Cell Transplantation Group Research Articles

Improvement of Relative Survival in Elderly Patients with Acute Myeloid Leukemia Emerging from Population-Based Cancer Registries in Switzerland from 2001-2013

Introduction: Acute Myeloid Leukaemia (AML) is a rare disease with increasing frequency in the elderly and an emerging impact on health care resources. Trends of classification, incidence, mortality, and survival of AML patients have not yet been reported for Switzerland. Given the demographic ageing, we were mainly interested to investigate whether elderly patients have benefited on a population-based level from the recent changes in AML management.Methods: We performed a population-based, observational analysis of AML cases reported to Cantonal Cancer Registries in Switzerland. Data was aggregated by the National Institute for Epidemiology and Cancer Registration and stratified for the two time-periods 2001-2007/2008-2013. The Swiss Federal Statistics Office provided canton-specific mid-year estimates of the size of the general population and mortality statistics. Data from transplanted patients was provided from the registry of the Swiss Blood Stem Cell Transplant Group .Results: 2'351 new AML cases were registered within the observation time at a median age of 68/67 yrs (range 0-96 yrs). The extrapolated mean annual frequency increased from 275 to 305 AML cases (+10.8%) in the two time periods. In contrast, the age-standardized incidence and mortality rates remained stable (3.0 [95 CI: 2.8-3.2] and 1.9-2.0 [1.8-2.1] per 100'000 person-years, respectively). Incidence was up to 1.4-fold more frequent in males and increased up to 7 fold in patients >75 yrs of age. The fraction of non-classifiable AML cases decreased over time (54.6%/41.8%), but remained high in elderly patients (75-84 yrs: 54.1%, 85+ yrs: 59.1%). As expected, 5-year relative survival (RS) correlated directly with AML risk classes (favorable: 61.7-68.4%, intermediate: 14.9%-27.3%, adverse: 11.4%-20.4%, non-classifiable: 11.4%-14.7%) and inversely with age (<65 yrs: 42.6-42.7%, 65-74 yrs: 5.2%-13.5%, 75-84 yrs: 2-3%; 85+ yrs: 0%). RS improved only modestly in all age classes over time (19.2%/23.3%). Most interestingly, a trend towards improved RS was found in AML patients aged 65-74 yrs (5.2%/13.5%) and with intermediate (20.4%/27.3%) as well as adverse risk (11.4%/ 21.9%). This trend was multifactorial and only partially explained by an increased referral to allogeneic HSCT (1.4%/7%) or inclusion in clinical trials of patients ≥65 yrs.Conclusions: AML incidence remained stable during the observation period, indicating that the 10.8% raise in annual case-frequency is mainly related to population growth and ageing and not to an increase of age-specific risk. AML classification improved over time, but non-classifiable AML cases remained high in elderly patients, suggesting that diagnostics and reporting is less accurate with increasing age. A trend towards improved RS was found in AML patients aged 65-74 yrs and with intermediate as well as adverse risk. These trends were caused by multiple factors and mainly based on the general changes of treatment management of AML patients ≥65 yrs. Survival of elderly AML patients remains dismal. Nevertheless, recent progress in clinical management of elderly AML patients resulted in an emerging improvement of survival on a population-based level in Switzerland. The demographic trend will further increase AML burden and has to be taken into account for structural as well as financial considerations for future health care systems. [Display omitted] DisclosuresGregor:AbbVie: Other: Personal Fees; Celgene: Other: Personal Fees, Non-Financial Support; Gilead: Other: Personal Fees; GlaxoSmithKline: Other: Personal Fees; Janssen: Other: Personal Fees, Non-Financial Support; Mundipharma: Other: Personal Fees; Novartis: Other: Personal Fees, Non-Financial Support; Roche: Other: Personal Fees, Non-Financial Support.

Clinical analysis of B-cell lymphoma treatment with autologous hematopoietic stem cell transplantation

