Mesenchymal stromal cells in the treatment of pediatric hematopoietic cell transplantation-related complications (graft vs. host disease, hemorrhagic cystitis, graft failure and poor graft function): a single center experience

Objectives To describe mesenchymal stromal cells (MSCs) in the treatment of hematopoietic stem cell transplantation (HSCT) complications and to assess its safety and efficacy. Methods Single-center retrospective study (2016–2023). Patients under 20 years who received MSCs for the treatment of HSCT-related complications were included. Results Thirty patients (53.7% boys), median age at transplant 11 years (range 2–19) were included. MSCs indications were: graft-vs.-host disease (GVHD) in 18 patients (60%), of them 13 had acute GVHD (43.3%) and 5 chronic GVHD (16.7%); Grade 3–4 hemorrhagic cystitis (HC) in 4 (13.3%); poor graft function (PGF) in 6 (20%), 5 of them receiving MSCs with a CD34 stem cell-boost coinfusion; graft failure (GF) in 2 (6.7%), to enhance engraftment after a subsequent HSCT. Infusion-related-adverse-events were not reported. Overall response (OR) was 83% (25/30); 44% of responders (11/25) showed complete response (CR). OR for GVHD, HC, PGF and GF was 83.3%, 100%, 66.7% and 100% respectively. Response rate was 40% (95% CI: 20–55) and 79% (95% CI: 57–89) at 15 and 30 days. With a median follow-up of 21 months (IQR11–52.5), overall survival (OS) was 86% (95% CI: 74–100) and 79% (95% CI: 65–95) at 6 and 12 months post-MSCs infusion. Conclusion In our study, the most frequent indication of MSCs was refractory aGVHD (43.3%). Response rates were high (OR 83%) and safety profile was good.


Introduction
Advanced Therapy Medicinal Products (ATMP) represent a new category of medicines with a wide therapeutic potential for treating different types of hereditary and acquired diseases.ATMP encompass gene therapy, somatic cell therapy (SCT), and tissue-engineered products (1,2).SCT refers to autologous or allogeneic cellular material that has been manipulated to change their biological characteristics and then is transferred into a patient for medical purposes (3)(4)(5).One type of SCT are mesenchymal stromal cells (MSCs).MSCs are multipotent, non-hematopoietic stem cells able to differentiate into various cell types, such as adipocytes, osteocytes, chondrocytes, and cells present in other connective tissues (6,7).MSCs, which are present in adult and fetal tissues, can be derived from numerous sources such as adult bone marrow (BM), umbilical cord blood (UCB) or adipose tissue (8).However, BM and UCB are the most common sources for clinical use (9,10).MSCs exhibit plasticity, self-renewal, immunomodulation, and anti-inflammatory properties.For example, they are able to secrete cytokines and growth factors at sites of tissue injury and inflammation or induce immunosuppressive effects by direct cell-to-cell interaction and paracrine signalling (11,12).Additionally, they play an essential role in the BM niche, as they have the ability to support hematopoietic stem cell survival and proliferation (6).These properties have prompted their clinical use in the setting of regenerative medicine and hematopoietic stem cell transplantation (HSCT) (7,8,10).

Study aims
The aim of this study was to describe the clinical use of allogenic MSCs in the treatment of HSCTs complications (GVHD, HC, GF or PGF) in the real-world practice.
The secondary objectives were to determine: -The safety profile of MSCs during and immediately after infusion [infusion-related adverse events (AE)].-The efficacy of MSCs therapy in terms of response rate [overall response (OR), complete response (CR), partial response (PR), probability of response, no response (NR)] and overall survival (OS).

Study design and inclusion criteria
Retrospective descriptive study between January 2016 and January 2023.Patients under 20 years of age who had received MSCs for the treatment of the above-mentioned HSCT-related complications were included.They all had at least 3 months of follow-up after MSCs infusions.All patients were treated in a single-center Pediatric Bone Marrow Transplant Unit.Neither patient received MSCs within a clinical trial, but off label.Given that the efficacy of MSCs in the transplantation setting is still unclear, they received MSCs when first line-therapies failed, which is the standard of care in our center.
For acute and chronic GVHD, MSCs were used in patients diagnosed with steroid-refractory (SR) GVHD after at least two previous lines of treatment.During MSCs treatment, previous immunosuppressive therapies were not discontinued, unless clinically indicated.
For patients with HC, MSCs therapy was indicated in case of grade 3 or 4 HC.As with GVHD, previous supportive therapies were not discontinued during MSCs treatment.
For patients diagnosed with PGF or GF, indication of MSC therapy was made based on the definitions below (24, 25).

