Phase I clinical trial of intra‐bone marrow cotransplantation of mesenchymal stem cells in cord blood transplantation

Abstract Mesenchymal stem cells (MSCs) have immunomodulatory properties and support hematopoiesis in the bone marrow (BM). To develop a new strategy to not only prevent graft‐vs‐host disease (GVHD) but also to enhance engraftment, a phase I trial of cord blood transplantation (CBT) combined with intra‐BM injection of MSCs (MSC‐CBT) was designed. Third‐party BM‐derived MSCs were injected intra‐BM on the day of CBT. The conditioning regimen varied according to patient characteristics. GVHD prophylaxis was tacrolimus and methotrexate. The primary endpoint was toxicity related to intra‐BM injection of MSCs. Clinical outcomes were compared with those of six controls who received CBT alone. Five adult patients received MSC‐CBT, and no adverse events related to intra‐BM injection of MSCs were observed. All patients achieved neutrophil, reticulocyte, and platelet recoveries, with median times to recoveries of 21, 35, and 38 days, respectively, comparable with controls. Grade II‐IV acute GVHD developed in three controls but not in MSC‐CBT patients. No patients developed chronic GVHD in both groups. At 1 year after transplantation, all MSC‐CBT patients survived without relapse. This study shows the safety of MSC‐CBT, and the findings also suggest that cotransplantation of MSCs may prevent GVHD with no inhibition of engraftment. This trial was registered at the University Hospital Medical Information Network Clinical Trials Registry as number 000024291.


| INTRODUCTION
Cord blood transplantation (CBT) is a curative treatment for various hematologic disorders. Previous studies have reported comparable outcomes of CBT and human leukocyte antigen (HLA)-matched unrelated donor transplantation in adult patients. 1,2 The incidence of chronic graft-vs-host disease (GVHD) is reported to be lower in CBT than in bone marrow transplant (BMT) and peripheral-blood stem-cell transplant (PBSCT); however, the incidence of acute GVHD in CBT is comparable with the other transplants. 1,[3][4][5] In addition to a relatively high incidence of acute GVHD, delayed hematologic recovery and a higher rate of graft failure after CBT lead to an increased risk of transplantrelated mortality in the early period after transplantation. 3,6 Several strategies, including double-unit CBT, 7 ex vivo expansion of cord blood (CB)-derived CD34+ cells, [8][9][10][11][12] and intra-bone marrow (BM) transplantation of CB cells, 13,14 have been explored in an effort to overcome these obstacles. In addition to these approaches, cotransplantation of CB and mesenchymal stem cells (MSCs) has been reported. [15][16][17][18] MSCs are a heterogeneous population of stromal stem cells that can be isolated from many tissues, such as BM, adipose tissue, CB, and placenta. MSCs have the capacity for self-renewal and can differentiate into mesodermal lineage cells. 19 In the BM, MSCs differentiate into BM stroma cells, osteocytes, osteoblasts, and endothelial cells.
These cells contribute to the formation of the BM microenvironment, known as the hematopoietic stem cell (HSC) niche, and they support hematopoiesis. 20,21 Besides this hematopoietic support capacity, MSCs can modulate immune responses by a cell-cell contact mechanism between MSCs and their target cells and by producing several soluble immunosuppressive factors. 19,22 These immunomodulatory effects of MSCs have already been clinically applied in the treatment of GVHD after allogeneic hematopoietic cell transplantation (HCT). [23][24][25][26][27] MSCs express a low level of HLA class I and are negative for HLA class II and costimulatory molecules such as CD80, CD86, and CD40, and, therefore, they are able to evade allogeneic rejection. 28,29 Furthermore, culture expansion of MSCs is relatively easy, and they can be stored by cryopreservation. Therefore, ex vivo expanded and cryopreserved MSCs derived from a third-party donor can be used for clinical treatment without considering HLA matching between patient and donor. Because of these properties, MSCs have been explored for enhancing engraftment and preventing GVHD after allogeneic HCT.
In previous clinical studies, the feasibility and safety of CBT with intravenous cotransplantation of MSCs were observed in pediatric patients. [15][16][17][18] However, to date, the cotransplantation of MSCs and CB cells has yet to be evaluated in adult patients, who have a greater risk of graft failure because of a lower CB cell dose per patient body weight. Concern regarding the route of MSC administration remains an issue because several animal model experiments have demonstrated that MSCs infused intravenously were trapped in lung, 30,31 and direct intra-BM injection of MSCs could enhance the engraftment of transplanted CB cells more than intravenous injection. 32 Additionally, intra-BM injection of MSCs has been reported to be safe in previous clinical studies. 33,34 Based on these properties of MSCs and experimental and clinical findings, to develop a new strategy not only to enhance engraftment but also to prevent GVHD after CBT, a phase I trial of CBT combined with intra-BM injection of ex vivo expanded MSCs (MSC-CBT) was designed. 35 The aim was to assess the safety of this treatment in adult patients with hematologic disorders.