Objective To investigate the clinical efficacy, safety and prognostic factors of autologous hematopoietic stem cell transplantation (auto-HSCT) in treatment of B-cell lymphoma. Methods From March 2001 to December 2014, a total of 50 patients with B-cell lymphoma who underwent auto-HSCT at Tangdu Hospital, the Fourth Military Medical University were selected as the study subjects. Among them, 24 patients were suffering from diffuse large B-cell lymphoma (DLBCL), 2 patients were suffering from follicular lymphoma (FL), 3 patients were suffering from B lymphoblastic lymphoma, 21 patients were suffering from B-cell lymphoma (undifferentiated). According to number of times of receiving auto-HSCT, these 50 patients were divided into two groups: single auto-HSCT group (n=28, only receiving auto-HSCT once) and double autologous hematopoietic stem cell transplantation (DAHSCT) group (n=22, the second auto-HSCT was planned in 2 to 6 months after the first auto-HSCT). Retrospective method was used to collect related clinical data from all patients. The severity of post-transplant adverse events was assessed according to the Common Criteria for Terminology of Incidents, version 4.0 (CTCAE v4.0). The efficacy of auto-HSCT was evaluated according to revised International Working Group (IWG) lymphoma efficacy criteria and the overall response rate of auto-HSCT was calculated. Survival analysis was performed using Kaplan-Meier method. Overall survival (OS) and progression-free survival (PFS) rates were compared between two groups using log-rank test. Cox regression analysis was used to univariate analyze the influencing factors that may influence the OS peroid of B-cell lymphoma patients in this study. According to available research results and clinical experience, multivariate Cox regression analysis was performed on the factors that were statistically significant in the univariate analysis. Results ①The differences of composition ratios of clinical stage and age-adjusted international prognostic index (aaIPI) score in single auto-HSCT group and DAHSCT group were significantly (composition ratio of patients with clinical stage Ⅰ-Ⅱ: 25.0% vs 54.5%, χ2=4.565, P=0.033; Composition ratio of patients with aaIPI 0-2: 35.7% vs 86.4%, χ2=12.974, P 0.05). ④ In single auto-HSCT group, 18 patients achieved complete remission (CR) and 8 patients achieved partial remission (PR), with the total response rate of auto-HSCT of 92.9% (26/28). In DAHSCT group, 20 patients achieved CR and 2 patients achieved PR, with the total response rate of DAHSCT of 100.0% (22/22). The median follow-up time was 54.0 months (24.8-76.3 months) in this study, and OS rate was 68.0%, PFS rate was 52.0%. OS rate and PFS rate were 77.3% and 68.2% in the DAHSCT group, which were both higher than that of 60.7% and 39.3% in the single auto-HSCT group respectively, and the differences were statistically significant (χ2=8.073, 4.250; P=0.004, 0.039). ⑤ The results of univariate Cox regression analysis showed that level of serum lactate dehydrogenase (LDH) and number of times of auto-HSCT were the risk factors influencing OS period in patients with B-cell lymphoma (P=0.033, 0.045). And results of multivariate Cox regression analysis showed that serum LDH levels>245 U/L and single auto-HSCT were independent risk factors for OS period of patients with B-cell lymphoma (HR=0.237, 95%CI: 0.062-0.905, P=0.035; HR=3.063, 95%CI: 0.970-9.675, P=0.047) in this study. Conclusions Auto-HSCT treatment of patients with B-cell lymphoma were with good efficacy, tolerability and safety, which is worth further clinical application. Key words: Lymphoma, B cells; Hematopoietic stem cell transplantation; Transplantation, autologous

Therapeutic effect of adipose‑derived stem cell transplantation on optic nerve injury in rats.

Adipose-derived stem cells (ADSCs) are mesenchymal stem cells in adipose tissue. ADSCs may differentiate into cells and tissues of different embryonic germ layers under specific inducing conditions. Previous studies have demonstrated that Schwann cells transformed from ADSCs can express nerve growth factors, generate nerve fibers and promote axonal regeneration in models of peripheral nerve injury. Stem cell transplantation is one of the potential techniques for the treatment of retinal ganglion cell injury. The present study developed an optic nerve crush injury rat model to investigate the therapeutic effect of fat stem cell transplantation on optic nerve injured rats. Retinal morphological changes were observed on day 3, 7, 14, 21 and 28 following optic nerve injury. It was determined that the number of retinal ganglion cells (RGCs) decreased with time following optic nerve injury; however, the number of RGCs in the stem cell transplantation group was higher compared with the buffer control group. The results of reverse transcription-quantitative polymerase chain reaction also demonstrated that the growth associated protein 43 mRNA expression level of the stem cell transplantation group was higher compared with the buffer control group. Apoptosis tests demonstrated that the stem cell transplantation group was able to resist the apoptosis of retinal cells. The present study demonstrated that invivo transplantation of ADSCs can improve the survival rate of RGCs in rats with optic nerve injury and to resist the apoptosis of retinal cells.

Therapeutic effect and prognostic factors of adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia

