Infusion of haploidentical hematopoietic stem cells combined with mesenchymal stem cells for treatment of severe aplastic anemia in adult patients yields curative effects.

BACKGROUND AIMS: Despite the great advances in immunosuppressive therapy for severe aplastic anemia (SAA), most patients are not completely cured. Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) has been recommended as an alternative treatment in adult SAA patients. However, haplo-HSCT presents a higher incidence of graft failure and graft-versus-host disease (GVHD). The authors designed a combination of haplo-HSCT and umbilical cord-derived mesenchymal stem cells (UC-MSCs) for treatment of SAA in adult patients and evaluated its effects. METHODS: Adult patients (≥18 years) with SAA (N = 25) were given HLA-haploidentical hematopoietic stem cells (HSCs) combined with UC-MSCs after a conditioning regimen consisting of busulfan, cyclophosphamide, fludarabine and anti-thymocyte globulin and intensive GVHD prophylaxis, including cyclosporine, basiliximab, mycophenolate mofetil and short-term methotrexate. Additionally, the effects of the protocol in adult SSA patients were compared with those observed in juvenile SAA patients (N = 75). RESULTS: All patients achieved myeloid engraftment after haplo-HSCT at a median of 16.12 days (range, 11-26). The median time of platelet engraftment was 28.30 days (range, 13-143). The cumulative incidence of grade II acute GVHD (aGVHD) at day +100 was 32.00 ± 0.91%. No one had grade III-IV aGVHD at day +100. The cumulative incidence of total chronic GVHD was 28.00 ± 0.85%. The overall survival was 71.78 ± 9.05% at a median follow-up of 42.08 months (range, 2.67-104). Promisingly, the protocol yielded a similar curative effect in both young and adult SAA patients. CONCLUSIONS: The authors' data suggest that co-transplantation of HLA-haploidentical HSCs and UC-MSCs may provide an effective and safe treatment for adult SAA.

Successful desensitization of high level donor-specific anti-HLA antibody in patients with hematological diseases receiving haploidentical allografts.

Donor-specific anti-human leukocyte antigen (HLA) antibody (DSA) is associated with a higher incidence of graft failure and mortality in HLA-mismatched allograft settings. However, the optimal protocol of desensitization for patients with positive DSA remains uncertain. We investigated the effectiveness of a desensitization protocol, including rituximab, high-dose intravenous immunoglobulin (IVIG), and a single session of plasma exchange (PE), for haploidentical allograft recipients with a high mean fluorescence intensity (MFI) level of DSA (≥ 5,000). Eleven patients with hematological disease who had positive DSA (median, 11,676, range 5387-20,435) were desensitized by the protocol. All of the patients achieved hematopoietic recovery. The median times for neutrophil and platelet engraftment were 13 (range, 11-26) days and 19 (range, 11-90) days, respectively. Grade II-IV acute graft-versus-host disease (GVHD) was seen in one patient and was controlled completely. Chronic cutaneous GVHD was seen in eight patients. Nine patients are alive with good performance so far. One patient suffered extramedullary relapse, and one patient died of transplantation-associated thrombotic microangiopathy. The 1-year probability of overall survival was 81.8%. These results suggest that successful desensitization could be obtained by a combination of rituximab, high-dose IVIG, and PE for haploidentical allograft recipients with high MFI levels of DSA.

Exosomes originating from MSCs stimulated with TGF-ß and IFN-γ promote Treg differentiation.

Mesenchymal stem cells (MSCs) have been approved as a cellular drug for the treatment of a variety of immune-related diseases by the government of many countries'. Previous investigations, including ours, have shown that exosomes secreted by MSCs (MSC-ex) are one of the main factors responsible for the therapeutic effect of MSCs. However, the immune modulation activities and the contents of MSC-ex derived from cells under different incubation conditions differ dramatically. Therefore, the optimal way to ensure effectiveness is by identifying and preparing MSC-ex with confirmed potent immunosuppressive activity. The aim of this study was to investigate and analyze the composition and function of MSC-ex secreted by MSCs stimulated by different cytokines to obtain exosomes with more potent immunosuppressive activity. To achieve this aim, umbilical cord-derived MSCs were treated with PBS, TGF-ß, IFN-γ, or TGF-ß plus IFN-γ for 72 hr. Then, exosomes were isolated from the culture supernatants. Common exosome markers, such as CD9, CD63, and CD81, were detected and analyzed by FCM. At the same time, the TGF-ß, IFN-γ, IDO, and IL-10 content in exosomes was detected, and the influence of exosmes from defferent groups on the induction of mononuclear cell transformation into Tregs was analyzed via FCM. Our results show that the TGF-ß combined with IFN-γ exosome group more effectively promoted the transformation of mononuclear cells to Tregs, and the analysis showed that IDO may play an important role. This study might provide a novel strategy to treat GVHD as well as other immune-associated disorders.