| MATERIALS AND METHODS
The study protocol has been described in detail previously. 35 This study was registered with the University Hospital Medical Information Network Clinical Trials Registry (number 000024291).

Significance statement
Mesenchymal stem cells (MSCs), which were derived from the bone marrow of third-party donors, were injected into the bone marrow of the recipient 4 hours before cord blood transplantation. This study showed the safety of cord blood transplantation combined with intra-bone marrow injection of MSCs and also suggested that cotransplantation of MSCs may prevent graft-vs-host disease without inhibition of engraftment. This strategy may be applicable not only to cord blood transplantation but also to bone marrow transplantation or peripheral blood stem cell transplantation, leading to the prevention of severe graft-vs-host disease, especially in human leukocyte antigen-mismatched settings, and reduction of the burden imposed on hematopoietic stem cell donors by decreasing the required stem cell number.

Lessons learned
• Mesenchymal stem cells (MSCs) support hematopoiesis in the bone marrow and have immunomodulatory properties.
• MSCs have potencies to enhance engraftment and to prevent graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation.
• This study shows the safety of intra-bone marrow cotransplantation of MSCs in cord blood transplantation.
• All patients achieved engraftment without clinically important GVHD.
• Co-transplantation of MSCs may prevent GVHD without inhibition of engraftment. This was a single arm, nonrandomized, open-label, single-center, phase I trial at Nagoya University Hospital. The target sample size was five patients. Eligible patients were aged 20 years or older; had a hematologic disorder with an indication for CBT; did not have malignant cells accounting for 70% or more of all nucleated cells in the BM;

| Study design and participants
had an Eastern Cooperative Oncology Group performance status of 0-2; had adequate organ function as defined by an ejection fraction of 40% or greater, forced vital capacity of 50% or greater, forced expiratory volume in 1 second of 60% or greater, aspartate aminotransferase and alanine aminotransferase concentrations less than 5 times the upper limit of normal, and serum creatinine less than 3 times the upper limit of normal; and they had available CB units with serological HLA-A, B, and DR ≥4/6 matched and with a total nucleated cell (TNC) dose of 1.5 × 10 7 cells per kg or higher. Additionally, patients had to have at least one potential MSC donor aged 20-74 years from a spouse or relative within the fourth degree of relationship.
The inclusion and exclusion criteria of the MSC donors are listed in the study protocol in detail. 35 Patients were excluded if they had history of allogeneic HCT in the 1 year prior to enrollment, exposure to gemtuzumab ozogamicin in the 6 months prior to enrollment, and allergy to the drugs used for Patients and donors were registered in this study after independent review by the data center in the Department of Hematology and Oncology, Nagoya University Graduate School of Medicine.

| Preparation of MSCs
Human platelet lysates were prepared from single-donor platelet concentrate provided by the Japan Red Cross Blood Center by the Application for the use of blood donated in Japan based on the "Guidelines on the use of donated blood in R&D, etc." Platelet concentrate was frozen at −30 C and thawed twice and then stored at −30 C. The frozen platelet concentrate was thawed at 4 C and centrifuged to obtain supernatant as platelet lysate.

