Wharton’s jelly‐derived stromal cells and their cell therapy applications in allogeneic haematopoietic stem cell transplantation

Abstract For decades, mesenchymal stromal cells (MSCs) have been of great interest in the fields of regenerative medicine, tissue engineering and immunomodulation. Their tremendous potential makes it desirable to cryopreserve and bank MSCs to increase their accessibility and availability. Postnatally derived MSCs seem to be of particular interest because they are harvested after delivery without ethical controversy, they have the capacity to expand at a higher rate than adult‐derived MSCs, in which expansion decreases with ageing, and they have demonstrated immunological and haematological supportive properties similar to those of adult‐derived MSCs. In this review, we focus on MSCs obtained from Wharton's jelly (the mucous connective tissue of the umbilical cord between the amniotic epithelium and the umbilical vessels). Wharton's jelly MSCs (WJ‐MSCs) are a good candidate for cellular therapy in haematology, with accumulating data supporting their potential to sustain haematopoietic stem cell engraftment and to modulate alloreactivity such as Graft Versus Host Disease (GVHD). We first present an overview of their in‐vitro properties and the results of preclinical murine models confirming the suitability of WJ‐MSCs for cellular therapy in haematology. Next, we focus on clinical trials and discuss tolerance, efficacy and infusion protocols reported in haematology for GVHD and engraftment.

MSCs have been isolated from multiple adult tissues other than bone marrow, including skeletal muscle, adipose tissue, synovial membranes, saphenous veins, dental pulp, periodontal ligaments, lung, liver and skin. MSCs obtained from extra-embryonic tissues (such as placenta/umbilical cord, Wharton's jelly and amniotic membrane) share properties with their adult counterparts. They also retain characteristics of primitive stem cells, like the expression of the embryonic stem cell markers Oct-4, Nanog, Sox-2 and c-Kit, as well as Dnmt3b and hTERT, albeit at much lower levels than those of embryonic stem cells. 5 They have many advantages for cellular therapy applications: available after delivery, their collection and expansion raise no ethical issues. They usually expand with a higher proliferation rate 6 and a broader multipotency than MSCs from adult tissues, though donor age affects these characteristics in bone marrowderived MSCs (BM-MSCs). 7 Wharton's jelly mesenchymal stromal cells (WJ-MSCs), were first isolated in 1991. 8 Their immunomodulatory properties and their low cellular immunogenicity 9 make them remarkably interesting for the induction of tolerance in transplantation. A few in vitro studies have reported that WJ-MSCs are less immunogenic than MSCs from other sources, especially when cultivated in hypoxia. 10 Amongst MSCs, WJ-MSCs have the lowest level of MHC class II expression. Moreover, they express low levels of cellsurface MHC class I and costimulatory molecules (CD40, CD80, CD86). In a comparative study of foetal adnexa-derived caprine MSCs, WJ-MCs outperformed MSCs from other sources of foetal adnexa (amniotic fluid, amniotic sac, cord blood) in terms of growth kinetics, relative messenger ribonucleic acid (mRNA) expression of surface antigens, pluripotency markers, and tri-lineage differentiation potential. 11 In allogeneic haematopoietic stem cell transplantation (allo-HSCT), BM-MSCs were the first described cells of potential interest. They have been mainly (and widely) studied in the treatment of steroid-refractory graft versus host disease (GVHD), with results varying depending on the study and the methods used. [12][13][14] Another topic of research has been their haematopoietic support functions since they are key players in the haematopoietic niche and can enhance haematopoietic stem cell (HSC) engraftment and function. [15][16][17] However, in this area of research, their efficacy remains to be established. 15,18 Several studies, which we describe below, have compared MSCs isolated from the most common sources in terms of their functional differences in immune modulation and haematopoietic support.

| Immunosuppression
MSCs derived from amnion, placenta, Wharton's jelly and umbilical cord blood elicit a similar degree of immunosuppression in vitro compared with that of BM-MSCs, but Manochantr et al. have demonstrated that WJ-MSCs have a higher proliferative capacity. 19 MSCs derived from WJ and adipose tissue have potent immunosuppressive effects on T cells, similar 20 or even superior to that observed with BM-MSCs. 21 WJ-MSCs have a capacity to suppress neutrophil adhesion to inflamed endothelium similar to that of BM-MSCs, but conserve their immune-suppressive properties after passage 7, unlike BM-MSCs. 22 In a murine model of experimental sepsis, mice treated with WJ-MSCs had a higher survival rate than mice treated with BM-MSCs. 23 In another study, by Karaöz et al, WJ-MSC co-culture with activated T cells led to a higher concentration of IL-17A, which would be of particular interest in a GVHD context. 24 In a recent work, Shin et al. analysed the protein secretome of four sources of MSCs: adipose tissue, bone marrow, placenta, and WJ. Each MSC secretome profile had distinct characteristics, depending on the source. The secretome of foetal-derived MSCs (placenta and WJ) had a more diverse composition than those of adipose tissue and BM-MSCs, and the authors assumed that their therapeutic potential was greater because of these properties. 25