miR-17-92 promotes leukemogenesis in chronic myeloid leukemia via targeting A20 and activation of NF-κB signaling.

miR-17-92 cluster are overexpressed in hematological malignancies including chronic myeloid leukemia (CML). However, their roles and mechanisms that regulate BCR-ABL induced leukemogenesis remain unclear. In this study, we demonstrated that genomic depletion of miR-17-92 inhibited the BCR-ABL induced leukemogenesis by using a mouse model of transplantation of BCR-ABL transduced hematopoietic stem cells. Furthermore, we identified that miR-19b targeted A20 (TNFAIP3). A20 overexpression results in inactivation of NF-κB activity including decrease of phosphorylation of P65 and IκBα, leads to induce apoptosis and inhibit proliferation and cycle in CML CD34 + cells. Thus we proved that miR-17-92 is a critical contributor to CML leukemogenesis via targeting A20 and activation of NF-κB signaling. These findings indicate that miR-17-92 will be important resources for developing novel treatment strategies of CML and better understanding long-term disease control.

Hepatocyte growth factor enhances the inflammation-alleviating effect of umbilical cord-derived mesenchymal stromal cells in a bronchiolitis obliterans model.

BACKGROUND AIMS: Specific and effective therapy for prevention or reversal of bronchiolitis obliterans (BO) is lacking. In this study, we evaluated the therapeutic effect of hepatocyte growth factor (HGF) gene modified mesenchymal stromal cells (MSCs) on BO. METHODS: A mouse model of experimental BO was established by subcutaneously transplanting the tracheas from C57BL/6 mice into Balb/C recipients, which were then administered saline, Ad-HGF-modified human umbilical cord-MSCs (MSCs-HGF) or Ad-Null-modified MSCs (MSCs-Null). The therapeutic effects of MSCs-Null and MSCs-HGF were evaluated by using fluorescence-activated cell sorting (FACS) for lymphocyte immunophenotype of spleen, enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (rt-PCR) for cytokine expression, and histopathological analysis for the transplanted trachea. RESULTS: The histopathologic recovery of allograft tracheas was improved significantly after MSCs-Null and MSCs-HGF treatment and the beneficial effects were particularly observed in MSCs-HGF-treated mice. Furthermore, the allo-transplantation-induced immunophenotype disorders of the spleen, including regulatory T (Treg), T helper (Th)1, Th2 and Th17, were attenuated in both cell-treated groups. MSCs-HGF treatment reduced expression and secretion of inflammation cytokines interferon-gamma (IFN-γ), and increased expression of anti-inflammatory cytokine interleukin (IL)-4 and IL-10. It also decreased the expression level of the profibrosis factor transforming growth factor (TGF)-ß. CONCLUSION: Treatment of BO with HGF gene modified MSCs results in reduction of local inflammation and promotion in recovery of allograft trachea histopathology. These findings might provide an effective therapeutic strategy for BO.

Comparison of Clinical Efficacy of Cytarabine with Different Regimens in Postremission Consolidation Therapy for Adult t(8;21) AML Patients: A Multicenter Retrospective Study in China.

