Xiao-Li Zheng

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.

Characterization of IFNγ-producing natural killer cells induced by cytomegalovirus reactivation after haploidentical hematopoietic stem cell transplantation.

During human cytomegalovirus (CMV) infection after umbilical cord blood or HLA-matched hematopoietic stem cell transplantation (HSCT), a population of NKG2C-expressing natural killer (NK) cells expand and persist. The expanded NK cells express high levels of inhibitory killer immunoglobulin-like receptors (KIR) specific for self-HLA and potently produce IFNγ. However, it remains unknown whether similar events would occur after haploidentical HSCT (haplo-HSCT). Here, we demonstrated that IFNγ-producing NK cells were expanded in haplo-HSCT patients with CMV reactivation. We then identified these expanded cells as a subset of CD56dim NK cells that expressed higher levels of both NKG2C and KIR, but lower level of NKG2A. Functionally, the subset of NK cells expressing NKG2C and self-KIR in patients with CMV reactivation accounted for IFNγ production in response to K562 cells. However, these phenomena were not observed in patients without CMV reactivation. We therefore characterized a subset of NK cells with the CD56dim, NKG2C+, and self-KIR+ phenotype that expanded and were responsible for IFNγ production during CMV infection after haplo-HSCT. Together, these findings support a notion that CMV reactivation induces expansion of more mature NK cells with memory-like features, which contributes to long-term control of both CMV infection and leukemia relapse after haplo-HSCT.

Haploidentical hematopoietic stem cell transplant with umbilical cord-derived multipotent mesenchymal cell infusion for the treatment of high-risk acute leukemia in children

Abstract In this study, 25 children with high-risk acute leukemia received haploidentical hematopoietic stem cell transplant (haplo-HSCT) with co-transfusion of umbilical cord multipotent mesenchymal cells (UC-MSCs). Adverse effects, hematopoietic recovery, complications and outcome were observed during a median follow-up of 12.8 months (range: 3–25 months). Myeloid engraftment was rapid, and the median time to neutrophil and platelet recovery was 15.12 days and 20.08 days, respectively. Eight patients developed grade I skin acute graft-versus-host disease (aGVHD) that responded well to standard steroid therapy. Of note, cytomegalovirus viremia was observed in most patients (23/25 cases). Patients died mainly of leukemia relapse and pulmonary complication. Fourteen patients are currently alive and remain with full donor chimerism at the time of reporting. The present results suggest further clinical trials to testify the effectiveness of UC-MSCs to prevent aGVHD in haplo-HSCT for treating children with high-risk leukemia.

Functional mesenchymal stem cells remain present in bone marrow microenvironment of patients with leukemia post-allogeneic hematopoietic stem cell transplant

Abstract Mesenchymal stem cells (MSCs) and their progenies are important supporting cells in the bone marrow (BM) microenvironment. However, the function and kinetics of MSCs post-hematopoietic stem cell transplant (HSCT) remain unknown. In the present study, MSCs were cultured from a total of 76 BM samples from 15 patients receiving HSCT. Colony-forming unit fibroblasts in BM before pre-conditioning and 1, 3, 6 and 9 months post-HSCT were cultured and counted to quantify MSCs. Hematopoiesis-supporting activity of MSCs was observed with long-term culture of hematopoietic progenitors. An inhibitory effect of MSCs on in vitro lymphocyte proliferation was also observed. Results showed that post-HSCT MSCs supported in vitro hematopoiesis and inhibited lymphocyte growth. Moreover, the quantity of MSCs was reduced at an early stage and restored to baseline level 9 months post-transplant. The results indicate that functional MSCs remain present in the BM microenvironment, and these findings shed light on the understanding of BM microenvironment reconstitution post-HSCT.

The epigenetically-regulated miR-34a targeting c-SRC suppresses RAF/MEK/ERK signaling pathway in K-562 cells

Previous reports show that miR-34a suppressed K-562 cell proliferation and contributed to megakaryocytic differentiation of K-562 cells. Here, we reported that miR-34a, a tumor suppressor gene, is down-regulated in the K-562 cells and chronic myeloid leukemia (CML) patients due to aberrant DNA hypermethylation. c-SRC is a target of miR-34a. Restoring miR-34a expression resulted in down-regulation of c-SRC and phosphorylated (Tyr416) c-SRC protein in K-562 cells, which consequently triggered suppression of the RAF/MEK/ERK signaling pathway to decrease cell proliferation.