| Haematopoiesis
One study found more proteins related to tissue development and the differentiation of haematopoietic cells in the secretome of WJ-MSCs compared with adipose tissue and BM-MSCs. 25 Moreover, CD117 (c-kit) the receptor for stem cell factor (SCF) harboured by haematopoietic stem/progenitor cells (HSPCs), has been repeatedly detected in WJ-MSCs. 26 WJ-MSCs express osteopontin and are able to secrete hyaluronic acid. 27 Interestingly, both these molecules are amongst the main constituents of the HSPC niche. Osteopontin is a critical regulator of HSPC localization and proliferation. 27,28 Human WJ-MSCs have been compared with BM-MSCs and show similar haematopoiesis-supportive functions in vitro, when co-cultured with CD34 + umbilical cord blood cells. 29 In a murine model, cotransplantation of either WJ-MSCs or BM-MSCs with CD34 + HSCPs from cord blood resulted in similarly enhanced recoveries of human platelets and CD45 + cells in the peripheral blood and a 3-fold higher engraftment in the bone marrow, blood, and spleen 6 weeks after transplantation when compared with transplantation of CD34 + cells alone. 30 Considering their low immunogenicity, their immunomodulatory and haematopoietic supportive properties in vitro, their growth kinetics, and their proteome diversity, WJ-MSCs are remarkably interesting cells for potential use in GVHD and haematopoietic engraftment ( Figure 1).

| WJ -MSC S SUS TAIN HAEMATOP OIE S IS
In the setting of sustaining haematopoiesis, MSCs from placenta and umbilical cord display interesting properties in vitro. 30 They promote growth and preserve the stemness of haematopoietic stem cells (HSCs) from autologous or allogeneic cord blood in twodimensional cultures, 31,32 and in three-dimensional scaffolds. 33 WJ-MSCs have been recently exploited as a feeder layer to expand haematopoietic stem cells, providing secreted proteins 34 and cytokines involved in the regulation of haematopoiesis, including interleukin (IL)-6, SCF, Fms-like tyrosine kinase 3-ligand (Flt-3L), macrophage colony-stimulating factor, granulocyte colonystimulating factor and granulocyte-monocyte colony-stimulating factor (Table 1). 35

| Preclinical studies
Studies in NOD/SCID/IL2Rγnull (NSG) mice suggest that WJ-MSCs may increase haematologic recovery. 36 Six weeks after allogeneic stem cell transplantation, NSG mice co-transplanted with 1 × 10 6 WJ-MSCs demonstrated a significantly higher median number of human CD45 + cells engrafting in the peripheral blood and bone marrow than those transplanted without WJ-MSCs: respectively, 28.2% (range, 24.6-33.1%) versus 5.3% (range, 4.2-6.5%) and 6.9% (range, 5.9-7.3%) versus 1.7% (range, 1.5-2.3%). WJ-MSCs were obtained from umbilical cords as follows: the main vessels were removed and the jelly was digested using 1 mg/ml collagenase. Cell culture was normoxic, and cells were cryopreserved before use. WJ-MSCs were selected if they matched at least three HLA alleles with recipients, then expanded and cryopreserved a F I G U R E 1 WJ-MSCs support haematopoiesis and modulate immunity via soluble factors and cell-cell contact. Upper: WJ-MSCs secrete growth factors that may enhance haematopoietic cell renewal or stemness, and they may create a fibronectin network that supports haematopoietic cell homeostasis. Thus, they are of interest in the treatment of poor graft function after HSCT. IL-6: Interleukin-6, SCF: stemcell factor, M-CSF: macrophage colony-stimulating factor, G-CSF: granulocyte colony-stimulating factor, GM-CSF: granulocyte macrophage colony-stimulating factor, Flt3: Fms-like tyrosine kinase 3; Lower: WJ-MSCs secrete cytokines and other molecules that decrease activated T-cell proliferation, or induce Tregs, and act on other immune cells. They also produce cytosolic IDO, an enzyme that depletes tryptophan in the medium and converts tryptophan into secreted metabolites (like kynurenine) that prevent T-cell proliferation. WJ-MSCs also express several membrane molecules that interact with activated T cells to induce exhaustion or apoptosis, or to prevent T-cell activation. The expression of soluble and membrane factors varies according to the level of inflammation in the environment. These properties make WJ-MSCs good candidates for GVHD prophylaxis or cure, for graft rejection prophylaxis, and for some disorders of uncontrolled inflammation, such as haemorrhagic cystitis. PGE2, prostaglandin E2; HGF, hepatic growth factor; IL, interleukin; TGF β1, transforming growth factor β1; HLA, human leukocyte antigen; PDL (1/2), programmed-death ligand; VCAM, vascular cell adhesion molecule; ICAM, intercellular adhesion molecule second time. They were infused into patients immediately after thawing, at an average dose of 1.2 × 10 6 / kg (range, 0.27-2.5 × 10 6 / kg). None of the patients experienced graft rejection, and all had rapid engraftment (mean times for neutrophil and platelet recovery were 13.95 days and 20.27 days, respectively). No severe acute GVHD (aGVHD) and no chronic GVHD (cGVHD) were observed.