BACKGROUND: The survival of patients with acute myeloid leukemia (AML) with t(8;21) was reported to be shorter in China than in other countries. PATIENTS: We analyzed the correlation between different cytarabine (Ara-c) regimens and outcome in 255 t(8;21) AML patients in China who received postremission consolidation chemotherapy only. RESULTS: The 5-year overall survival (OS) of the high-dose Ara-c group (HDAC; 2≤ Ara-c ≤3 g/m2), intermediate-dose Ara-c group (MDAC; 1.0≤ Ara-c <2.0 g/m2), low-dose Ara-c group (LDAC; 0.2< Ara-c <1.0 g/m2) and standard-dose Ara-c group (SDAC; 0.1≤ Ara-c ≤0.2 g/m2) were 65.3, 39.4, 25.2 and 27.9%, respectively (p = 0.003). In the HDAC group, but not in the MDAC group, the 5-year OS of patients who achieved 3-4 cycles of chemotherapy was superior to those who underwent 1-2 cycles (84.4 vs. 43.6%, p < 0.05), and the 3-year OS of patients who achieved an accumulated 36 g/m2 of Ara-c was significantly higher compared to those who did not (85.3 vs. 39.2%, p < 0.05). Multivariate analysis indicated that factors such as WBC >3.5 × 109/l, PLT ≤30 × 109/l, and extramedullary infiltration were associated with a poor prognosis. CONCLUSION: The survival of t(8;21) AML patients treated with high-dose Ara-c (≥2 g/m2) was superior to other dose levels in postremission consolidation chemotherapy. Patient survival was improved by 3-4 cycles of chemotherapy with an accumulated concentration of 36 g/m2 of Ara-c. WBC >3.5 × 109/l, PLT ≤30 × 109/l and extramedullary infiltration could be indicative of a poor clinical prognosis.

Hematopoietic stem cell transplantation with umbilical cord multipotent stromal cell infusion for the treatment of aplastic anemia--a single-center experience.

BACKGROUND AIMS: The purpose of this study was to observe the outcome of co-transfusion of umbilical cord multipotent stromal cells (UC-MSC) and allogeneic hematopoietic stem cells in the treatment of heavily-transfused patients with severe aplastic anemia. METHODS: Of the 22 patients, eight cases received haploidentical hematopoietic stem cells from granulocyte colony-stimulating factor-primed bone marrow and peripheral blood grafts; the other patients received granulocyte colony-stimulating factor-mobilized peripheral blood grafts from human leukocyte antigen-matched related (six cases) and unrelated donors (eight cases). MSCs were intravenously infused at a mean dose of 1.2 × 10(6)/ kg (range, 0.27-2.5 × 10(6)/kg). Fludarabine-based conditioning was conducted, and graft-versus-host disease prophylaxis containing cyclosporine A, methotrexate and mycophenolate mofetil with or without addition of anti-CD25 monoclonal antibody was performed. Hematopoietic engraftment, the occurrence of graft-versus-host disease (GVHD) and infections and overall survival were documented. RESULTS: All patients had rapid engraftment; mean time for neutrophil and platelet recovery was 13.95 d and 20.27 d, respectively. No acute toxicity associated with UC-MSC transfusion was observed. Acute GVHD developed in seven cases (grade I-II), and none had development of chronic GVHD. Cytomegalovirus reactivation was observed in 11 cases. One patient died of pulmonary complication 6 months after transplantation. Twenty-one patients are currently alive, at a median follow-up of 15 months; they are transfusion-independent and reached full donor chimerism at the time of reporting. CONCLUSIONS: UC-MSC infusion might be an alternative option to promote hematopoietic engraftment and reduce the occurrence of GHVD in hematopoietic stem cell transplantation in the treatment of heavily transfused patients with severe aplastic anemia.

Clinical analysis of the treatment of spinal cord injury with umbilical cord mesenchymal stem cells.

BACKGROUND AIMS: The purpose of this study was to observe the clinical effect and safety of umbilical cord mesenchymal stem cells (UC-MSCs) in treating spinal cord injury (SCI) by intrathecal injection. METHODS: From January 2008 to October 2010, we treated 22 patients with SCI with UC-MSCs by intrathecal injection; dosage was 1 × 10(6) cells/kg body weight once a week given four times as a course. Four patients received two courses, one patient received three courses and all other patients received one course. American Spinal Injury Association scoring system and International Association of Neurorestoratology Spinal Cord Injury Functional Rating Scale were used to evaluate neural function and ability to perform activities of daily living. RESULTS: Treatment was effective in 13 of 22 patients; nine patients had no response. Among patients with incomplete SCI, the response to treatment was 81.25%; there was no response to treatment among six patients with complete SCI. Five patients with a response to treatment received two to three courses of therapy, and effects in these patients were further enhanced. In most patients in whom treatment was effective, motor or sensory functions, or both, were improved, and bowel and bladder control ability was improved. In 22 patients 1 month after therapy, algesia, tactile sensation, motion and activity of daily living scale were significantly improved (P < 0.01). During therapy, common adverse effects were headache (one case) and low back pain (one cases); these disappeared within 1-3 days. No treatment-related adverse events occurred during a follow-up period ranging from 3 months to 3 years. CONCLUSIONS: UC-MSC therapy by intrathecal injection is safe and can improve neurologic function and quality of life in most patients with incomplete SCI.