The absolute number of regulatory T cells in unmanipulated peripheral blood grafts predicts the occurrence of acute graft-versus-host disease post haplo-identical hematopoietic stem cell transplantation

CD4+CD25+Foxp3+ regulatory T cells (Tregs) have been reported to play a central role in suppressing acute graft-versus-host disease (aGVHD), but whether the Treg contents of grafts correlates with the occurrence of aGVHD post haplo-identical hematopoietic stem cell transplantation (haplo-HSCT) remains unclear. In the present study, changes in Tregs in peripheral blood (PB) were followed before and after granulocyte colony-stimulating factor (G-CSF) mobilization. In addition, functional analyses of Tregs in PB grafts and bone marrow (BM) grafts were performed. To probe the prognostic value of Tregs for aGVHD, an analysis of Tregs in haplo-identical grafts was conducted in 61 patients. Moreover, univariate and multivariate analyses of both clinical variables and cellular subsets were performed. The results showed that both the percentage Tregs of CD4+ T cells and absolute numbers of Tregs per 106 total nucleated cells significantly increased after G-CSF administration. Additionally, Tregs in PB grafts exhibited a stronger inhibitory effect on antigen-specific T cell proliferation than did Tregs in BM grafts. Strikingly, patients receiving more Tregs in PB grafts had a lower cumulative incidence of aGVHD II-IV (36% versus 58%, P=0.046). Further, in a multivariate analysis, the number of Tregs in PB grafts was significantly associated with a lower occurrence of aGVHD II-IV (P=0.02). In contrast, the number of Tregs in BM grafts was not associated with the occurrence of aGVHD in the current study. Further analysis showed that the Treg content in grafts did not affect Treg reconstitution, infection rate, or incidence of chronic GVHD after haplo-HSCT. Moreover, no significant correlations between cell types in grafts and the survival end points or relapse rates were found in the present study. In conclusion, our results suggest that a high number of Tregs in PB grafts is associated with reduced risk of aGVHD in haplo-HSCT in our transplant setting.

Co-infusion of mesenchymal stromal cells has no effect on relapse and infection in patients with leukemia undergoing haploidentical hematopoietic stem cell transplant

