| Differentiation assay
Fresh CD34 + cells from human umbilical cord blood were cultured in expansion culture medium for 7 days in the presence of TNFSF15 at 37°C with 5% CO 2 . Then cells were collected, respectively, washed and stained with PE.Cy7-conjugated anti-CD3 (BD; 557851), APC.

| SRC assay
CD34 + cells from human umbilical cord blood were cultured in expansion culture medium for 7 days in the presence of TNFSF15 at 37°C with 5% CO 2 . Six-week-old female NOD/SCID (Institute of Laboratory Animals, Beijing, China) was irradiated with X-ray (200 cGy) 12 hours before transplantation. Then, the cells after treatment of TNFSF15 were collected, washed and calculated. The NOD/SCID mice were randomized and injected with cultured CD34 + cells with or without TNFSF15 treatment at different cell concentrations (2000, 5000, 10 000 and 20 000 initialling cells, respectively) intravenously. Bone marrow (BM) cells were collected, stained with FITC-conjugated anti-human CD45 (BD, 561865) and analysed for human cell engraftment by flow cytometry in 12 weeks. After 16 weeks, the percentage of engraftment was detected using human CD45 staining.

| Cell apoptosis assay
Cell apoptosis assay was performed according to the manufacture`s advice. CD34 + cells from human umbilical cord blood were cultured in expansion culture medium for 7 days in the presence of TNFSF15 at 37°C with 5% CO 2 . The cells were collected and washed with PBS.
Then resuspended with 1× loading buffer, and stained with Annexin Ⅴ-FITC and PI for 15 minutes at room temperature in dark. After that the cells were analysed by flow cytometry.

| Cell cycle assay
CD34 + cells from human umbilical cord blood were cultured in expansion culture medium for 7 days in the presence of TNFSF15 at 37°C with 5% CO 2 . The cells were collected, washed with PBS and fixed with cold 70% ethanol overnight. After washed with PBS twice, the cells were incubated with propidium iodide buffer in the dark for 30 minutes. Then cell cycle distribution was determined by flow cytometry. As to G0 phase detection, the cells were collected, fixed with cold 70% ethanol for 1 hour on ice and stained with FITCconjugated anti-Ki67 (eBioscience, 11-5698-82) antibody at room temperature in dark for 60 minutes. The cells were incubated with propidium iodide buffer in the dark for 30 minutes. The percentage of G0 phase was determined by flow cytometry.

| Western blot assay
CD34 + cells from human umbilical cord blood were cultured in expansion culture medium for 7 days in the presence of TNFSF15 at 37°C with 5% CO 2 . The cells were collected in lysis buffer. The BCA assay was used to determine the protein concentration. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. After blocked by nonfat milk, the membranes were incubated with antibodies specific for c-myc, hes1, Notch1, NCID and β-actin overnight. Then washed with PBST for five times, incubated with anti-HRP secondary antibody and visualized by the Tanon Chemiluminescent Imaging System.

| TNFSF15 increases the number of primitive human CD34 + CD49f + haematopoietic stem cells
Notta and colleagues reported that CD49f was a unique cell surface marker of HSCs that contributed greatly to the separation of HSCs from multi-potent progenitors (MPPs). 19 Therefore, we used CD34 and CD49f as HSC enrichment markers to validate the HSC expansion effect. We collected human umbilical cord blood and first isolated CD34 + bulk cells for a dose response assay of TNFSF15 and the purity of CD34 + cells after magnetic sorting guaranteed at about 95% ( Figure S1A). We found that TNFSF15 could significantly increase the percentage and the total number of CD34 + CD49f + cells with slightly inhibition of total mononuclear cells ( Figure 1A