The first suspected disseminated Hormographiella aspergillata infection in China, diagnosed using metagenomic next-generation sequencing: a case report and literature review.

Hormographiella aspergillata is a rare and emerging cause of invasive mould infections in patients with haematological malignancies, with a mortality rate of approximately 70%. Here, we present the first reported case of suspected disseminated H. aspergillata infection in China. The patient experienced a second relapse of acute myeloid leukaemia and developed neutropenia, fever, discrepant blood pressure between limbs, and cutaneous lesions limited to the left upper extremity. Since lung tissue biopsy was not feasible, metagenomic next-generation sequencing (mNGS) and panfungal polymerase chain reaction (PCR) analysis of bronchoalveolar lavage fluid and blood samples were performed, which indicated probable H. aspergillata pulmonary infection. Histopathology of cutaneous lesions revealed numerous fungal hyphae within dermal blood vessels. mNGS of a skin biopsy sample identified H. aspergillata sequences, and the fungi was subsequently recovered from fungal culture, proving cutaneous H. aspergillata infection. Despite combined antifungal therapy, the patient died owing to disease progression. Additionally, 22 previously reported cases of invasive H. aspergillata infection were reviewed in patients with haematological malignancies. Thus, mNGS is a powerful diagnostic tool for the early and effective detection of invasive H. aspergillata infections, with the advantage of sequencing all potential pathogens, and providing results within 24 h.

[Clinical Analysis of Post-Transplant Lymphoproliferative Disorder after Hematopoietic Stem Cell Transplantation in Severe Aplastic Anemia Patients].

OBJECTIVE: To analyze the clinical characteristics of SAA patients with post-transplantation lymphoproliferative disease (PTLD) after allogeneic hematopoietic stem cell transplantation, and to improve diagnosis and treatment of PTLD. METHODS: The clinical data of 192 patients with SAA patients who underwent HSCT in a single center from September 2010 to September 2017 were analyzed retrospectively. All patients were received antithymocyte globulin（ATG） conditioning regimen and mesenchymal stem cell(MSC) infusion. RESULTS: Among 192 cases, PTLD occurred in 14 cases, the incidence was 7.29％， 9 of them were diagnosed by pathology， and 5 were diagnosed clinically. EBV infection occurred with a median time of 72（35-168） days, Viral load higher than 1×104 copies/ml occured in all PTLD patients. The incidence of probable PTLD in patients ≤12 years old and ＞12 years old was 11.11%, 2.38%, respectively (P<0.01）. Univariate and multivariate analysis that the EBV infection, patients age≤12 years old, HLA-mismatch in URD-HSCT, grade II to IV aGVHD were the independent risk factors for PTLD. All PTLD patients were treated with rituximab(RTX) when EBV-DNA load higher than 1×104 copies/ml, or reducted the use of immunosuppression(RIS), patients with poor therapeutic effect were treated combined with EBV-specific CTLs(EBV-CTL) and chemotherapy. All patients were treated effectively, and the total effective rate was 100％. The median follow-up time was 65(62-115) months, and the overall survival rate was 92.85%. One patients died of cerebral hemorrhage, 7 months after PTLD curred. CONCLUSION: The incidence of PTLD after HSCT with SAA who used ATG and MSC in conditioning regimen closely relates to EBV infection, age of patients≤12 years, HLA-mismatch in URD-HSCT, grade II to IV GVHD. Rituximab combined with RIS may reduce the incidence of PTLD, combined EBV-CTL and chemotherapy may be the useful and most important treatment for PTLD.

[Haploidentical Hematopoietic Stem Cell Transplantation with Co-Infusion of Mesenchymal Stromal Cells for Acquired Severe Aplastic Anemia: A Report of 127 Cases].