Objective To explore the outcome and prognostic factors of adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL). Methods Forty-nine newly diagnosed adult patients with Ph+ ALL were analyzed retrospectively, and the treatment effect and the impact of different factors on prognosis were explored. Results In 49 patients, there were 24 males and 25 females; the median age was 38 years (range 15-77 years). Hematologic complete remission (CR), major molecular response (MMR) and complete molecular remission (CMR) rate in patients received tyrosine kinase inhibitors (TKI) plus chemotherapy were higher than those in patients received chemotherapy only (96.8% vs. 72.2%, χ2 = 4.308, P = 0.038; 64.5% vs. 16.7%, χ 2 = 10.468, P = 0.001; 25.8% vs. 11.1%, χ 2 = 4.250, P = 0.039). With a median overall survival (OS) of 24 months (3-70 months), the 3-year OS and relapse-free survival (RFS) rates were 32.7% and 21.4%, respectively. The 3-year OS rate and 1-year RFS rate in TKI plus chemotherapy group were 40.3% and 67.8% respectively, which were higher than those in chemotherapy group (11.1% and 11.1%) (χ 2 = 12.725, χ 2 = 17.401, both P < 0.001). The 3-year OS and RFS rates in the allogeneic hematopoietic stem cell transplantation (allo-HSCT) group were higher than those in the group without allo-HSCT (62.5% vs. 25.7%, χ2 = 6.196, P = 0.013; 41.7% vs. 15.0%, χ2 = 8.032, P = 0.005). The 3-year OS and RFS rates in patients achieved MMR after 2 circles treatment were higher than those in the others (45.1% vs. 17.6%, χ2 = 5.446, P = 0.020; 28.9% vs. 11.7%, χ2 = 6.484, P = 0.011). Multivariate analysis showed that received TKI (HR = 0.227, 95% CI 0.094-0.550, P = 0.001) was an independent prognostic factor for OS; received TKI (HR = 0.225, 95% CI 0.082-0.618, P = 0.004) and allo-HSCT (HR = 0.275, 95% CI 0.077-0.983, P = 0.047) were independent prognostic factors for RFS. Conclusions TKI can increase CR, MMR and CMR rates, improve outcome, and give more chance to receive HSCT. In TKI era, allo-HSCT is still the important treatment for Ph+ ALL, especially for patients without MMR. Key words: Leukemia, acute; Philadelphia chromosome; Prognosis; Hematopoietic stem cell transplantation; Imatinib

Purified Human Skeletal Muscle-Derived Stem Cells Enhance the Repair and Regeneration in the Damaged Urethra.

Postoperative damage of the urethral rhabdosphincter and nerve-vascular networks is a major complication of radical prostatectomy and generally causes incontinence and/or erectile dysfunction. The human skeletal muscle-derived stem cells, which have a synchronized reconstitution capacity of muscle-nerve-blood vessel units, were applied to this damage. Cells were enzymatically extracted from the human skeletal muscle, sorted using flow cytometry as CD34/45 (Sk-34) and CD29/34/45 (Sk-DN/29) fractions, and separately cultured/expanded in appropriate conditions within 2 weeks. Urethral damage was induced by manually removing one third of the wall of the muscle layer in nude rats. A mixture of expanded Sk-34 and Sk-DN/29 cells was applied on the damaged portion for the cell transplantation (CT) group. The same amount of media was used for the non-CT (NT) group. Urethral pressure profile was evaluated via electrical stimulation to assess functional recovery. Cell engraftments and differentiations were detected using immunohistochemistry and immunoelectron microscopy. Expression of angiogenic cytokines was also analyzed using reverse transcriptase-polymerase chain reaction and protein array. At 6 weeks after transplantation, the CT group showed a significantly higher functional recovery than the NT group (70.2% and 39.1%, respectively; P < 0.05). Histological analysis revealed that the transplanted human cells differentiated into skeletal muscle fibers, nerve-related Schwann cells, perineuriums, and vascular pericytes. Active paracrine angiogenic cytokines in the mixed cells were also detected with enhanced vascular formation in vivo. The transplantation of Sk-34 and Sk-DN/29 cells is potentially useful for the reconstitution of postoperative damage of the urethral rhabdosphincter and nerve-vascular networks.

Bone marrow derived mesenchymal stem cell transplantation for treatment of denervated intrinsic paw muscular atrophy: an experimental research in rats

Objective To investigate the feasibility of bone marrow derived mesenchymal stem cell (BMSC) transplantation in the treatment of denervated intrinsic paw muscle atrophy. Methods Twelve adult male Sprague-Dawley rats (24 sides) were randomly divided into ulnar nerve transection group, stem cell transplantation group, culture medium injection group and normal control group. GFP-transfected BMSCs cultured in vitro were injected into the paw muscles of rats in the stem cell transplantation group. After 1 month, intrinsic paw muscles were tested by histology, immunofluorescence and PCR. Results The muscle wet weight and muscle fiber cross-sectional area of stem cell transplantation group were 66% and 63% of normal control group, respectively, and twice as much as that of ulnar nerve transection and culture medium injection groups (P<0.05). GFP-positive mesenchymal stem cells still could be detected at injection site. The expression of VEGF, HGF, CNTF, bFGF in stem cell transplantation group were higher than that in ulnar nerve transection and culture medium injection groups, and the apoptotic index (apoptotic cells/total cells) in stem cell transplantation group was smaller (P<0.05). Conclusion BMSCs can effectively delay the denervated intrinsic paw muscular atrophy. BMSCs may have the capbility of secreting neurotrophic factor and reducing muscle tissue apoptosis. Key words: Mesenchymal stem cell transplantation; Muscular atrophy; Intrinsic paw muscles; Animal experimentation

Transplantation of Human Dental Pulp-Derived Stem Cells or Differentiated Neuronal Cells from Human Dental Pulp-Derived Stem Cells Identically Enhances Regeneration of the Injured Peripheral Nerve.