Mesenchymal stromal cells
Non-crossed matched allogenic MSCs were used in all cases.MSCs doses were manufactured in Banc de Sang i Teixits (BST) Barcelona, Spain.The dose was the same for all indications.However, there could be a little variation of the viable cells/kg dose (between 0.7 and 1.3 × 10 6 ), which depended on the manufacturing and culture expansion process.In this study, the median dose that patients received was 1.1 viable cells/kg (range 0.8-1.3× 10 6 cells/kg).
Considering that we treated different HSCT-complications and a standardized dose and infusion schedule for MSC therapy has not yet been established the dosing and infusion schedule was based on previous publications (25)(26)(27)(28).MSCs infusion scheme for each post-HSCT complication is summarized in Table  Wharton's Jelly (WJ)-MSCs were derived, expanded and characterized following Good Manufacturing Practice (GMP)compliant procedures and appropriate donor informed consent as reported extensively elsewhere (30)(31)(32).Briefly, a fragment of umbilical cord tissue was cut longitudinally and the WJ was scrapped with a surgical scalpel, spread uniformly over the plastic surface of a T-flask with re-closable lid (TPP, Trasadingen, Switzerland), and incubated for 30 min at 37°C.After the incubation, 10 ml of Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) containing 2 mmol/L glutamine was added and supplemented with 2 × 10 4 UI/ml penicillin (Invitrogen, New York, NY, USA), 20 mg/ml streptomycin (Invitrogen), 120 µg/ml amphotericin B (Invitrogen), and 10%-20% human serum B (hSerB, Banc de Sang i Teixits, Barcelona, Spain).After 2-5 days, a washing step with saline solution was performed, and 10 ml of fresh medium was added.From this point, the culture medium was replaced every 3-4 days.Cells were further expanded in vitro by seeding cell culture flasks at (1-3) × 10 3 cell/cm 2 .When the total number of cells reached at least 5 × 10 6 , they were frozen in cryovials producing the master cell bank (MCB).Further expansion was performed after thawing for the generation of either working cell bank (WCB) or drug product (DP) directly.Final product (FP) was defined as the cell suspension resulting from thawing a DP, washed and conditioned for administration in patients.
Bone Marrow (BM)-MSCs were obtained under GMP conditions using a bioprocess that included a derivation step of MSC from BM aspirates and ex vivo expansion in an approximately 21-days.BM samples were harvested from the posterior iliac crest of donors and nucleated cells (NC) were isolated using an automated Sepax device (Biosafe) and Ficoll-Paque reagent (GE Healthcare).Then NC were washed and plated at 2 × 10 5 cells/cm 2 onto cell culture vessels (CellSTACK) with DMEM supplemented with 10%.All cultures were maintained at 37°C and 5% CO 2 in humidified incubators.The medium was changed every 3-4 days and at day 10, cells were trypsinized using 0.05% Trypsin/ethylenediaminetetraacetic acid (EDTA; Gibco, Life Technologies), when 70%-90% confluence was reached.Then cells were reseeded at 1 × 10 3 cells/cm 2 .Finally on day 21, cells were harvested and washed with a saline solution (Plasmalyte; Baxter) and were frozen in cryovials.
During the process, intermediate products and finally doses were analyzed to verify compliance with the established criteria according to GMP regulations (33).All MSC products tested negative for bacteria and Mycoplasma, and endotoxin levels were always below 0.5 EU/ml.

Definitions
Acute GVHD (aGVHD) was graded according to MAGIC criteria (34), which goes from 1 to 4, depending on the number of organs involved [skin, liver, lower gastrointestinal tract (GI) and upper GI] and its severity.Chronic GVHD (cGVHD) was graded according to the 2014 NIH criteria (35,36).The overall severity of cGVHD was classified as mild, moderate, or severe based on organ-specific grading.
Primary graft failure was defined as the lack of achievement of an absolute neutrophil count (ANC) 0.5≥ × 10 9 /L by day +28 after peripheral blood/bone marrow allo-HSCT and by day +42 after umbilical cord blood transplantation (24,25,38).