OBJECTIVE: To analyze the prognostic factors of haplo-HSCT combined with MSC in the treatment of SAA. METHODS: 127 SAA patients who had undergone haplo-HSCT with co-infusion of MSC in our center from January 2014 to August 2019 were analyzed retrospectively. Median age was 11 (1-37) years, and median follow-up time was 39.8 (1-74) months. RESULTS: The median time for neutrophil and platelet engraftment was 14 d and 18 d respectively. The cumulative incidences of grade III-IV aGVHD was 4.4%±1.9% at day +100. The 2-year cumulative incidence of extensive cGVHD was 8.3%± 2.7%. The estimated 3-year OS was 86.1%±3.1%. Univariate analysis showed that high-dose CD34+ cells (>6.69×106/kg) could promote the engraftment of neutrophil (97.9%±0.05% vs 88.6%±0.13% at day +21, P=0.0006) and platelet (81.2%±0.33% vs 70.8%±0.26% at day +28, P=0.002) and did not increase the incidence of aGVHD (10.4%±0.1% vs 18.9%±0.1% at day +100, P=0.18). More nucleated cells (>12.78×108/kg) caused a lower incidence of grade II-IV aGVHD (8.6%±0.13% vs 21.7%±0.25% at day+100, P=0.04) and a higher incidence of 3-year OS (91.3%±3.2% vs 78.1%±6.5%, P=0.03) than less nucleated cells (≤12.78×108/kg). Younger patients (age≤12 y) had faster neutrophil engraftment (94.9%±0.06% vs 87.5%±0.24% at day+21, P=0.02), higher 3-year OS (93.6%±2.8% vs 75.9%±6.4%, P=0.006) and higher 3-year FFS (93.6%±2.8% vs 68.3%±7.1%, P=0.000) than older patients (age>12 y). The shorter the time from diagnosis to HSCT (≤29.5 months), the higher the 3-year FFS of patients (88.8%±3.5% vs 74.2%±7.2%, P=0.028). Male patients with female donors had higher cumulative incidence of extensive cGVHD than others (20.0%±0.8% vs 4.6%±0.1%, P=0.01). CONCLUSION: In the haplo-HSCT of SAA, the prognosis of children patients is better than that of adults patients. More CD34+ cells and nucleated cells can promote engraftment, reduce the incidence of aGVHD and improve OS. HSCT should be performed as early as possible, and the occurrence of cGVHD should be reduced in male patients by avoiding female donors.

Infusion of haploidentical HSCs combined with allogenic MSCs for the treatment of ALL patients.

Although haploidentical stem cell transplantation (haplo-HSCT) offers almost all acute lymphoblastic leukaemia (ALL) patients an opportunity for immediate transplantation, it exhibits a higher incidence of graft failure and graft versus host disease (GVHD). Mesenchymal stem cells (MSCs) are characterised by their haematopoiesis-promoting and immunomodulatory capacity. Thus, we designed a combination of haplo-HSCT and MSCs for ALL patients. ALL patients (n = 110) were given haploidentical HSCs combined with allogenic MSCs, and ALL patients without MSC infusion (n = 56) were included as controls. The 100-day cumulative incidences of grade ≥2 acute GVHD (aGVHD) and grade ≥3 aGVHD were 40.00% and 9.09% compared to 42.32% (P = 0.79) and 22.79% (P = 0.03) in patients without MSC infusion, respectively. The 3-year cumulative incidences of chronic GVHD (cGVHD) and extensive cGVHD were 22.27% and 10.27% compared to 32.14% (P = 0.19) and 22.21% (P = 0.04) in patients without MSC infusion, respectively. No significant differences in the 3-year relapse incidence, nonrelapse mortality, leukaemia-free survival or overall survival in groups with and without MSC cotransplantation were observed. Multivariate analysis showed that MSC infusion contributed to a lower risk of developing extensive cGVHD. Our data suggested that haplo-HSCT combined with MSCs may provide an effective and safe treatment for ALL patients.

[Clinical Safety of NK Cell in the Prevention of Leukemia Relapse Post-transplantation and in Treatment of the Elderly Leukemia Patients].