Human dental mesenchymal stem cells isolated from the dental follicle, pulp, and root apical papilla of extracted wisdom teeth have been known to exhibit successful and potent neurogenic differentiation capacity. In particular, human dental pulp-derived stem cells (hDPSCs) stand out as the most prominent source for in vitro neuronal differentiation. In this study, to evaluate the in vivo peripheral nerve regeneration potential of hDPSCs and differentiated neuronal cells from DPSCs (DF-DPSCs), a total of 1 × 106 hDPSCs or DF-hDPSCs labeled with PKH26 tracking dye and supplemented with fibrin glue scaffold and collagen tubulization were transplanted into the sciatic nerve resection (5-mm gap) of rat models. At 12 weeks after cell transplantation, both hDPSC and DF-hDPSC groups showed notably increased behavioral activities and higher muscle contraction forces compared with those in the non-cell transplanted control group. In immunohistochemical analysis of regenerated nerve specimens, specific markers for angiogenesis, axonal fiber, and myelin sheath increased in both the cell transplantation groups. Pretransplanted labeled PKH26 were also distinctly detected in the regenerated nerve tissues, indicating that transplanted cells were well-preserved and differentiated into nerve cells. Furthermore, no difference was observed in the nerve regeneration potential between the hDPSC and DF-hDPSC transplanted groups. These results demonstrate that dental pulp tissue is an excellent stem cell source for nerve regeneration, and in vivo transplantation of the undifferentiated hDPSCs could exhibit sufficient and excellent peripheral nerve regeneration potential.

Clinical observation on limbal stem cell transplantation and amniotic membrane transplantation for the treatment of pterygium

Objective To observe the clinical effect of amniotic membrane transplantation and autologous corneal limbal stem cell transplantation in the treatment of primary pterygium. Methods Seventy-five eyes of 68 cases of pterygium were randomly divided into two groups: the stem cell transplantation groups, 38 eyes of 35 cases, underwent pterygium excision combined with autologous limbal stem cell transplantation; amniotic membrane transplantation group, 37 eyes of 33 cases, underwent pterygium excision combined with amniotic membrane transplantation. The postoperative recurrence, corneal wound healing time and complications of two groups were observed. Results The recurrence rate of stem cell transplantation group was 2.6%, the recurrence rate of amniotic membrane transplantation group was 18.9%, and the recurrence rate of stem cell transplantation group was lower than that of amniotic membrane transplantation group. In the stem cell transplantation group, corneal wound healing time was average (3.49±0.50) days, the average time of corneal wound healing time was (5.28±1.37) days. The corneal wound healing time of the stem cell transplantation group was shorter than that of the amniotic membrane transplantation group, the difference was statistically significant (t=7.550, P=0.000). Conclusion The effect of autologous corneal limbal stem cell transplantation for primary pterygium, with shorter healing time, lower recurrence rate and fewer complications, is better than that of amniotic membrane transplantation. Key words: Pterygium; Transplantation; Stem cell; Amniotic membrane

Survival, differentiation and migration of the spinal cord derived neural stem cells from transgenic mice transplanted into mouse models of spinal cord injury

Objective To observe the mice motion recovery and neural stem cell (NSCs) survival, migration and differentiation after the green fluorescent protein (GFP)+ -positive NSCs which was isolated and cultivated from mouse embryonic spinal cord was transplanted into mouse models of spinal cord injury lesions. Methods mice were randomly divided into control group (n=10), injury group (n=30) and cell transplanted group (n=30). The NSCs were transplanted into spinal cord injury after establishing the injured models. The Basso-Beattie-Bresnahan (BBB) function scores, inclined plane test and immunohistochemistry testing were respectively evaluated and recorded 1, 2, 4, 6, 8 weeks after the operation. Results The mouse hind limbs were paralyzed after hitting and BBB scores were 0. The transplanted group increased to 4.50 ± 1.04, injury group was 2.17 ± 0.75, scores began to appear differences at 2 weeks (P=0.023). The scores of transplanted group was 8.17±1.47 and injury group was 5.33±1.03 at 4 weeks (P=0.015). The scores of two groups were increased with the passage of time after 4 weeks. The scores of transplanted group was 10.8±1.47 and injury group was 8.33±0.81 at 6 weeks (P=0.017). The difference was biggest at 8 weeks, transplanted group was 15.50±1.37 and injury group was 11.17±1.16 (P=0.016). The angle of transplanted group was (17.17±3.18)°and injury group was (14.83±3.06)° at 1 weeks. At 2 weeks, the angle of transplanted group was (23.33±4.27)°and injury group was (18.17±2.40)°. The two groups’ results appeared significant differences at 4 weeks in inclined plane test (P=0.024). The transplanted group was (33.83±6.30)°, injury group was (23.50±1.76)°(P=0.029). The small difference of The difference was biggest at 8 weeks, transplanted group was (46.83±6.05)° and injury group was (36.33±7.23)°(P=0.019). The transplanted NSCs can survive and migrate in the body, some of which can remain undifferentiated state, a small part of which can differentiate into glia, less of which differentiate into neurons. Conclusion There was a promoting effect of NSCs for the recovery of spinal cord injury. It was forming a micro-environment conducive to resume by transplanting cells and increasing the proportion of neurons to promote recovery. Key words: Green fluorescent protein; Neural stem cells; Spinal cord injury; Cell transplantation; Mouses

