Enhanced angiogenic properties of umbilical cord blood primed by OP9 stromal cells ameliorates neurological deficits in cerebral infarction mouse model.

Umbilical cord blood (UCB) transplantation shows proangiogenic effects and contributes to symptom amelioration in animal models of cerebral infarction. However, the effect of specific cell types within a heterogeneous UCB population are still controversial. OP9 is a stromal cell line used as feeder cells to promote the hematoendothelial differentiation of embryonic stem cells. Hence, we investigated the changes in angiogenic properties, underlying mechanisms, and impact on behavioral deficiencies caused by cerebral infarction in UCB co-cultured with OP9 for up to 24 h. In the network formation assay, only OP9 pre-conditioned UCB formed network structures. Single-cell RNA sequencing and flow cytometry analysis showed a prominent phenotypic shift toward M2 in the monocytic fraction of OP9 pre-conditioned UCB. Further, OP9 pre-conditioned UCB transplantation in mice models of cerebral infarction facilitated angiogenesis in the peri-infarct lesions and ameliorated the associated symptoms. In this study, we developed a strong, fast, and feasible method to augment the M2, tissue-protecting, pro-angiogenic features of UCB using OP9. The ameliorative effect of OP9-pre-conditioned UCB in vivo could be partly due to promotion of innate angiogenesis in peri-infarct lesions.

Safety and efficacy of amnion-derived mesenchymal stem cells (AM01) in patients with steroid-refractory acute graft-versus-host disease after allogeneic haematopoietic stem cell transplantation: a study protocol for a phase I/II Japanese trial.

INTRODUCTION: Regenerative medicine and cell therapies have been gaining much attention among clinicians. Therapeutic infusion of mesenchymal stromal cells (MSCs) is now a leading investigational strategy for the treatment of acute graft-versus-host disease (aGVHD). Bone marrow MSCs are approved for manufacture and marketing as a cell therapy for aGVHD. Our non-clinical studies confirmed that human amnion-derived MSCs had immunomodulatory activity equal to or higher than that of human bone marrow MSCs. This study will aim to evaluate the safety and efficacy of amnion-derived MSCs (AM01) in patients with steroid-refractory aGVHD. METHODS AND ANALYSIS: This study will be a multicentre, single-arm, open-label trial (an interventional study). This clinical trial will begin with a low-dose group, and when safety has been confirmed in at least three cases in the low-dose group, treatment will begin for the high-dose group, for which the safety will also be verified. The primary endpoint is to assess the safety of intravenous infusion therapy of AM01 within 24 hours after intravenous infusion of AM01. The secondary endpoint is to explore the efficacy of intravenous infusion therapy with AM01. ETHICS AND DISSEMINATION: The institutional review boards of all participating hospitals approved this study protocol (latest V3.3.0, 3 August 2018). Final data will be publicly announced. A report releasing the study results will be submitted for publication to an appropriate peer-reviewed journal. TRIAL REGISTRATION NUMBER: UMIN000029945.

Low incidence of HHV-6 reactivation in haploidentical hematopoietic stem cell transplantation with corticosteroid as graft-vs-host disease prophylaxis compared with cord blood transplantation.

BACKGROUND: Human leukocyte antigen (HLA) mismatch and the administration of immunosuppressive agents are considered risks for human herpesvirus 6 (HHV-6) reactivation after stem cell transplantation (SCT). However, the incidence of HHV-6 reactivation in HLA-mismatched related SCT remains unknown. METHODS: We monitored plasma HHV-6 DNA loads weekly using real-time quantitative polymerase chain reaction for 5 weeks after SCT and compared serum IL-6 levels in HLA-mismatched SCT groups. RESULTS: Compared with detection in all 11 umbilical cord blood transplantation (CBT) patients (100%), plasma HHV-6 DNA was detected in only 3 of 42 haplo-SCT patients (7.1%) despite the use of methylprednisolone and antithymocyte globulin as graft-vs-host disease prophylaxis and a reduced-intensity conditioning regimen, respectively. Correspondingly, serum IL-6 levels in haplo-SCT patients were significantly lower than those in CBT patients. No HHV-6-associated encephalitis developed in either groups. CONCLUSIONS: Neither HLA disparity nor the use of methylprednisolone and antithymocyte globulin were risk factors for HHV-6 reactivation in our haplo-SCT patients. Rather than increasing risk, the administration of immunosuppressive agents potentially prevented HHV-6 reactivation after haplo-SCT by suppressing IL-6 production.