OBJECTIVE: To observe the safety of donor NK cell infusions in the settings of hematopoietic stem cell transplantation and after consolidation chemotherapy in elderly patients with acute myeloid leukemia (AML). METHODS: Forty patients with AML were included, in which 21 patients aged over 60 years were at the stage of complete remission (CR) and 19 patients that received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mononucleated cells were isolated from peripheral blood from the donors (for allo-HSCT) or healthy immediate family members (elderly AML). The cells were seeded into the flasks pre-coated with NK cell specific activators, and expanded in media containing recombinant human IL-15 and IL-2 for 14 days. The cells were transfused intravenously after the identification of quality control. Trypan blue exclusion test was used for the determination of cell viability and counting. Flow cytometry analysis was performed to assess the surface antigenic profile. Seventy-eight infusions of the cell products were received by the elderly patients with AML after consolidation chemotherapy, 11 infusions were received by the patients during allo-HSCT and 32 infusions 3 moths after transplantation. The safety of cell therapy, body temperature, blood pressure and other indexes were observe during and 48 hours after cell transfusion. Meanwhile, the occurrence and severity of acute graft-versus-host disease (GVHD) were documented. RESULTS: Flow cytometry analysis showed that the proportion of NK cells (CD3-CD56+) in the mononucleated cells before culture was (14.10±4.22)% (n=121), and the proportion increased dramatically up to (87.29±8.75)% (n=121) after culture for 14 days, the number of NK cells increased to 753.47±140.13 times (n=121). The doses of the infused NK cells was (7.58±2.50)×107/kg per infusion. Moderate fever occurred in three cases after multiple infusions, and the temperature restored to normal on the same day after treatment. Fever was observed in one patient after every infusion of four times in total. The temperature reached to 38.5-39.0 ℃ and returned to normal within 1-2 hours after adequate antipyretic treatment, and then there was no discomfort. No GVHD was observed in the elderly AML patients, while 6 cases that received allo-HSCT developed moderate acute GVHD, among them grade I in 5 cases and grade II in 1 case. No other severe toxicities were observed. CONCLUSION: NK cell products with a high-purity could be obtained by ex vivo expansion with this protocol. The transfusion of these expanded cells is generally safe in the elderly patients with AML that have received chemotherapy or patients that received hematopoietic stem cell transplantation.

Outcomes of haploidentical bone marrow transplantation in patients with severe aplastic anemia-II that progressed from non-severe acquired aplastic anemia.

Severe aplastic anemia II (SAA-II) progresses from non-severe aplastic anemia (NSAA). The unavailability of efficacious treatment has prompted the need for haploidentical bone marrow transplantation (haplo-BMT) in patients lacking a human leukocyte antigen (HLA)-matched donor. This study aimed to investigate the efficacy of haplo-BMT for patients with SAA-II. Twenty-two patients were included and followed up, and FLU/BU/CY/ATG was used as conditioning regimen. Among these patients, 21 were successfully engrafted, 19 of whom survived after haplo-BMT. Four patients experienced grade II-IV aGvHD, including two with grade III-IV aGvHD. Six patients experienced chronic GvHD, among whom four were mild and two were moderate. Twelve patients experienced infections during BMT. One was diagnosed with post-transplant lymphoproliferative disorder and one with probable EBV disease, and both recovered after rituximab infusion. Haplo-BMT achieved 3-year overall survival and disease-free survival rate of 86.4% ± 0.73% after a median follow-up of 42 months, indicating its effectiveness as a salvage therapy. These promising outcomes may support haplo-BMT as an alternative treatment strategy for patients with SAA-II lacking HLA-matched donors.

A study of human leukocyte antigen-haploidentical hematopoietic stem cells transplantation combined with allogenic mesenchymal stem cell infusion for treatment of severe aplastic anemia in pediatric and adolescent patients.

The clinical applications of human leukocyte antigen (HLA) haploidentical hematopoietic stem cells transplantation (haplo-HSCT) have offered most of the young severe aplastic anemia (SAA) patients an opportunity to accept curative therapy at the early stage of bone marrow lesions. However, the outcome of juvenile SAA patients received haplo-HSCT remain to be improved due to high incidence of graft failure and graft vs host disease (GVHD). Mesenchymal stem cells (MSCs) have been characterized by their hematopoiesis-supporting and immunomodulatory properties. In the current study, we designed a combination of haplo-HSCT with allogenic MSC for treatment of SAA in pediatric and adolescent patients and evaluated its effects. Juvenile patients (<18 years) with SAA (n = 103) were given HLA-haploidentical HSC combined with allogenic MSC after a conditioning regimen consisting of busulfan, cyclophosphamide, fludarabine, and antithymocyte globulin and an intensive GVHD prophylaxis, including cyclosporine, short-term methotrexate, mycophenolate mofetil, and basiliximab. Neutrophil engraftment was achieved in 102 of 103 patients in a median time of 14.3 days (range 9-25 days). The median time of platelet engraftment was 25.42 days (range 8-93 days). The cumulative incidence of II-IV acute GVHD at day +100 was 26.32% ± 0.19% and III-IV acute GVHD was 6.79% ± 0.06% at day +100, respectively. The cumulative incidence of chronic GVHD was 25.56% ± 0.26%. The overall survival was 87.15% ± 3.3% at a median follow-up of 40 (1.3-98) months. Our data suggest that cotransplantation of HLA-haploidentical HSC and allogenic mesenchymal stem cell may provide an effective and safe treatment for children and adolescents with SAA who lack matched donors.