Human Muse Cells, Nontumorigenic Phiripotent-Like Stem Cells, Have Liver Regeneration Capacity through Specific Homing and Cell Replacement in a Mouse Model of Liver Fibrosis

Muse cells, a novel type of nontumorigenic pluripotent-like stem cells, reside in the bone marrow, skin, and adipose tissue and are collectable as cells positive for pluripotent surface marker SSEA-3. They are able to differentiate into cells representative of all three germ layers. The capacity of intravenously injected human bone marrow-derived Muse cells to repair an immunodeficient mouse model of liver fibrosis was evaluated in this study. The cells exhibited the ability to spontaneously differentiate into hepatoblast/hepatocyte lineage cells in vitro. They demonstrated a high migration capacity toward the serum and liver section of carbon tetrachloride-treated mice in vitro. In vivo, they specifically accumulated in the liver, but not in other organs except, to a lesser extent, in the lungs at 2 weeks after intravenous injection in the liver fibrosis model. After homing, Muse cells spontaneously differentiated in vivo into HepPar-1 (71.1±15.2%), human albumin (54.3±8.2%), and anti-trypsin (47.9±4.6%)-positive cells without fusing with host hepatocytes, and expressed mature functional markers such as human CYP1A2 and human Glc-6-Pase at 8 weeks after injection. Recovery in serum, total bilirubin, and albumin and significant attenuation of fibrosis were recognized with statistical differences between the Muse cell-transplanted group and the control groups, which received the vehicle or the same number of a non-Muse cell population of MSCs (MSCs in which Muse cells were eliminated). Thus, unlike ESCs and iPSCs, Muse cells are unique in their efficient migration and integration into the damaged liver after intravenous injection, nontumorigenicity, and spontaneous differentiation into hepatocytes, rendering induction into hepatocytes prior to transplantation unnecessary. They may repair liver fibrosis by two simple steps: expansion after collection from the bone marrow and intravenous injection. A therapeutic strategy such as this is feasible and may provide significant advancements toward liver regeneration in patients with liver disease.

Establishment of intraperitoneal germ cell transplantation for critically endangered Chinese sturgeon Acipenser sinensis

Recent progress in germ cell transplantation techniques in fish has paved the way for the conservation of endangered species. Here, we developed an intraperitoneal germ cell transplantation procedure using Chinese and Dabry's sturgeon as donor and recipient species, respectively. Histological analysis revealed that primordial germ cells migrated on the peritoneal wall at 16 days post-hatch (dph) in Dabry's sturgeon. The genital ridges of Dabry's sturgeon (recipient) first formed at 28 dph, suggesting that for successful colonization of donor germ cells in the recipient gonads, the transplantation should be performed earlier than this age. Sexual dimorphism of gonadal structure was first observed at 78 dph. Gonadal germ cell proliferation was not seen in either sex during this period. Immunohistochemistry using the anti-Vasa antibody found that donor testes from 2-year-old Dabry's sturgeon mainly consisted of single- or paired-type A spermatogonia, while donor ovaries from 11.5-year-old Chinese sturgeon had perinucleolus stage oocytes and clusters of oogonia. Donor cells isolated from Dabry's sturgeon testes or Chinese sturgeon ovary labeled with PKH26 fluorescent dye were transplanted into the peritoneal cavity of the 7- or 8-dph Dabry's sturgeon larvae. More than 90% and 70% of transplanted larvae survived after 2 days post-transplantation (dpt) and 51 dpt, respectively. At 51 dpt, PKH26-labeled cells exhibiting germ cell-specific nuclear morphology and diameter were observed in excised recipient gonads by fluorescent and confocal microscopy. The colonization rate of allogeneic testicular germ cell transplantation (Group 1) was 70%, while that of two batches of xenogeneic ovarian germ cell transplantation (Group 2 and Group 3) were 6.7% and 40%, respectively. The ratio of colonized germ cells to endogenous germ cells was 11.96%, 5.35% and 3.56% for Group 1, Group 2 and Group 3, respectively. Thus, we established a germ cell transplantation technique for the critically endangered Chinese sturgeon using the most closely related species as a recipient and demonstrated the successful preparation of transplantable female germ cells from aged adult Chinese sturgeon.