Response and survival
OR was defined as the proportion of patients who presented a PR or CR to MSC therapy.For GVHD CR was reached when the disease resolved completely and no other treatments after MSC infusion were needed.PR was defined as improvement in staging of at least one involved organ, grade or score (in case of cGVHD) and at least steady state on other symptoms (39).For GF, CR was achieved when ANC ≥0.5 × 10 9 /L was recovered.PGF CR was defined as ANC >1 × 10 9 /L, platelet count >30 × 10 9 /L, Hb >10 g/dl (20).Response in at least 1 hematopoietic cell

Safety assessment
Patients were monitored throughout the MSC infusion and 4 h after the procedure; during the infusion, non-invasive continuous cardiac and respiratory monitoring was performed, and vital signs were recorded hourly for up to 4 h after infusion.Premedication was not administered to any patient.
Since this was not a clinical trial, but an observational retrospective study, only AEs occurring during 4 h after MSCs infusion were considered, as this information was specifically documented in the patient clinical charts.However, patients' medical charts were carefully reviewed in search of other possible AEs during the follow-up period.

Statistical analyses
Statistical analyses were performed in SPSS (version 28.0).Significance was set at a p level of 0.05 (two-tailed).Qualitative results were expressed as absolute and relative frequencies and quantitative variables, as median and interquartile range (IQR) or range.To test if quantitative data was normally distributed, Shapiro-Wilk Test was used.Mann-Whitney U-test was applied to analyze if time from 1st MSCs infusion to OR was associated with better response (PR or CR).Kaplan Meier (KM) estimator was used to evaluate and graphically display OS.Cumulative incidence was used to estimate the probabilities of partial and complete response.Probabilities were reported as percent with a 95% confidence interval (CI).

Patient characteristics
Thirty patients (53.7% boys, 46.3% girls), median age at transplant 11 years (range 2-19) treated at a single institution were included.One patient was included twice (patient 2 Tables 2, 3), as she received MSCs for 2 different indications at separated time-periods; first she received two MSC infusion due to GF, and 2 months later, 3 weekly MSCs infusions were administered to treat aGVHD.

MSCs indications, characteristics and dosage
MSCs therapy indications and infusion scheme are summarized in Table 1.MSCs therapy and patients characteristic for each specific indication is summarized in Supplementary Table S1 (HC), Supplementary Table S2 (GF and PGF) and Table 2 (GVHD).There were two patients with GVHD that did not strictly followed the infusion scheme for his/her specific MSCs indication (patients 2 and 4, Table 2); patient 4 received 3 doses instead of 4, as he showed CR shortly after the second MSC infusion.Patient 2 received 2 MSCs doses, as she has previously got 2 MSCs infusion due to graft failure.
MSCs indications were: GVHD in 18 patients (60%), 13 of them had aGVHD and 5 cGVHD; Grade 3-4 HC in 4 patients (13.3%), all cases being due to BK virus; PGF in 6 patients (20%), of whom 5 were co-infused with a CD34 + stem cell boost; primary GF in 2 (6.7%), to enhance engraftment after a subsequent HSCT.All patients receiving a boost of CD34 cells were non-conditioned and the CD34 boost was administered on the same day of first MSC infusion.For both patients with GF, MSC first infusion was administered on the same day of the HSCT.The second MSC infusion was administered 2 weeks later (please see dosing schedule for patients with GF in Table 1).
Patient 1 (Supplementary Table S2) had previously undergone 3 allo-HSCT.Due to GF diagnosis, she received a 4th allo-HSCT from a different donor (unmanipulated haploidentical donor) and same stem cell source (peripheral blood).Conditioning consisted of fludarabine, cyclophosphamide, anti-thymocyte globulin and rituximab.Graft vs. host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate.Patient 2 (Supplementary Table S2) had undergone a previous allo-HSCT.After GF diagnosis, she received a second transplant from a different donor (mismatched unrelated donor, 9/10) and same source (peripheral blood).Conditioning consisted of fludarabine, cyclophosphamide and antithymocyte globulin.GVHD prophylaxis consisted of cyclosporine and mophetil mycophenolate (MMF).
Regarding previous lines of treatment, in case of GVHD, median lines were 4 (range 2-5).Of note, cyclosporine (CsA) was not considered a line of treatment, as it was part of GVHD prophylaxis during HSCT in all patients.MMF was neither considered a new line of treatment when referring to "previous lines of treatment" if patients had received it as GVHD prophylaxis (patients 2, 3, 8, 13 and 17).MSCs therapy for GVHD is summarized in Table 2.
Before the indication of MSCs therapy for PGF, one patient received a boost of donor CD34 + cells 50 days before first MSC infusion and another was treated with Eltrombopag but none of them responded.Eltrombopag was discontinued one month before MSCs infusion.