Co-transfusion of haplo-identical hematopoietic and mesenchymal stromal cells to treat a patient with severe aplastic.

A 3-year-old girl with severe aplastic anemia (SAA) that was unresponsive to steroid, cyclosporine and filgrastim treatments received bone marrow (BM) mesenchymal stromal cells (MSC; 1.25 x 10(6)/kg), granulocyte colony-stimulating factor (G-CSF)-mobilized BM and peripheral blood stem cell grafts from her father. Prior to stem cell transplantation, she had experienced repeated bacterial infections and received 44 blood transfusions during 8 months after diagnosis. The conditioning regimen consisted of fludarabine, cyclophosphamide and busulfan, and prophylaxis of acute graft-versus-host disease (GvHD) was performed by administration of anti-CD25 monoclonal antibody, cyclosporine A, methotrexate, mycophenolate mofetil and anti-thymocyte globulin. The patient achieved rapid hematopoietic engraftment of donor origin and no acute or chronic GvHD was observed. She is now alive with a good performance status, and the dose of cyclosporine A is being tapered. The novel regimen described here might be a suitable option for children with SAA who lack immediate access to HLA-matched sources.

Thrombopoietin enhances hematopoietic stem and progenitor cell homing by impeding matrix metalloproteinase 9 expression.

We reported a novel function of recombinant human thrombopoietin (TPO) in increasing hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow (BM). Single doses of TPO treatment to the recipients immediately after BM transplantation showed significantly improved homing of HSPCs to the BM, which subsequently resulted in enhanced short- and long-term engraftment of HSPCs in mice. We found that TPO could downregulate the expression and secretion of matrix metalloproteinase 9 in BM cells. As a result, SDF-1α level was increased in the BM niche. Blocking the interaction of SDF-1α and CXCR4 on HSPCs by using AMD3100 could significantly reverse the TPO-enhanced HSPC homing effect. More importantly, a single dose of TPO remarkably promoted human HSPC homing and subsequent engraftment to the BM of nonobese diabetic/severe combined immunodeficiency mice. We then performed a clinical trial to evaluate the effect of TPO treatment in patients receiving haploidentical BM and mobilized peripheral blood transplantation. Surprisingly, single doses of TPO treatment to patients followed by hematopoietic stem cell transplantation significantly improved platelet engraftment in the cohort of patients with severe aplastic anemia (SAA). The mean volume of platelet and red blood cell transfusion was remarkably reduced in the cohort of patients with SAA or hematological malignancies receiving TPO treatment. Thus, our data provide a simple, feasible, and efficient approach to improve clinical outcomes in patients with allogenic hematopoietic stem cell transplantation. The clinical trial was registered in the Chinese Clinical Trial Registry website (www.chictr.org.cn) as ChiCTR-OIN-1701083.

Tumor necrosis factor α in aGVHD patients contributed to the impairment of recipient bone marrow MSC stemness and deficiency of their hematopoiesis-promotion capacity.