Spousal hematopoietic stem cell transplantation.

We report a pilot series of five patients who received stem cell transplantation (SCT) from a spouse for post-transplant relapse or rejection. The inclusion criterion regarding HLA disparities was three or fewer antigen mismatches in the graft-versus-host direction at the HLA-A, B, and DR loci. Four patients received spousal SCT as a third transplant attempt after post-transplant relapse and one as rescue for graft rejection. The reduced intensity conditioning (RIC) regimen consisted of fludarabine, melphalan, and anti-thymocyte globulin (ATG) with 3 Gy of total body irradiation (TBI) for relapse cases and ATG plus 4 Gy of TBI for the rejection case. Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus, methylprednisolone, and mycophenolate mofetil. Peripheral blood stem cells were transplanted. Granulocyte engraftment was achieved in all cases between days 9 and 11 (median, 10) with complete spousal chimerism. In three of the five patients, no acute GVHD was observed, while one case developed grade III GVHD and one case grade IV. All four patients evaluable for the anti-leukemic effect achieved complete remission; however, all relapsed between 106 and 334 day post-transplant, and died between days 152 and 548. We suggest that spousal SCT can be performed as a repetitive SCT using a RIC regimen with low-dose ATG and steroid-containing GVHD prophylaxis.

Transplantation of hematopoietic stem cells: intra-arterial versus intravenous administration impacts stroke outcomes in a murine model.

Based on results of hematopoietic stem cell transplantation in animal models of stroke, clinical trials with hematopoietic stem cells administered intra-arterially or intravenously have been initiated in patients. Although intra-arterial injection is expected to deliver transplanted cells more directly to the ischemic tissue, the optimal route for enhancing clinical outcomes has not been identified in the setting of stroke. In this study, we compared the therapeutic potential of intra-arterial versus intravenous injection of bone marrow derived-mononuclear cells (BM-MNCs) and CD133-positive (CD133(+)) cells in a murine stroke model. We have found that intra-arterial injection of BM-MNCs exaggerates inflammation with accompanying loss of microvascular structures in poststroke brain and no improvement in cortical function. In contrast, intravenous injection of BM-MNCs did not similarly enhance inflammation and improved cortical function. Our results indicate that the optimal route of cell transplantation can vary with different cell populations and highlight possible issues that might arise with intra-arterial cell administration for acute ischemic cerebrovascular disease.

Allogeneic stem cell transplantation for X-linked agammaglobulinemia using reduced intensity conditioning as a model of the reconstitution of humoral immunity.

BACKGROUND: We herein report the first case of X-linked agammaglobulinemia (XLA) that underwent allogeneic stem cell transplantation using reduced intensity conditioning (RIC). We chronologically observed the reconstitution of humoral immunity in this case. CASE PRESENTATION: The patient was a 28-year-old Japanese male with XLA who previously had life-threatening infectious episodes and was referred for the possible indication of allogeneic stem cell transplantation. After a thorough discussion within specialists from different backgrounds, we decided to perform allogeneic peripheral stem cell transplantation from his HLA-identical elder brother. Due to the non-malignant nature of XLA, we selected RIC consisting of fludarabine, cyclophosphamide, anti-thymocyte globulin, and 3 Gy of total body irradiation. Neutrophil engraftment was achieved on day 11 with complete donor chimerism. No major complications, except for stage 1 skin graft-versus-host disease, were observed. The patient was discharged on day 75 and has been followed as an outpatient without any infectious episodes for more than 500 days. CONCLUSIONS: Regarding immune reconstitution, CD19(+) cells, IgA, and IgM, which were undetectable before allogeneic stem cell transplantation (allo-SCT), started to increase in number 10 days after allo-SCT and continued to increase for more than 1 year. Anti-B antibodies appeared as early as day 10. Total IgG levels decreased after the discontinuation of IgG replacement and spontaneously recovered after day 350. However, most anti-viral IgG titers, except EB virus-virus capsid antigen IgG, disappeared after the discontinuation of IgG replacement. A seasonal vaccination to influenza was performed on day 148, with neither anti-influenza type A nor type B being positive after the vaccination. The transient transfer of allergic immunity to orchard grass was observed. Similar Bruton's tyrosine kinase (BTK) expression levels in monocytes and B-cells were observed between the patient and healthy control. B-cells in the peripheral blood (PB) of the patient on day 279 showed sufficient proliferation after a CD40L and IL-21 or CD40L and CpG stimulation. Effective immunoglobulin production and class switching were also observed after a CD40L and IL-21 or CpG stimulation. Signal joint kappa-deleting recombination excision circles (sjKRECs) became positive 16 days post-SCT, increased to 6300 copies/µg DNA at 42 days, and were maintained at a high level thereafter. The recovery of T-cell receptor excision circles (TRECs) was slow, but became detectable 1 year post-hematopoietic stem cell transplantation (HSCT).