Safety
No patients experienced adverse events during or immediately after MSC infusion.No other AEs were retrospectively associated with MSCs therapy during the follow-up period.
Overall 7 patients (24%) died, but in none of them death was related to MSC therapy.No deaths were observed in the CR group (Figure 3).Causes of death are summarized in Table 4.

Discussion
In the present study, we describe MSCs as a therapy for HSCTrelated complications in a series of 30 pediatric patients.The most frequent indication was refractory aGVHD (43.3%, n = 13/30).In
Regarding GVHD, clinical studies of MSC therapy in patients with SR-aGVHD have demonstrated favorable clinical response rates with an acceptable safety profile (40)(41)(42)(43)(44)(45).For example, Kurtzberg et al. (46) recently reported on 241 children with grade II-IV SR-aGVHD who were on additional second-line treatments.The 28-day OR rate was 65% with a 14% CR.The 100-day OR was not assessed.In another study (47), the same group analyzed 54 children with primary SR-aGVHD who were naive to other immunosuppressant therapies and were treated with MSC product (remestemcel-L).A 70.4% OR was achieved, and response was sustained through day 100, including an increase in CR from 29.6% at day 28 to 44.4% at day 100.In the present study, patients with aGVHD had comparable response rates, with 84% OR and 36.4% CR.Nonetheless, 20% of all patients (4 of them with aGVHD) presented loss of response.Of note, two patients who received MSCs due to aGVHD developed severe cGVHD and died of respiratory failure.Although they had previously shown PR to MSCs, they were certainly at risk of developing cGVHD, since both of them had prior severe aGVHD (grade III and IV) (48).As opposed to the 2nd study of Kurtzberg et al. (42), patient population in our study were highly pretreated and refractory to previous treatments, which might have worsened response.Some authors (42,44,49) have also showed that, when MSCs were applied in an earlier stage of SR-aGVHD, the CR rates were higher.For example, Le Blanc et al. (42), in their study of a group of SR grade II-IV patients, described 55% CR and 16% PR.For that reason, taking into account the side effects of second line immunosuppression for GVHD and the good safety profile of MSCs (even in severely affected children), earlier use of MSCs therapy could be considered for patients with acute SR-GVHD.
Compared to aGVHD, there is limited use of MSCs therapy in cGVHD, probably because, in many cases, the chronic fibrotic processes of cGVHD are irreversible (49).In our study, MSCs therapy was more effective in aGVHD than in cGVHD (84% OR with 36.4% CR vs. 80% OR with no CR).Similar to previous report (49)(50)(51)(52), most patients showed response, although only PR were observed.
Another indication for MSC is HC (22, 53, 54).Although there is limited clinical experience and it is mainly used in adults, some pediatric studies have shown promising results; for example, Tong et al. ( 22) reported thirteen pediatric patients with severe BKV-HC (grade 3-4) who presented 100% OR with rapid symptoms improvement.These results are consistent with our series, where OR was 100% (50% CR) and no loss of response was observed.
MSCs have also been used to reverse graft failure and enhance engraftment both in children and adults (18-21, 55, 56).In a meta-analysis by Li et al. (18), studies comparing MSC cotransplantation in allo-HSCT with allo-HSCT alone where analyzed (19 clinical trials), showing that MSC co-infusion generally improved engraftment without increasing mortality or relapse.In another study by Servais et al.,(20).MSC were administered as a single i.v.infusion at a dose of 1-2 million(s) cells/kg body weight in patients with PGF.Within 90 days post-MSC infusion, OR was 53% with 37% CR, observing a response rate increase to 67% OR and 53% CR within 180 days after MSC infusion.In our series, all patients with GF and MSC cotransplantation presented CR.Compared to Servais et al., most Kaplan-Meier curves by type of response.This graph represents the survival time (in months) from 1st MSC infusion, in relation to each type of treatment response.For patients with NR, OS was 53% (95% CI: 21-100) at 6 months of follow-up onwards.In the PR group, OS was 80% (95% CI: 60-100) at 6 months of follow-up and 60% (95% CI: 36-100) from 12 months onwards.Since no death were observed in the CR group, the survival probability was 100%.Kaplan-Meier plot of overall survival (OS) or probability of survival.The x-axis is months of follow up after the first MSC infusion until death occurs, while the