| Conditioning regimen and GVHD prophylaxis
The conditioning regimen was not defined in this study. in vivo purging of T cells using treatments such as anti-thymocyte globulin was prohibited. GVHD prophylaxis consisted of the combination of tacrolimus and short-term methotrexate.

| Cotransplantation of MSCs and CB cells
On the day of CBT, MSCs were thawed, washed, and resuspended in 2-10 mL of a saline solution. Premedication with hydrocortisone 100 mg and chlorpheniramine 10 mg was administered approximately 30 minutes before injection of MSCs. After local anesthesia, a standard BM aspiration needle was inserted into the iliac bone on one side. To ensure that the needle was securely inserted into the BM cavity, aspiration of <0.5 mL BM was done. Then, approximately 5 mL of MSC suspension were injected slowly. This procedure was repeated on the iliac bone on the contralateral side. Four hours after MSC injection, CB was infused intravenously with the standard procedure. Granulocyte colony-stimulating factor (G-CSF) was administered from 7 days after transplant to neutrophil engraftment.

| Follow-up and assessment
Adverse events were graded by Common Terminology Criteria for Adverse Events version 4.0. Safety was assessed by monitoring and recording of all adverse events and serious adverse events. The study was monitored by an independent data and safety monitoring committee, and serious adverse events were reviewed and judged to determine whether an adverse event was attributable to treatment.
Periodic monitoring was done according to the Japanese clinical trial guideline at least annually. The study stopping rules included graft failure, transplant-related mortality before engraftment, or grade 4-5 adverse event in three patients.
Patients had routine clinical assessments and laboratory investigations such as blood cell counts, tacrolimus levels, and cytomegalovirus antigenemia. Patients were planned to be followed up for at least 1 year after transplant or less if they satisfied one of the discontinuation criteria. Chimerism analyses were done at the National Hospital Organization Nagoya Medical Center using short tandem repeats by PCR assay in peripheral CD3+ T cells on days 14, 28, and 56 after transplantation as previously described. 36  To evaluate cytokine and chemokine kinetics, serum concentrations of IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17,   IL-21, IFN-γ, tumor

| Outcomes
The primary endpoint of this study was toxicity related to intra-BM injection of MSCs within 14 days after transplantation, which was defined as adverse events that could not be explained by other complications, such as regimen-related toxicity or infection, that generally occur after transplantation. Secondary endpoints included the rate of engraftment, the time to hematopoietic recoveries, the incidences and severities of acute and chronic GVHD, the incidences of regimenrelated toxicities and infection, and the probabilities of nonrelapse mortality (NRM), relapse, disease-free survival, and overall survival at 1 year after transplantation.

| Control comparison
The study patients were compared with a control group of six patients who received CBT without MSCs during the same time period, between May 2017 and May 2018, in Nagoya University Hospital with respect to hematopoietic recoveries, clinical outcomes, lymphocyte subsets, and cytokine/chemokine kinetics. The control patients did not join this study because of patient decisions (n = 1), not enough time to prepare MSCs (n = 1), the difficulty of BM aspiration due to BM fibrosis (n = 1), not meeting the criteria for release of MSCs (n = 1), or the close of registration for this study (n = 2).

| Definitions and statistical analysis
Engraftment was defined as neutrophil recovery to greater than 0.5 × 10 9 /L for 3 consecutive days. The time to neutrophil engraftment was defined as the first day of achieving an absolute neutrophil count greater than 0.5 × 10 9 /L for 3 consecutive days. The times to platelet and reticulocyte recoveries were defined as the first days of achieving a platelet count greater than 20 × 10 9 /L or 50 × 10 9 /L and a reticulocyte count greater than 1% for 3 consecutive days without transfusions. Primary graft failure was defined as lack of neutrophil engraftment in patients surviving at least 60 days, and secondary graft failure was defined as neutrophil engraftment followed by a decline in the neutrophil count to less than 0.5 × 10 9 /L for 3 consecutive days.
Acute GVHD was diagnosed and graded according to the consensus T A B L E 2 Characteristics of patients and outcomes of cord blood transplantation combined with intra-bone marrow injection of MSCs