Host Foxp3+CD4+ Regulatory T Cells Act as a Negative Regulator of Dendritic Cells in the Peritransplantation Period.

Host Foxp3+CD4+ regulatory T cells (Tregs) have been shown to suppress graft-versus-host disease (GVHD) in experimental bone marrow transplantation (BMT) models; however, the detailed mechanism is unknown. To address this issue, we established a murine MHC-haploidentical BMT model (BDF1 (H-2b/d) → B6C3F1 (H-2b/k)), in which transplantation following conditioning with high-dose (13 Gy) or low-dose (5 Gy) total body irradiation corresponds to myeloablative stem cell transplantation (MAST) or reduced-intensity stem cell transplantation (RIST) BMT. All MAST recipients died of GVHD within 70 d, whereas RIST recipients developed almost no GVHD and survived for at least 3 mo. In this BMT model, we investigated the kinetics of immune cells in the mesenteric lymph nodes because GVHD was most prominent in the intestines. Host Tregs that survived after total body irradiation could proliferate transiently by day 4. Comparing the kinetics of immune cells among MAST, RIST, and anti-CD25 mAb-treated RIST, we found that the transiently surviving host Tregs were fully functional, closely contacted with host dendritic cells (DCs), and significantly restrained the maturation (CD80 and CD86 expression) of DCs in a dose-dependent manner. There was a positive correlation between the ratio of DCs to host Tregs and the extent of maturation of DCs. Host Tregs suppressed alloresponse mainly by contact inhibition. Host Tregs are already active in lymph nodes before transplantation and restrain the maturation of host DCs, thereby dampening the ability of DCs to activate allogeneic donor T cells and consequently reducing the magnitude of graft-versus-host reaction. Thus, host Tregs are negative regulators of host DCs that act in the peritransplantation period.

Intravenous Autologous Bone Marrow Mononuclear Cell Transplantation for Stroke: Phase1/2a Clinical Trial in a Homogeneous Group of Stroke Patients.

The goal of this clinical trial was to assess the feasibility and safety of transplanting autologous bone marrow mononuclear cells into patients suffering severe embolic stroke. Major inclusion criteria included patients with cerebral embolism, age 20-75 years, National Institute of Health Stroke Scale (NIHSS) score displaying improvement of ≤ 5 points during the first 7 days after stroke, and NIHSS score of ≥ 10 on day 7 after stroke. Bone marrow aspiration (25 or 50 mL; N = 6 patients in each case) was performed 7-10 days poststroke, and bone marrow mononuclear cells were administrated intravenously. Mean total transplanted cell numbers were 2.5 × 10(8) and 3.4 × 10(8) cells in the lower and higher dose groups, respectively. No apparent adverse effects of administering bone marrow cells were observed. Compared with the lower dose, patients receiving the higher dose of bone marrow cells displayed a trend toward improved neurologic outcomes. Compared with 1 month after treatment, patients receiving cell therapy displayed a trend toward improved cerebral blood flow and metabolic rate of oxygen consumption 6 months after treatment. In comparison with historical controls, patients receiving cell therapy had significantly better neurologic outcomes. Our results indicated that intravenous transplantation of autologous bone marrow mononuclear cells is safe and feasible. Positive results and trends favoring neurologic recovery and improvement in cerebral blood flow and metabolism by cell therapy underscore the relevance of larger scale randomized controlled trials using this approach.