Peripheral blood autologous CD34+ cell transplantation promotes angiogenesis in elderly patients with atherosclerotic ischemia: study protocol for a prospective, single-center, open-label, randomized, controlled, clinical trial

Background: The main clinical manifestation of senile arteriosclerosis obliterans is lower limb ischemia, which is currently difficult to treat. One method is autologous stem cell transplantation into the muscles of ischemic limbs to improve the formation of new capillaries and restore lower limb blood flow. Endothelial progenitor cell marker CD34+ cell transplantation has been shown to promote angiogenesis in ischemic limbs. Therefore, we propose that peripheral blood autologous CD34+ cell transplantation in older adult patients with atherosclerotic ischemia could effectively promote angiogenesis. Methods/Design: We propose to conduct a prospective, single-center, open-label, randomized, and controlled clinical trial at the Qingdao No. 9 People's Hospital, China. Twenty older adult patients with atherosclerotic lower limb ischemia will be randomized into two groups. In the cell transplantation group (n = 10), peripheral blood CD34+ cells transfected with vascular endothelial growth factor 165 (VEGF165) gene will be transplanted into the muscles of ischemic limbs in older adult patients with atherosclerotic lower limb ischemia. In the control group (n = 10), physiological saline will be injected into the muscles of ischemic limbs. Patients will be followed up for 6 months. The primary outcome will be ankle-brachial indices before and 6 months after transplantation to assess lower limb ischemia in both groups. The secondary outcomes will be the number of microvessels in the lower limb muscles before and 6 months after transplantation, the morphology of new blood vessels revealed by CT angiography, the number of VEGF-immunoreactive cells 6 months after transplantation and the incidence of adverse reactions. Discussion: This trial will begin in January 2018 and finish in December 2019. We aim to quantify the effects of VEGF165 gene-modified CD34+ cell transplantation in the treatment of older adult patients with atherosclerotic ischemia to develop a new effective treatment of lower limb ischemia. Trial registration: ClinicalTrials.gov identifier: NCT03098771. Ethics: The study protocol has been approved by the Ethics Committee of Qingdao No. 9 People's Hospital of China. All protocols will be in accordance with the Declaration of Helsinki, formulated by the World Medical Association. Informed consent: Written informed consent will be provided by participants.

FLT4 Expressing CD34+CD38- Leukemic Stem Cells in Bone Marrow of Refractory Patients are Protected by Receptor Internalization under Abundant VEGF-C Condition

So far, the cause of relapse or refractoriness in acute myeloid leukemia (AML) is regarded as the persistence of chemoresistant leukemic stem cells (LSCs). Even VEGF-C/FLT4 axis mediates blast proliferation and survival, understanding of the dynamic behavior of LSCs in bone marrow remains elusive. Especially, FLT4 endocytosis by VEGF-C is related to various biologic functions including signal activation. Based on our previous study, which addressed the potential of FLT4 as a marker for LSCs, we showed that surface FLT4 can be internalized by VEGF-C exposure in bone marrow (BM) derived LSCs. It resulted in resistant to chemotherapy, suggesting LSCs protection. In addition, data for apoptosis revealed that inhibition of FLT4 using MAZ51 with cytosine arabinoside (Ara-C) can effectually increase cell death under abundant VEGF-C in refractory patients. It indicated that targeting FLT4 allows investigators to establish advanced therapeutic strategy with conventional Ara-C in refractory patients with high VEGF-C. In clinic, low level of surface FLT4 in de novo AML-BM LSCs, but not PB cells were inclined to undergo refractory status after induction chemotherapy. To investigate whether FLT4 expressing CD34+CD38- LSCs can be protected by VEGF-C via internalization, FACS analysis, western blotting and immunocytochemistry were performed. Meanwhile, total 66 newly diagnosed (ND) AML patients were used to analyze for clinical correlation. (BM, n=37; PB, n=19). FACS analysis showed that surface FLT4 expressing CD34+CD38- LSCs in BM, but not PB, were significantly decreased by VEGF-C (In BM; without VEGF-C & with VEGF-C, 38.7±8.1% & 16.0±5.1%). Similar with surface FLT4 in PB cells, both intracellular FLT4 in BM and PB was highly sustained, regardless of VEGF-C (In BM; without VEGF-C & with VEGF-C, 62.9±17.2% & 69.7±14.9%, In PB; without VEGF-C & with VEGF-C, 77.4±15.6% & 70.4±21.8%). Immunocytochemistry and western blotting also showed internalization of surface FLT4 by VEGF-C treatment. FLT4+CD34+CD38- cells both in BM and PB were significantly higher in refractory patients than in post allogeneic stem cell transplantation (SCT) group, implying clinical correlation of FLT4 with refractory patients. Consistent with findings in ND-AML and refractory groups, FLT4+CD34+CD38- cells were highly sustained in complete remission group, showing drug resistant population (ND-AML, n=30; refractory AML, n=9; Complete remission AML, n=18; post-allogeneic stem cell transplantation, n=9). High level of VEGF-C was detected in refractory patients, compared to that of normal donors (Refractory AML, n=9, normal donors, n=7). Apoptosis results showed that high number of apoptotic CD45dimCD34+CD38- cells in MAZ51 (FLT4 antagonist) and Ara-C dual treatment under VEGF-C exposure, compared to no treatment of both MAZ51 and Ara-C (without VEGF-C; 0.5-1.3 folds, with VEGF-C; 2.7-43.3 folds) or single exposure either to MAZ51 or Ara-C (In Ara-C only; without VEGF-C; 1.0-9.5 folds, with VEGF-C; 0.1- 1.3 folds, In MAZ51 only; without VEGF-C; 1.1-1.7 folds, with VEGF-C; 1.3-39.3 folds), suggesting that Ara-C induced-blast apoptosis can be augmented by FLT4 inhibition even in the presence of VEGF-C in a sub-group of either refractory or relapsed AML patients. VEGF-C/FLT4 axis in AML is involved in PI3K and AKT pathway.Collectively, we demonstrated that FLT4 internalization under VEGF-C in BM leads to protection of FLT4 expressing LSCs and is clinically relevant to refractory subgroup. This data could suggest some clues to develop therapeutic strategies in targeting FLT4 expressing refractory LSCs. DisclosuresNo relevant conflicts of interest to declare.