We observed the outcome of co-transplant of umbilical cord-derived, ex vivo-expanded mesenchymal stromal cells (MSCs) in patients with leukemia undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT), and compared the relapse and infection rates with those in patients receiving traditional haplo-HSCT. The procedure was intended to assess the influence of MSCs on the relapse and infection rates of patients with leukemia after HSCT. From February 2010 to December 2012, 41 patients with leukemia received co-transplant of haplo-HSCT with MSCs (MSC  HSCT group). They were followed up for 21 months (2–50 months). From June 2008 to March 2010, 42 patients with leukemia received traditional haplo-HSCT (HSCT group). They were followed up for 35 months (2–70 months). This study was approved by the ethics and technological committees of the General Hospital of the Air Force, and all patients provided informed consent before MSC use. We found that the mean time for neutrophil engraftment in the MSC  HSCT group was significantly shorter than in the HSCT group (16.90  0.63 days vs. 18.83  0.63 days, p  0.03). There was no significant difference in the cumulative incidence of II–IV acute graft versus host disease (aGVHD) or chronic GVHD (cGVHD) between MSC  HSCT and HSCT groups (p  0.109 and p  0.556, respectively). In addition, no notable effect of MSC co-infusion was found on overall survival (p  0.573), relapse-free survival (p  0.424) and event-free survival (p  0.222) or non-relapse mortality (p  0.853) in patients who underwent traditional haplo-HSCT. Also, there was no significant difference in the rate of leukemia relapse (p  0.736), pulmonary infection (p  0.182), cytomegalovirus (CMV) infection (p  0.652) or pneumonia-associated death (p  0.396) within 1 year after haplo-HSCT between MSC  HSCT and HSCT groups. Our results indicate that co-infusion of MSCs can shorten the time of neutrophil engraftment, but does not increase the rates of relapse and infection in haplo-HSCT. Eighty-three patients received a regimen consisting of busulfan at 4 mg/kg/day orally or 0.8 mg/kg/day intravenously (IV) (days  6,  7 and  8), cytarabine at 4 g/m2/ day (days  4 and  5) and cyclophosphamide at 55 mg/ kg/day (days  2 and  3). Stem cell collection and MSC preparation were performed as previously reported [1,2]. The MSCs were passaged, and cells at passage 3 were used for therapy after phenotypic analysis with fluorescence activated cell sorting (FACS) (FACSCalibur; BD, Franklin Lakes, NJ) (CD44, CD73, CD166, CD45, HLA-DR, CD14, CD31). At day 0, under monitoring of vital signs, patients were given MSCs IV via a central venous catheter before hematopoietic graft infusion. GVHD prophylaxis was conducted using a combination of immunosuppressive drugs including cyclosporine A (CSA), methotrexate (MTX), mycophenolate mofetil (MMF) and rabbit anti-thymocyte globulin (ATG; Fresenius AG, Oberursel, Germany). Briefly, before transplant, CSA was administered IV at 1.5 mg/kg/day from day  7 to day  2, and the dose was increased to 2.5 mg/kg/day from then until recovery of intestinal function, when it was given orally at 5 mg/kg/day. ATG was administered at a dose of 5 mg/kg/day from day  4 to day  1. MTX was administered IV at 15 mg/m2 on day  1 and at 10 mg/m2 on days  3,  6 and  11. MMF was administered orally at 0.25–0.5 g/day from day  7 to day  30 (Supplementary Figure 1, to be found at online http://informahealthcare. com/doi/abs/10.3109/10428194.2015.1020061). All patients were monitored for engraftment and post-transplant adverse events, including GVHD, relapse and infection. Hematopoietic stem cell engraftment was defined as maintenance of an absolute peripheral neutrophil count above 0.5  109/L or an unsupported platelet count above 20  109/L for 3 consecutive days. Hematopoietic chimerism was identified by blood type analysis (blood type mismatch), sex chromosome determination (sex mismatch) and/or polymerase chain reaction (PCR)-DNA fingerprinting (short tandem repeat) to confirm Leukemia & Lymphoma, October 2015; 56(10): 2965–2968

Mesenchymal Stem Cells in Grafts Failed to Engraft in the Bone Marrow Microenvironment of a Leukemia Patient Post HLA-match and Haplo-Identical Allogeneic Hematopoietic Stem cell Transplantations

Mesenchymal stem cells (MSCs) are an important cellular component of the bone marrow microenvironment (BM-ME) [1–3]. After hematopoietic stem cell transplantation (HSCT), the normal rebuilding of the recipient’s BM-ME is vital for the reconstitution of the immune system and hematopoiesis. However, it remains controversial whether donor MSCs in grafts can aid in the rebuilding of the patient’s BM-ME [4,5]. Here, we report a unique case that the BM-MSCs from a patient who received tandem HSCTs remain host-origin. A 17-year-old male with chronic myeloid leukemia was admitted to our hospital 5 years after receiving human leukocyte antigen (HLA)matched peripheral blood stem cell (PBSC) transplantation because he was experiencing a relapse of his primary disease. The HLA-matched grafts were from his brother, and the pre-conditioning regimen was myeloablative, consisting of busulfan and cyclophosphamide. In our hospital, he was treated with a Hyper-CVMD A and B chemotherapy regimen combined with nilotinib, and the treatment resulted in complete remission. Then, he received a haplo-identical HSCT with a myeloablative pre-conditioning regimen consisting of busulfan (12 mg/kg), cyclophosphamide (110 mg/kg), and cytarabine (8 g/m2). The haplo-identical grafts included PBSCs (8 × 108/kg of nucleated cells) and BM cells (2.72 × 108 /kg of nucleated cells) from his mother. The transplantation was successful, and the chimerism was analyzed using short-tandem repeats (STR). The