| TNFSF15 sustained selfmaintenance and multi-lineage differentiation potential of haematopoietic stem cells
Haematopoietic colony-forming unit (CFU) assays represent a classical tool for quantifying and evaluating haematopoietic progenitor content which was produced by haematopoietic stem cell within samples. The dysregulation of HSC biology may lead to unbalanced differentiation and could be detected by the presence of single dominant colonies in the CFU assay. Thus, we determined the capacity of TNFSF15 to promote the expansion of haematopoietic progenitor cells by using short-term haematopoietic colony-forming unit (CFU) assays. 20 We cultured CD34 + cells from human cord blood for 7 days before plating into semi-solid culture media for 14 days. All kinds of colonies Burst-forming unit-erythroid (BFU-E), Colony-forming unit-erythroid (CFU-E), Colony-forming unit-granulocyte (CFU-G), Colony-forming unit-macrophage (CFU-M), (Colony-forming unit-granulocyte, F I G U R E 1 TNFSF15 promotes in vitro expansion of primitive human CD34 + CD49f + haematopoietic stem cells. A, Number of total mononuclear cells after being treated with TNFSF15 for 7 d at 2 µg/mL in expansion medium (n = 3). B, Percentage of CD34 + CD49f + cells after being treated with TNFSF15 for 7 d at 2 µg/mL in expansion medium on the same human umbilical cord blood sample (n = 3). 1 × 10 4 CD34 + human UCB cells were seeded in the beginning. The experiment was repeated for three times. C, Absolute number and representative pictures (D) of CD34 + CD49f + cells after the treatment of TNFSF15 at 2 µg/mL for 7 d in expansion medium on the same human umbilical cord blood sample (n = 3). E, Percentages and absolute number of CD34 + CD49f + cells in CD34 + cells treated with various concentrations of TNFSF15 (0, 0.2, 1, 2 and 4 µg/mL) for 3 d in expansion medium with 1 × 10 4 initiating CD34 + cells. F, Percentages and absolute number of CD34 + CD49f + cells in CD34 + cells treated with various concentrations of TNFSF15 (0, 0.2, 1, 2 and 4 µg/mL) for 7 d in expansion medium with 1 × 10 4 initiating CD34 + cells (n = 3). G, The percentage and total number of CD34 + CD49f + cells per sample of 33 cases of human umbilical cord blood samples cultured in the presence or absence of TNFSF15 (2 µg/mL) for 7 d in expansion medium (n = 33); horizontal bar, mean value. H, Representative flow cytometry images of CD34 + CD90 + , CD34 + CD45RA − , CD34 + CD45RA − CD90 + CD38 − and CD34 + CD45RA − CD90 + CD38 − CD49f + cells after being treated with TNFSF15 (2 µg/mL) in the presence or absence of TNFSF15 and TNFSF15-neutralizing antibody 4-3H. Each experiment was repeated for three times. I, The number of CD34 + CD90 + , CD34 + CD45RA − , CD34 + CD45RA − CD90 + CD38 − and CD34 + CD45RA − CD90 + CD38 − CD49f + cells after being treated with TNFSF15 (2 µg/mL) in the presence or absence of TNFSF15 and TNFSF15-neutralizing antibody 4-3H (n  (Figure 2A and B).
Noticeably, the number of colonies formed by CFU-GEMM cells, which represent more primitive and multi-potent progenitor cells derived from primary HSC, significantly increased in response to TNFSF15 treatment. This indicated that treatment with TNFSF15 significantly increased the haematopoietic stem and progenitor cells by the colony-forming capacity assay.
We also assessed the long-term self-maintenance capacity of TNFSF15-treated HSC by using the cobblestone area-forming cell (CAFC) assay. 21 We treated CD34 + cells for 7 days with TNFSF15

| TNFSF15 showed synergistic effect in combination with SR1
Small molecules are emerging as valuable tools for regulating stem cell fates. Recent studies showed that aryl hydrocarbon receptor (AhR) antagonist named SR1 24 and UM171 25 with unclear mechanism showed excellent effect on haematopoietic stem cell expansion. TNFSF15 showed almost the same effective on CD34 + CD49f + cell expansion compared with SR1 and UM171, two commonly used reagents for this purpose ( Figure 3A). Moreover, TNFSF15 also exhibited a synergistic effect with SR1 ( Figure 3B and C) while showed antagonistic effect with UM171 ( Figure 3B and D). To further reveal the combination of TNFSF15 and SR1, we detected the percentage and absolute number of CD34 + CD49f + CD45RA − CD90 + after cultured for 7 days which showed a synergistic effect with SR1 ( Figure 3E and F).