Analysis of Therapy and Efficacy after Remission of Acute Myeloid Leukemia

To investigate the therapy and efficacy after remission of patients with acute myeloid leukemia (AML). The clinical data of 110 patients diagnosed as AML treated from 2008 to 2013 were analyzed retrospectively. According to different consolidation therapy regimens, the patients were divided into 4 groups:1 ID-Ara-C group, 2 ID-Ara-C group, 3-4 ID-Ara-C group, allogeneic hematopoietic stem cell transplantation (allo-HSCT) group. The disease-free survival (DFS) and overall survival (OS) were analyzed retrospectovely. 110 patients were completely remittend by 1-2 couses. The median follow-up time was 26.8 months, 35 cases relapsed and 58 cases died, the median DFS and OS were 20.5 and 26.8 months. The 3-year DFS of 1, 2, 3-4 ID-Ara-C and allo-HSCT groups were 0%, 36.1%, 37.5% and 67.9% respectively. The 5-year DFS of 1, 2, 3-4 ID-Ara-C and allo-HSCT groups were 0%, 30.1%, 37.5% and 63.0%. The 3-year OS rates of 1, 2 ID-Ara-C groups, 3-4 ID-Ara-C group and allo-HSCT group were 24.0%, 36.0%, 58.3% and 67.8%. The 5-year OS of 1, 2 ID-Ara-C grous, 3-4 ID-Ara-C and allo-HSCT group were 24.0%, 36.0%, 58.3% and 67.8% respectively. The 5-year OS rates of 1 ID-Ara-C group, 2 ID-Ara-C group, 3-4 ID-Ara-C group were 0%,30.0%,35.0% and 62.9% respectively. The multifactor analysis indicated that the courses of ID-Ara-C and allo-HSCT were independent risk factors for DFS and OS. ≥2 ID-Ara-C regimen may be used as one regimen of consolidation therapy for patients with AML after remission.

Cell therapy for cerebral hemorrhage: Five year follow-up report.

The aim of the study was to examine treatment of cerebral hemorrhages with bone-marrow or human umbilical cord-derived mesenchymal stem cells (BMSCs or Hu-MSCs) and conventional surgical approaches, and determine and compare the effectiveness, feasibility, safety and reproducibility of each method. A retrospective analysis was performed on a cohort of cell-treated cerebral hemorrhage patients from October 1, 2007 to October 1, 2009. A total of 24 patients, all of whom received conventional surgical treatment, were classified as follows: i) The control group consisted of 8 patients who received only hematoma removal surgery, ii) the autologous group consisted of 7 patients who received additional autologous bone marrow mononuclear cell transplantation, and iii) the allograft group consisted of 9 patients who received additional umbilical cord mononuclear cell transplantation. After conventional hematoma removal surgery and X-ray supervision within 24 h and at 7 days, neurological disability and function tests were completed 3, 6, 12, 36 and 60 months later. The T-cell marker plasma levels were analyzed after 60 months. The results showed that, at approximately 3.5 months after graft the hematomas in all the groups were completely reabsorbed as observed on computed tomography scans. However, the functional outcomes in the cell-transplanted groups were better than in the control group after 5 years. While the National Institutes of Health Stroke Scale, modified Rankin score and modified Barthel index scores were simliar in the cell-transplanted groups, patients in the allograft group had better outcomes than those in the autologous graft group starting at 3 months and until the end of the follow-up period. The serum levels of T-cell markers CD4, CD56 and human leukocyte antigen-DR in the allograft group showed no signs of immunogenic graft complications and there were no significant differences in T-cell subtypes among the patient groups. The results of the present study suggest that, treatment of cerebral hemorrhage patients can be safely and effectively accomplished using Hu-MSC grafting and larger clinical trials should be considered in the future.

In Vivo Dynamic Metabolic Changes After Transplantation of Induced Pluripotent Stem Cells for Ischemic Injury.