Decreased levels of circulating CD34(+) cells are associated with coronary heart disease in Japanese patients with type 2 diabetes.

AIMS/INTRODUCTION: Circulating progenitor cells, including CD34 positive (CD34(+)) cells, play a key role in neovascularisation and the maintenance of vascular endothelial function. Several lines of evidence show an association between decreased levels of circulating CD34(+) cells and cardiovascular disease. However, the contribution of circulating CD34(+) cells to the occurrence of cardiovascular events in diabetic patients remains unclear. MATERIALS AND METHODS: In the present study with a median follow up of 4.6 years, we analyzed the level of circulating CD34(+) cells in 192 patients with type 2 diabetes. The outcome variables were coronary heart disease (CHD) events (cardiovascular death, unstable angina, myocardial infarction, percutaneous coronary intervention or coronary artery bypass grafting) and cerebrovascular disease events (cerebral infarction, cerebral hemorrhage or transient ischemic attack). RESULTS: Decreased levels of circulating CD34(+) cells were associated with a significantly higher incidence of CHD based on Kaplan-Meier analysis (P = 0.0052). After adjusting for age, sex, dyslipidemia, hypertension, glycated hemoglobin, history of cardiovascular disease, body mass index, and statin and renin angiotensin system inhibitors use, decreased levels of CD34(+) cells were significantly associated with the incidence of CHD events (hazard ratio of low tertile 2.61, 95% confidence interval 1.22-5.96; P = 0.013, reference; high tertile). CONCLUSIONS: Decreased levels of circulating CD34(+) cells might predict CHD events in patients with diabetes, and this could be useful for identifying patients with diabetes at high risk of cardiovascular events.

Early detection of cytomegalovirus-specific cytotoxic T lymphocytes against cytomegalovirus antigenemia in human leukocyte antigen haploidentical hematopoietic stem cell transplantation.

Human leukocyte antigen (HLA)-haploidentical stem cell transplantation (haplo-SCT) is associated with a high incidence of cytomegalovirus (CMV) infection, probably originating from the delayed reconstitution of CMV-specific T cell immunity. There have been few reports on the presence of CMV-specific cytotoxic T lymphocytes (CMV-CTLs) after haplo-SCT. We have studied CMV-specific immune reconstitution by measuring the absolute number of CMV-CTLs using a flow cytometry method with HLA-A2-restricted NLVPMVATV peptide dextramers. We examined the association between reconstitution patterns of CMV-CTLs and the duration of CMV antigenemia in 15 patients who underwent first allogeneic SCT from HLA-haploidentical-related donors with HLA-A2. In seven and eight patients, CMV antigenemia consecutively resolved for more than 4 weeks (the CMV antigenemia 'resolved' group) and intermittently persisted (the CMV antigenemia 'persistent' group) during a 100-day observation period, respectively. The group of the seven patients, in whom levels of CMV antigenemia were reduced to zero, had a significantly lower maximum level of CMV antigenemia than the CMV antigenemia persistent group. In contrast, the CMV antigenemia persistent group had a significantly higher maximum level of CMV-CTLs, but the levels took longer to peak. Despite no difference in general lymphocyte recovery between the two groups, the CMV antigenemia resolved group had significantly higher median CMV-CTL counts than the CMV antigenemia persistent group at 6 weeks after onset of CMV infection. Flow cytometry analysis of CMV-CTLs is a convenient method of monitoring reconstitution of CMV-specific lymphocyte immunity following haplo-SCT.

Soluble interleukin-2 receptor level on day 7 as a predictor of graft-versus-host disease after HLA-haploidentical stem cell transplantation using reduced-intensity conditioning.