| Clinical studies
With a median follow-up of 15 months, 21 patients were alive and transfusion-independent with full donor chimerism. This report has been confirmed in 17 SAA adult patients treated with haploidentical HSCT after a reduced-intensity conditioning regimen and with co-infusion of culture-expanded third-party donor-derived WJ-MSCs 4 hours before allo-HSCT. 38 In this study, WJ-MSCs were obtained after the cord was sectioned, without enzymatic digestion.
Cells were thawed only once, in autologous plasma from cord blood, and re-expanded before their use at day 0 of HSC transplantation.
Moreover, 17 children and adolescents with SAA were treated with haploidentical HSCT after myeloablative conditioning and coinfusion of culture-expanded third-party donor-derived WJ-MSCs

| GVHD treatment
The first phase 1 trial published with WJ-MSC administration in the treatment of acute GVHD was performed in the USA. WJ-MSCs were used immediately after thawing but the culture conditions were not described. Ten patients with de novo high risk or steroid-refractory acute GVHD received WJ-MSCs intravenously on days 0 and 7 (low-dose cohort, 2 × 10 6 /kg, n = 5; high-dose cohort, 10 × 10 6 /kg, n = 5). No infusion-related toxicity, treatmentrelated adverse events, or ectopic tissue formation was observed in either cohort. Clinical response was suggested at day 28, as the overall response rate (ORR) was 70%, with a complete response in

| GVHD prophylaxis
In

| WJ -MSC S MODUL ATE IMMUNE RECONS TITUTION AF TER TR AN S PL ANTATI ON
Only a few studies have sought to determine the impact of WJ-MSC co-injection on immune reconstitution during the few months following allo-HSCT. 53 In a controlled, randomized multicentre study, four monthly injections of 3 × 10 7 WJ-MSCs after haploidentical HSCT for chronic GVHD prevention led to a decrease in total NK cells.
Although the numbers of CD3 + CD4 + cells did not differ significantly between the two groups, the number of CD4 + CD25 + CD127 low regulatory T (Treg) cells in the MSC group was higher than that in the non-MSC group. And although the numbers of CD19 + B cells were not significantly different, the CD27 + memory B-lymphocyte numbers were significantly increased after MSC infusion. Moreover, during the first month post HSCT, the Th1 (interferon γ): Th2 interleukin (IL)-4 + cell ratio increased. 53 Another study of WJ-MSC injections in 24 patients with refractory GVHD treatment showed that patients had lower levels of mature dendritic cells (CD83 + , CD86 + , and HLA-DR + cells) after MSC infusion (between days +14 and days +56) than before MSC infusion 54 (Table 1).

| DISCUSS ION
In this review we have presented the reasons why WJ-MSCs are attracting interest in haematology (summarized in Table 1). They have  Figure 1), since they may undergo polarization to either MSC1s or MSC2s according to the environment. 68 EVs do not have this plasticity, and the priming of WJ-MSCs with inflammatory cytokines should be considered before exosome collection in the field of GVHD. 69 Second, WJ-MSC phagocytosis by monocytes in vivo has been described as a key mechanism of immunomodulation, 45,59 and nothing is known about that phenomenon in regard to MSC-derived EVs.
Third, cell preparation is an important variable that must be better defined before WJ-MSC use in human therapy. For instance, since WJ-MSCs are banked, the impact of freezing and thawing these cells before use must be carefully studied. Some authors have noted that BM-MSCs need a few days of culture after thawing to recover their full immunological properties. [70][71][72][73] This was not confirmed in a preclinical pig model of septic shock where thawed human WJ-MSCs were immediately infused and provided significant clinical and biological improvements. 74 The culture medium 75 and culture atmospheric conditions are also correlated with WJ-MSC functions. 19 Hypoxia has been described as a favourable condition in maintaining HSC stemness, 76 but its impact on WJ-MSC immune properties is not well characterized to date. The only published means of enhancing their immune capacities is to licence WJ-MSCs with pro-inflammatory cytokines , 77 for instance IFNγ, which improves their function in preventing GVHD in mouse models. 49,78 In the haematopoietic support setting as well, the cell preparation protocol appears to be of great importance to full WJ-MSC potency. Threedimensional culture of MSCs with HSCs enhances the expansion of cord blood CD34 + cells. 79 Ex vivo expansion of HSCs without eliminating the long-term repopulating capacity of more primitive HSCs is more feasible when haematopoietic niches are mimicked. 80 In this niche, cell contact between WJ-MSCs and HSCs seems to be preferable. 34 The surface structure of the microenvironment has also been shown to modify the cytokine secretion profile of MSCs. 81 Even the stiffness of polydimethylsiloxane substrates for BM-MSC culture can lead to a change in HSPC phenotype. 82  in Belgium, NCT02032446 in Italy, NCT03847844 in Malaysia and NCT04738981 and NCT04213248 in China).

ACK N OWLED G EM ENTS
Not applicable.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Not applicable.