BACKGROUND: Though accumulated evidence has demonstrated visceral organ involvement in acute graft-versus-host disease (aGVHD), how aGVHD influences the bone marrow (BM) niche and the reconstitution of hematopoiesis post-hematopoietic stem cell transplantation remains largely unknown. METHODS: In the current study, the cell morphology, immunophenotype, multi-differentiation capacity, self-renewal capacity, and hematopoiesis promotion of the MSCs from aGVHD and non-aGVHD patients were investigated. Additionally, the stemness and hematopoiesis-promoting property of healthy donor-derived MSCs were evaluated in the presence of BM supernatant from aGVHD patients. Mechanistically, antibodies targeting inflammatory cytokines involved in aGVHD were added into the MSC culture. Furthermore, a recombinant human tumor necrosis factor (TNF-α) receptor-Ig fusion protein (rhTNFR:Fc) was used to protect healthy donor-derived MSCs. Moreover, mRNA sequencing was performed to explore the underlying mechanisms. RESULTS: The aGVHD MSCs exhibited morphological and immunophenotypic characteristics that were similar to those of the non-aGVHD MSCs. However, the osteogenic and adipogenic activities of the aGVHD MSCs significantly decreased. Additionally, the colony formation capacity and the expression of self-renewal-related genes remarkably decreased in aGVHD MSCs. Further, the hematopoiesis-supporting capacity of aGVHD MSCs significantly reduced. The antibody neutralization results showed that TNF-α contributed to the impairment of MSC properties. Moreover, rhTNFR:Fc exhibited notable protective effects on MSCs in the aGVHD BM supernatants. The mRNA sequencing results indicated that the TNF-α pathway and the Toll-like receptor pathway may be activated by TNF-α. CONCLUSIONS: Thus, our data demonstrate MSCs as cellular targets of aGVHD and suggest a potential role of TNF-α blockage in maintaining the BM niche of aGVHD patients.

Impact of prophylactic/preemptive donor lymphocyte infusion and intensified conditioning for relapsed/refractory leukemia: a real-world study.

Prophylactic/preemptive donor lymphocyte infusion (p/pDLI) and intensified conditioning have shown promising results in experimental studies of refractory/relapsed acute leukemia (RRAL), but real-world data remain scarce. We conducted a multicenter, population-based analysis of 932 consecutive patients. The three-year leukemia-free survival (LFS) rates were 56% for patients receiving both p/pDLI and intensified myeloablative conditioning (MAC) (intenseMAC) and 30% for those who received neither therapy per landmark analysis. Multivariable analyses were run separately for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), and p/pDLI treatment was linked to significantly higher LFS than non-DLI for both AML and ALL patients without increasing the nonrelapse mortality. IntenseMAC was associated with significantly lower relapse and higher LFS than nonintensified MAC despite higher nonrelapse mortality rates in ALL, while there was no impact of intenseMAC observed in AML. p/pDLI achieved superior outcomes in both matched-sibling donor (MSD) and haploidentical donor transplantation, while intenseMAC only influenced MSD outcomes. Data suggest that RRAL patients receiving "total therapy" by way of p/pDLI and intensified conditioning treatment have an improved chance for LFS, with p/pDLI being safer with a more extensive impact relative to intenseMAC. Patients with RRAL can tolerate both interventions and achieve a reasonable outcome.

In vitro and in vivo immunosuppressive characteristics of hepatocyte growth factor-modified murine mesenchymal stem cells.

BACKGROUND: Previous data have proven that hepatocyte growth factor (HGF) is able to maintain the survival of mesenchymal stem cells (MSCs), rendering HGF gene modification as an optional strategy for MSC therapy. However, the question about whether gene-transferred MSCs (MSC/HGFs) exhibit more potent immunosuppressive activity remains elusive. MATERIALS AND METHODS: Murine MSCs were isolated, culture-expanded and transfected by an adenovirus carrying human HGF cDNA (Ad-HGF). The transfection efficiency was evaluated by measuring HGF concentrations in the culture supernatants. An MHC-incompatible skin grafting model (C57BL-->BALB/c) was used to observe if MSC/HGF transfusion could prolong the survival time of skin transplants compared to MSCs. Furthermore, their inhibitory effects on the proliferation of T lymphocytes elicited by Con A and the activation of CD11b+ cells in mixed lymphocyte reaction were compared with carboxyfluorescein diacetate succinmidyl ester labeling and flow cytometric techniques. RESULTS: Ad-HGF was able to transfect mouse MSCs at high efficiency and administration of MSC/HGFs remarkably prolonged the mean survival time of skin grafts (16.73 +/- 0.57 days, p<0.01), compared with mice receiving MSCs (14.27 +/- 0.63 days), or saline (10.92 +/- 0.73 days). However, the presence of MSC/HGFs exhibited little additive impact on the suppression of T lymphocyte proliferation and activation of CD11b-positive and -negative cells in comparison with MSCs, though the inhibitory effects were evidently greater than with NIH3T3 cells and their Ad-HGF-modified counterparts. CONCLUSION: MSC/HGFs inhibit in vitro immune responses in a pattern similar to MSCs, but this gene modification might have beneficial effects for transplanted cells and damaged tissue.