| TNFSF15-induced enhancement of haematopoietic stem cell engraftment in NOD/ SCID or NOG mice
To determine the effect of TNFSF15 on the engrafting capability of TNFSF15-treated UCB-HSC, we sorted 300 CD34 + CD38 -CD45RA − CD90 + cells with or without TNFSF15 treatment for 4 days and transplanted them into the tibia of NOD/Shi-SCID/ IL2Rg null (NOG) mice, using 300 initiating cells per injection.
Simultaneously, some NOG mice injected with freshly sorted CD34 + CD38 − CD90 + CD45RA − cells (not cultured) were considered as uncultured control. We found that the percentage of CD45 + cells in periphery blood was statistically higher in TNFSF15-treated group compared to uncultured or vehicle-treated control groups when analysed after 12 weeks post-transplantation ( Figure 4A and B), and that the percentage of CD45 + cells in the injected tibiae in TNFSF15 group was approximately 1.75-fold of that in the vehicle group ( Figure 4A and C). The percentage of CD45 + cells in the untreated tibiae was also higher, though marginally, in TNFSF15 group than in vehicle-treated group ( Figure 4D). Furthermore, the percentage of CD34 + CD38 − cells in CD45 + cells exhibited a 1.8-fold increase in TNFSF15 group ( Figure 4E). These data suggested that TNFSF15treated UCB-HSC maintains HSC's haematopoietic reconstitution potential and increased the engraftment of UCB-HSC.
To measure the frequency of HSC present after culture, we performed a limiting dilution analysis with CD34 + cells cultured in TNFSF15 or vehicle control into NOD-SCID mice. Haematopoietic cells with the capable of haematopoietic reconstitution were considered as NOD-SCID repopulating cells (SRCs) which represented candidate human HSCs. We found that, 12 weeks post-transplantation, the frequency of SCID mice repopulation cells (SRC) isolated from the tibiae of the TNFSF15 group was 3.14-fold of that of the control group ( Figure 4F). The SRC in the opposite tibiae in TNFSF15 group was 1.17-fold of that in the control group ( Figure 4G).
To determine whether TNFSF15-expanded cells are capable of long-term engraftment, we then performed secondary transplant experiments 26 with human cells isolated from the bone marrow of the NOG mice previously transplanted with TNFSF15-treated cells and found a rate of successful secondary engraftment of 0.71 compared with 0.25 and 0.29, respectively, for the uncultured and vehicle groups ( Figure 4H and I), a completely successful rate being 1.00.
Additionally, the percentage of re-engrafted TNFSF15-treated cells is 3.33-fold of that of the uncultured cells ( Figure 4J). There is no significant difference in multi-lineage differentiation including myeloid (CD33 + ), lymphocyte cell (CD19 + ), T lymphoid cell (CD3 + ), erythroid cell (CD235a) and NK cell (CD56) after transplantation without significant difference compared to uncultured group and control group ( Figure 4K, Figure S2).

| TNFSF15 facilitates human umbilical cord blood haematopoietic stem cell expansion by Notch signal pathway
To analysis the mechanism of TNFSF15 in HSC expansion, cell apoptosis and cell cycle assay were performed. We found TNFSF15 treatment of UCB-HSC did not impose any significant impact on cell apoptosis rates ( Figure 5A and B); rather, TNFSF15-treated cells exhibited a decrease of the G0/G1 phase and an increase of the G2/M phase when cell cycle distribution was analysed ( Figure 5C and D), indicating enhanced cell proliferation. We further performed experiment to distinguish the percentage of the G0 phase from G0/G1 with PI/Ki67 double staining. The results showed the G0 phase after TNFSF15 treatment was decreased clearly ( Figure 5E, Figure S1C).
To detect the change of genes related in proliferation and differentiation in human HSCs after TNFSF15 treatment, we performed single-cell PCR assay with 50 CD34 + CD38 − CD45RA − CD90 + CD49f + cells sorted from CD34 + cells cultured in expansion medium after treatment for 7 days. The expression of 95 genes was detected by PCR assay. The results showed that genes in Notch signal pathway including c-myc and Notch1 were significantly changed ( Figure 5F and G). Since the Notch signalling pathway is reported to be pivotal in the sustenance and proliferation of HSC, [27][28][29] we determined the relative abundance of each of the Notch signal pathway proteins C-myc, Hes1 and Notch1 in TNFSF15-treated UCB-HSC, and