In the present study, we analyzed the kinetics of serum soluble interleukin-2 receptor (sIL-2R) using data from 77 patients undergoing HLA-haploidentical transplantation using reduced-intensity conditioning (RIC), who were at an advanced stage or at high risk for relapse, to clarify the usefulness of sIL-2R as a biomarker of acute graft-versus-host disease (GVHD). Anti-T-lymphocyte globulin and methylprednisolone were used as GVHD prophylaxis. While the median sIL-2R in 38 patients not developing GVHD was suppressed at levels <740 U/ml, sIL-2R in 25 patients developing severe GVHD peaked on day 11 (1,663 U/ml), and thereafter decreased to <1,000 U/ml after day 30. The occurrence of GVHD was not limited to times of high sIL-2R level, but occurred at any time point on the sIL-2R curve. Most patients developing GVHD, however, experienced a higher sIL-2R level early in their transplant course. The combination of RIC and glucocorticoids sufficiently suppressed sIL-2R levels after HLA-haploidentical transplantation. In a multivariate analysis to identify factors associated with GVHD, day 7 sIL-2R >810 U/ml was the only factor significantly associated with the occurrence of severe GVHD (p = 0.0101).

Comparison of angiogenic, cytoprotective, and immunosuppressive properties of human amnion- and chorion-derived mesenchymal stem cells.

Although mesenchymal stem cells (MSCs) can be obtained from the fetal membrane (FM), little information is available regarding biological differences in MSCs derived from different layers of the FM or their therapeutic potential. Isolated MSCs from both amnion and chorion layers of FM showed similar morphological appearance, multipotency, and cell-surface antigen expression. Conditioned media obtained from amnion- and chorion-derived MSCs inhibited cell death caused by serum starvation or hypoxia in endothelial cells and cardiomyocytes. Amnion and chorion MSCs secreted significant amounts of angiogenic factors including HGF, IGF-1, VEGF, and bFGF, although differences in the cellular expression profile of these soluble factors were observed. Transplantation of human amnion or chorion MSCs significantly increased blood flow and capillary density in a murine hindlimb ischemia model. In addition, compared to human chorion MSCs, human amnion MSCs markedly reduced T-lymphocyte proliferation with the enhanced secretion of PGE2, and improved the pathological situation of a mouse model of acute graft-versus-host disease. Our results highlight that human amnion- and chorion-derived MSCs, which showed differences in their soluble factor secretion and angiogenic/immuno-suppressive function, could be ideal cell sources for regenerative medicine.

Detection of donor-derived CMV-specific T cells in cerebrospinal fluid in a case of CMV meningoencephalitis after cord blood stem cell transplantation.

Cytomegalovirus (CMV) meningoencephalitis is a rather rare complication after allogeneic stem cell transplantation. We describe here the case of a 59-year-old man with acute myeloid leukemia who developed CMV meningoencephalitis after cord blood transplantation. The patient presented with a sudden onset of neurological symptoms, such as convulsion, on day 37. The analysis of cerebrospinal fluid (CSF) sample revealed an increase in the number of cells, which were of donor (cord blood) origin, consisting mainly of T cells. No bacteria were detected in the CSF sample. Real-time PCR analysis revealed that the CSF sample was positive for CMV, but was negative for HHV-6, adenovirus, or BK virus. The patient was diagnosed with CMV meningoencephalitis and received cidofovir. His neurological symptoms were gradually improved and completely disappeared by day 60. CMV-specific dextramer-positive CD8(+) T cells were detected in the peripheral blood and CSF samples, with the frequency being much higher in the CSF. To our knowledge, this is the first report on the appearance of CMV-specific T cells in CSF samples from a patient with CMV meningoencephalitis. Cord blood-derived CMV-specific T cells may develop early after transplantation, enter the intrathecal compartment, and likely contribute to the regulation of CMV-meningoencephalitis.

Haploidentical hematopoietic stem cell transplantation for lymphoma with monosomy of chromosome 6 (loss of heterozygosity in the HLA region)--who should be a donor?