| Characteristics of patients, grafts, and MSC donors
Between February 2017 and June 2018, six patients were enrolled in this study, but one patient did not receive protocol treatment because the MSC product did not meet release criteria because of insufficient cell counts (data of MSC expansion are summarized in Table 1). The characteristics and outcomes of five patients who received MSC-CBT are summarized in

| Adverse events
No adverse events related to intra-BM injection of MSCs, including swelling and prolonged pain at the injection site, embolism, and osteomyelitis, within 14 days after transplantation (primary endpoint) were observed. Regimen-related toxicities within 28 days after transplantation are summarized in Table 3

| GVHD and survival
Only one patient experienced transient grade I acute GVHD of the skin, which improved without systemic immunosuppressive treatment. No patient developed grade II-IV acute GVHD ( Figure 1E), and no patient developed chronic GVHD. All patients were alive without relapse at 1 year after transplantation.

| Control comparison
The control group consisted of six adult patients who received CBT without MSC during the same time period at our institution (Table S2).
The comparisons of patient characteristics and transplant outcomes between MSC-CBT patients and controls are summarized in Table 4. There was no significant difference between patients who received MSC-CBT and controls in terms of patient characteristics.
One control patient died with relapse of leukemia without achievement of platelet recovery (Table S2). The cumulative incidences of neutrophil, reticulocyte, and platelet recoveries were similar in the two groups ( Figure 1A-D). For those who achieved hematopoietic recovery, the median time to neutrophil, reticulocyte, and platelet recoveries was not significantly different between the two groups (Table 4). Grade II-IV acute GVHD developed in three controls (50%); however, there was no grade II-IV acute GVHD in MSC-CBT patients. The cumulative incidence of grade II-IV acute GVHD was significantly lower in MSC-CBT patients compared with controls ( Figure 1E). Chronic GVHD did not develop in both groups. The incidence of relapse, transplant-related mortality, and overall survival were not significantly different between the two groups.

| Lymphocyte subset and cytokine/chemokine analysis
Flow cytometry analysis was performed to compare lymphocyte reconstitution after transplantation between MSC-CBT patients and controls. There were no significant differences in lymphocyte subsets at 28, 42, 56, and 84 days after transplantation between the two groups ( Figure S1 and Table S3). Cytometric bead array analysis was performed to detect changes in cytokine and chemokine production with the addition of intra-BM injection of MSCs. There were tendencies to decreases in IFN-γ, IL-1α, IL-2, IL-4, and IL-21 levels within 28 days after transplantation in MSC-CBT patients compared with controls ( Figure S2).

| DISCUSSION
The present study showed the safety of CBT combined with intra-BM injection of MSCs for adult patients with hematologic disorders.
Although several previous clinical studies had shown the safety and feasibility of cotransplantation of MSCs, [15][16][17][18][41][42][43][44][45][46] MSCs were intravenously infused in all except one study, 33  In the present study, the engraftment of cotransplanted MSCs was analyzed by chimerism analysis using short tandem repeats with a  32 This report also showed that significantly higher engraftment of CB cells was observed in not only BM into which MSCs were injected, but also the contralateral side BM without direct injection of MSCs. 32  In adult patients undergoing CBT in Japan, the incidences of grade II-IV and grade III-IV acute GVHD were reported to be 13%-41% and 8%-12%, respectively. 2,3,6 Although no development of grade II-IV acute GVHD in MSC-CBT patients suggests the potential that cotransplantation of MSCs may prevent GVHD, this should be confirmed by further studies, and a phase II trial to evaluate the efficacy of this strategy is now being planned.

| CONCLUSION
CBT combined with intra-BM injection of MSCs was found to be a safe and feasible therapeutic strategy. Furthermore, the present findings suggest the potential that intra-BM injection of MSCs may pre-

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author.