| D ISCUSS I ON
Haematopoietic stem cells from human umbilical cord blood (hUCB-HSCs) have been a significant source of haematopoietic stem cells due to tremendous promise for clinical HSC transplantation. 30 However, the relatively low number of HSCs in a UCB unit limits its successful widespread transplantation especially in adult recipients. One strategy to overcome this barrier was the application of two partially HLA-matched UCB units while this aggravated the difficult of HLA-matched, slow platelet and neutrophil engraftment, and made it much more impossible for auto-transplantation patients. 31,32 The other strategy was made effort in searching culture conditions that sustain hUCB-HSCs expansion ex vivo and developed cellular therapies to improve the outcomes of hUCB-HSCs transplantation. 30 Cytokine cocktails including SCF, TPO, FLt3, IL-3 and IL-6 were frequently used in hUCB-HSCs expansion ex vivo which resulted in rapid proliferation of partially differentiated HSC F I G U R E 5 TNFSF15 facilitates human umbilical cord blood haematopoietic stem cell expansion by Notch signal pathway. A, The representative plots of flow cytometry analysis and (B) the statistics of CD34 + cell apoptosis rates in the presence or absence of TNFSF15 (2 µg/mL). The experiment was repeated for three times (n = 3). C, The representative plots of flow cytometry analysis and (D) the statistics of CD34 + cell cycle distribution of G0/G1, S and G2-M phase in the presence or absence of TNFSF15 (2 µg/mL) for 7 d in expansion medium (n = 3). The experiment was repeated for three times. E, The percentage of G0 phase was detected in the presence or absence of TNFSF15 (2 µg/mL) for 7 d in expansion medium by Ki67/PI double staining assay (n = 3). F, The gene expression of c-myc in CD34 + CD38 − CD90 + CD45RA − CD49f + cells after the treatment of TNFSF15 (2 µg/mL) for 7 d in expansion medium (n = 3). G, The gene expression of hes1 in CD34 + CD38 − CD90 + CD45RA − CD49f + cells after the treatment of TNFSF15 (2 µg/mL) for 7 d in expansion medium (n = 3). H, Western blotting analysis of Notch signal pathway proteins including c-myc, hes1, Notch1 and NCID in CD34 + cells treated with TNFSF15 at indicated concentrations for 7 d in expansion medium. I, The absolute number of CD34 + CD49f + cells after cultured with combination of TNFSF15 (2 µg/mL) and DAPT (0.5 µmol/L) for 7 d in expansion medium (n = 3) compartment while losing the characteristic of long-term repopulating ability. 33 Small molecules such as SR1, 24 UM171, 25 CHIR-911, 34 valproic acid 35 and so on were of increasing concern with the effect of promoting HSC self-renewal, multi-lineage potency activities and homing, and as to SR1 the engraftment for neutrophils and platelets showed significantly faster than that in patients treated with unmanipulated UCB in clinical trial 36 which showed attractive prospects, while the effect and the safety needed further identification. 30 As a result, there were no sufficient means to achieve the expansion of human HSCs in the present culture conditions. TNFSF15, an endogenous vascular endothelial growth inhibitor (VEGI), is mainly produced by vascular endothelial cells in a normal tissue and modulated vascular homeostasis by inhibiting endothelial cells proliferation. Previously, our group reported that TNFSF15 treatment caused the number of HSC cells in the bone marrow to increase by about three-fold which prompt us to propose that TNFSF15 may promote HSC production. 16 To verify our hypothesis, herein we demonstrate that TNFSF15 has the ability to promote UCB-HSC expansion ex vivo in amplification cultures stimulated by combination of SCF, TPO and Flt3. Our data showed a significant increase of UCB CD34 + CD49f + cells after cultured for 7 days with the treatment of TNFSF15. Further characterization of the HSCs showed the percentage and absolute number of CD34 + CD45RA -, CD34 + CD90 + , CD34 + CD49f + CD90 + CD45RA − and CD34 + CD49f + CD90 + CD45RA − CD38 − were significantly more preserved after treatment of TNFSF15. The CFUs assay showed that TNFSF15 increased the number of colony-forming units especially CFU-GEMM which indicated that TNFSF15 promoted the expansion of multi-potent progenitors. Although the multiple differentiation potential is responsible to HSC for producing kinds of functional blood cells, the capacity of self-renewal is critical in maintaining the size of the HSC pool. 3 The CAFC assay demonstrated that TNFSF15 could promote the self-renewal of hUCB-HSCs. In consecutive transplant, experiments with NOG mice and NOD/SCID mice indicated that TNFSF15 promoted the expansion of hUCB-HSCs that retained multi-lineage long-term engraftment.
Furthermore, the mechanism study of TNFSF15 for the expansion of hUCB-HSCs showed that TNFSF15 affected mainly by activating Notch signal pathway.

| CON CLUS ION
We demonstrated that TNFSF15 has the ability to promote UCB-HSC expansion ex vivo through activating Notch signal pathway, and TNFSF15-treated HSCs are able to sustain bone marrow engraft.
Our research suggested that TNFSF15 may thus be useful for acquiring HSC from umbilical cord blood for the treatment of haematological malignancies and degenerative diseases.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

CO N S E NT FO R PU B LI C ATI O N
All authors reviewed and approved the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article. The data sets analysed during the current study available from the corresponding author on reasonable request.