Hematopoietic stem cell transplantation (HSCT) from an HLA haploidentical family donor is an option for patients who do not have a full HLA matched donor and lack the time to find an unrelated one [1,2]. Furthermore, it may facilitate a powerful graft versus leukemia/lymphoma (GVL) effect to help combat hematological malignancies by directly targeting the mismatched HLA expressed on leukemia/lymphoma cells [3]. On the contrary, leukemia/lymphoma cells escape from the surveillance of the donor-derived GVL effect by losing the target HLA (mismatched HLA in GVH direction). This mechanism has been called loss of heterozygosity (LOH) in the HLA gene region on chromosome 6 [4,5]. Taking the above into account, in this case report, we present a case of lymphoma with monosomy 6, which means natural LOH of HLA, and suggest that selection of a haploidentical family donor matched with the missing HLA haplotype seems to be very effective.

Feasibility of unmanipulated haploidentical stem cell transplantation using standard GVHD prophylaxis for HLA-homozygous patients.

HLA-haploidentical hematopoietic stem cell transplantation (haplo-SCT) in HLA-homozygous patients is accompanied by HLA mismatches only in the host-versus-graft vector, and thus theoretically could be performed with standard graft-versus-host disease (GVHD) prophylaxis. However, the risk of GVHD remains uncertain, and graft failure could be a problem. In this study, we assessed nine HLA-homozygous patients who underwent haplo-SCT. Preparative treatment was cyclophosphamide/total body irradiation-based regimen in five patients, fludarabine/busulfan-based regimen in two, and other regimens in two. GVHD prophylaxis consisted of cyclosporine and methotrexate in seven patients, cyclosporine and mycophenolate mofetil in one, and cyclosporine alone in one. Seven patients achieved neutrophil engraftment and platelet recovery. The median times to neutrophil engraftment and platelet recovery were 15 and 44 days, respectively. Two patients developed graft failure, including one who achieved engraftment with a second SCT from the same donor. Grade II GVHD was observed in half of the evaluable patients; grades III and IV were not observed. Two patients died from treatment-related causes. Five patients were alive after a median follow-up period of 563 days. The probability of overall survival at 5 years was 65 %. These findings may serve as a rationale for considering haplo-SCT as a treatment option for HLA-homozygous patients.

Incidence and treatment strategy for disseminated adenovirus disease after haploidentical stem cell transplantation.

Adenovirus (AdV) infection is an emerging complication in patients undergoing allogeneic stem cell transplantation (SCT) and is closely associated with delayed immune reconstitution. In particular, disseminated AdV disease accompanies a high mortality. We retrospectively examined the incidence of AdV infection in patients undergoing unmanipulated haploidentical SCT. Following 121 transplantations in 110 patients, three had asymptomatic AdV viremia, three had localized AdV disease (hemorrhagic cystitis, HC), and seven had disseminated AdV disease (HC + viremia). The median time from transplantation to the onset of AdV-associated HC was 15 days (range 4-39), and the median time to the onset of disseminated AdV disease was 23 days (range 7-38). The cumulative incidence of AdV-associated HC was 8.3 %, and that of disseminated AdV disease was 5.8 %. AdV group B (type 11, type 34, or type 35) was detected in plasma samples from all the patients with disseminated AdV disease. Among them, three patients who received either cidofovir or donor lymphocyte infusion (DLI) alone progressed to pneumonia and died. The remaining four patients were treated with the combination of cidofovir and low-dose unmanipulated DLI, and all survived. We showed that disseminated AdV disease is a significant complication after haplo-SCT and that the combination of cidofovir and DLI is a promising treatment option.

Human umbilical cord provides a significant source of unexpanded mesenchymal stromal cells.

BACKGROUND AIMS: Human mesenchymal stromal cells (MSC) have considerable potential for cell-based therapies, including applications for regenerative medicine and immune suppression in graft-versus-host disease (GvHD). However, harvesting cells from the human body can cause iatrogenic disorders and in vitro expansion of MSC carries a risk of tumorigenesis and/or expansion of unexpected cell populations. METHODS: Given these problems, we have focused on umbilical cord, a tissue obtained with few ethical problems that contains significant numbers of MSC. We have developed a modified method to isolate MSC from umbilical cord, and investigated their properties using flow cytometry, mRNA analysis and an in vivo GvHD model. RESULTS: Our study demonstrates that, using umbilical cord, large numbers of MSC can be safely obtained using a simple procedure without in vitro expansion, and these non-expanded MSC have the potential to suppress GvHD. CONCLUSIONS: Our results suggest that the combined banking of umbilical cord-derived MSC and identical cord blood-derived hematopoietic stem cell banking, where strict inspection of the infectious disease status of donors is performed, as well as further benefits of HLA-matched mesenchymal cells, could become one of the main sources of cells for cell-based therapy against various disorders.