y-axis is the proportion of subjects surviving.OS was 90% at 3 months (95% CI: 79-100), 86% at 6 months (95% CI: 74-100) and 79% at one year (95% CI: 65-95).cases of PGF received a CD34 + cells co-infusion, observing response rates were higher (OR 66% with 75% CR) and more rapid [26 days from first MSC infusion (range 13-34)].Certainly, the CD34 + boost or the subsequent HSCT have most probably improved response rates in our study.Therefore, more evidence is needed to confirm the real impact of MSCs on engraftment and graft function, alone (in case of PGF) or as a co-adjuvant therapy.Regarding survival analysis, and according to literature, OS rates varies between 50 and 80%, depending on MSCs indication and time-point of analysis, among others (18,20,22,46,47).In our study, OS was 90% at 3 months (95% CI: 79-100) and 79% at one year (95% CI: 65-95).Notably, similar to Kurtzberg et al. (47), we observed survival was higher in responders compared with non-responders [none of the responders died while, in the non-responder's group, OS was 53% (95% CI: 21-100) at 6 months of follow-up onwards].For that reason, especially in the case of aGVHD (the most frequent MSCs indication in our study), considering that early use of MSCs seems to improve response and consequently, survival, with no identified safety concerns (even in gravely ill children such as our cohort), we believe that, despite possibly being a promising therapeutic tool, there is certainly room for improvement in the use of MSCs.
Regarding the causes of death (Table 4), three patients died of respiratory failure and alveolar hemorrhage.We believe these deaths were not related to MSCs therapy since MSCs infusions occurred more than 6 months before either death (7, 17 and 54 months in patients 2, 7 and 4 respectively).Additionally, all these patients had previous risk factors of pulmonary bleeding.For example, the patient with dyskeratosis congenital (patient 7, Table 4) maintained platelet count between 30 and 50.000 and developed empyema and pulmonary hemorrhage after chest tube drainage, which prompted death.The other two patients (one with beta-thalassemia and the other with B-acute lymphoblastic leukemia) developed severe cytomegalovirus pneumonitis and diffuse alveolar hemorrhage as a consequence, which led to death.
Limitations of our study stem in the small number of patients, the sample heterogeneity with variable follow-up periods, the retrospective design of the study, the lack of control group against which to assess the efficacy and safety of MSCs and the fact that it was single-center.Additionally, the possibility that some patients might have responded to other concomitant therapies rather than MSCs cannot be excluded.There are some confounding factors when comparing results between studies that we should also acknowledge, such as variability in MSCs donor types, MSCs dose per infusion, and number of infusions per patient-even within the same trial in some instances.
In conclusion, our study supports that MSCs seem to be a safe and effective therapy in the treatment of pediatric patients with HSCT-related complications.Further adequately powered prospective studies and clinical trials are required to confirm efficacy and establish the place and infusion schedule of MSCs therapy in each specific HSCT-related complication.

Maximun response to MSCs
Patient's clinical situation with respect to the indication that led to MSC therapy at last FU Cause of death/months after last MSCs infusion

Funding
The author(s) declare no financial support was received for the research, authorship, and/or publication of this article.
Anonymized clinical data were collected from the clinical records at Vall d'Hebron Hospital (VHH).Patients and/or legal tutors gave written consent for the transplantation procedure and to record patient´s anonymized transplantation-related data for studies.Legal tutors and/or patients signed the inform consent to receive compassionate use MSCs therapy.Good clinical practice according to Helsinki Declaration were applied.The study was approved by the Ethics Committee of VHH.

TABLE 1
MSCs therapy indications and infusion scheme.Pérez-Torres Lobato et al. 10.3389/fped.2024.1375493lineage was considered as PR.For HC, CR was achieved when gross hematuria disappeared.If it persisted but improved in at least one grade, it was was considered PR (38).Overall response, overall survival and event-free survival were calculated from first MSC infusion.

TABLE 3
Evaluation of response to MSC therapy according to the indication of MSCs.
a Also referred as patient 2 in