Mouse ES cells maintained in different pluripotency-promoting conditions differ in their neural differentiation propensity.

Prior to differentiation, embryonic stem (ES) cells in culture are maintained in a so-called "undifferentiated" state, allowing derivation of multiple downstream cell lineages when induced in a directed manner, which in turn grants these cells their "pluripotent" state. The current work is based on a simple observation that the initial culture condition for maintaining mouse ES cells in an "undifferentiated" state does impact on the differentiation propensity of these cells, in this case to a neuronal fate. We point out the importance in judging the "pluripotency" of a given stem cell culture, as this clearly demonstrated that the "undifferentiated" state of these cells is not necessarily a "pluripotent" state, even for a widely used mouse ES cell line. We partly attribute this difference in the initial value of ES cells to the naïve-to-primed status of pluripotency, which in turn may affect early events of the differentiation in vitro.

Intrabone marrow transplantation of unwashed cord blood using reduced-intensity conditioning treatment: a phase I study.

The outcome of cord blood transplantation following reduced-intensity conditioning is suboptimal because of fatal infection triggered by prolonged neutropenia and graft-versus-host disease (GVHD) in addition to graft rejection. Intrabone marrow injection (IBMI) may improve the outcome by providing better hematopoietic engraftment and less GVHD. We therefore evaluated IBMI safety in reduced-intensity stem cell transplantation. Furthermore, we used unwashed cord blood to avoid stem cell loss. Ten patients (median age = 61 years old) were enrolled. Cord blood cells were thawed at the bedside and injected into 4 iliac bone sites (2 at each hemipelvis). The procedure was well tolerated with no injection-related complications. Nine patients achieved donor engraftment. The median time to neutrophil recovery (>0.5 × 10(9)/L) was 17 days, and platelet recovery was achieved in 8 patients. Early full donor chimerism was achieved (median of 15 and 20 days in T cells and myeloid cells, respectively). Three of 9 evaluable patients developed grade II to III GVHD, and 5 of 10 patients died of treatment-related toxicities. The probability of survival at 1 year was 46.7%. IBMI of unwashed cord blood following reduced-intensity conditioning is safe, well tolerated, and may lead to an increased donor engraftment rate.

Allogeneic stem cell transplantation as treatment for heavily treated, refractory acute graft-versus-host disease after HLA-mismatched stem cell transplantation.

OBJECTIVE: No effective treatment has been established for patients with steroid-refractory acute graft-versus-host disease (GVHD). Recently, we demonstrated in a murine tandem bone marrow transplantation model that life-threatening GVHD established by the first bone marrow transplantation was successfully treated by engraftment of a second donor graft after reduced-intensity conditioning. We named the effect by which allografts counteract GVHD "graft-versus-GVHD." MATERIALS AND METHODS: To investigate the efficacy of graft-versus-GVHD treatment clinically, 16 patients who developed, after human leukocyte antigen-mismatched stem cell transplantation, severe GVHD, refractory to three to five lines of GVHD-specific treatments, underwent 17 allogeneic stem cell transplantations using reduced-intensity conditioning regimens with grafts from a second donor. RESULTS: Among the 15 transplantations that could be evaluated, rescue donor grafts were engrafted in 11 cases and rejected in 4 cases. For patients who achieved rescue donor engraftment, the response rate was 90.9% (eight complete response, two partial response, and one stable disease). Six of the eight patients with complete response survived without GVHD symptoms, with a median follow-up of 2128 days. No new development of GVHD by the second graft was observed. No patients had recurrence of the original malignant disease. In contrast, no long-term survivors were observed in patients who rejected rescue donor grafts. CONCLUSIONS: We propose here a novel graft-versus-GVHD treatment to treat refractory GVHD, and these results strongly suggest that GVHD can be successfully treated by eliminating the harmful lymphocytes responsible for GVHD by a second allogeneic stem cell transplantation.