Serial 18F-FDG PET, echocardiographic, immunohistochemical, and immunofluorescence studies were performed after transplantation of iPSCs and iPSC-CMs and compared with embryonic stem cells (ESCs), ESC-CMs, and a phosphate-buffered saline control group of rats with myocardial infarction. Increased glucose metabolism in periinfarct areas and improved myocardial function were observed in the stem cell transplantation groups compared with the control group, and serial immunofluorescence and immunohistochemical results exhibited the survival and migration of stem cells during the study period. Serial 18F-FDG PET and echocardiographic imaging studies demonstrated the dynamic metabolic changes and recovery of myocardial function after stem cell transplantation. 18F-FDG PET could be a potential approach to evaluating spatiotemporal dynamic metabolic changes in vivo after transplantation of iPSCs or iPSC-CMs for ischemic injury.

The Protective Effect of Transplanting Liver Cells Into the Mesentery on the Rescue of Acute Liver Failure After Massive Hepatectomy.

Postoperative liver failure is one of the most critical complications following extensive hepatectomy. Although transplantation of allogeneic hepatocytes is an attractive therapy for posthepatectomy liver failure, transplanting cells via the portal veins typically causes portal vein embolization. The embolization by transplanted cells would be lethal in patients who have undergone massive hepatectomy. Thus, transplant surgeons need to select extrahepatic sites as transplant sites to prevent portal vein embolization. We aimed to investigate the mechanism of how liver cells transplanted into the mesentery protect recipient rats from acute liver failure after massive hepatectomy. We induced posthepatectomy liver failure by 90% hepatectomy in rats. Liver cells harvested from rat livers were transplanted into the mesenteries of hepatectomized rats. Twenty percent of the harvested cells, which consisted of hepatocytes and nonparenchymal cells, were transplanted into each recipient. The survival rate improved significantly in the liver cell transplantation group compared to the control group 7 days after hepatectomy (69 vs. 7%). Histological findings of the transplantation site, in vivo imaging system study findings, quantitative polymerase chain reaction assays of the transplanted cells, and serum albumin measurements of transplanted Nagase analbuminemic rats showed rapid deterioration of viable transplanted cells. Although viable transplanted cells deteriorated in the transplanted site, histological findings and an adenosine-5'-triphosphate (ATP) assay showed that the transplanted cells had a protective effect on the remaining livers. These results indicated that the paracrine effects of transplanted liver cells had therapeutic effects. The same protective effects were observed in the hepatocyte transplantation group, but not in the liver nonparenchymal cell transplantation group. Therefore, this effect on the remnant liver was mainly due to the hepatocytes among the transplanted liver cells. We demonstrated that transplanted liver cells protect the remnant liver from severe damage after massive hepatectomy.

Comparative effect of immature neuronal or glial cell transplantation on motor functional recovery following experimental traumatic brain injury in rats.

The present study evaluated the comparative effect of stereotaxically transplanted immature neuronal or glial cells in brain on motor functional recovery and cytokine expression after cold-induced traumatic brain injury (TBI) in adult rats. A total of 60 rats were divided into four groups (n=15/group): Sham group; TBI only group; TBI plus neuronal cells-transplanted group (NC-G); and TBI plus glial cells-transplanted group (GC-G). Cortical lesions were induced by a touching metal stamp, frozen with liquid nitrogen, to the dura mater over the motor cortex of adult rats. Neuronal and glial cells were isolated from rat embryonic and newborn cortices, respectively, and cultured in culture flasks. Rats received neurons or glia grafts (~1×106 cells) 5 days after TBI was induced. Motor functional evaluation was performed with the rotarod test prior to and following glial and neural cell grafts. Five rats from each group were sacrificed at 2, 4 and 6 weeks post-cell transplantation. Immunofluorescence staining was performed on brain section to identify the transplanted neuronal or glial cells using neural and astrocytic markers. The expression levels of cytokines, including transforming growth factor-β, glial cell-derived neurotrophic factor and vascular endothelial growth factor, which have key roles in the proliferation, differentiation and survival of neural cells, were analyzed by immunohistochemistry and western blotting. A localized cortical lesion was evoked in all injured rats, resulting in significant motor deficits. Transplanted cells successfully migrated and survived in the injured brain lesion, and the expression of neuronal and astrocyte markers were detected in the NC-G and GC-G groups, respectively. Rats in the NC-G and GC-G cell-transplanted groups exhibited significant motor functional recovery and reduced histopathologic lesions, as compared with the TBI-G rats that did not receive neural cells (P<0.05, respectively). Furthermore, GC-G treatment induced significantly improved motor functional recovery, as compared with the NC-G group (P<0.05). Increased cytokine expression levels were detected in the NC-G and GC-G groups, as compared with the TBI-G; however, no differences were found between the two groups. These data suggested that transplanted immature neural cells may promote the survival of neural cells in cortical lesion and motor functional recovery. Furthermore, transplanted glial cells may be used as an effective therapeutic tool for TBI patients with abnormalities in motor functional recovery and cytokine expression.

Combination of low-energy shock-wave therapy and bone marrow mesenchymal stem cell transplantation to improve the erectile function of diabetic rats.

Stem cell transplantation and low-energy shock-wave therapy (LESWT) have emerged as potential and effective treatment protocols for diabetic erectile dysfunction. During the tracking of transplanted stem cells in diabetic erectile dysfunction models, the number of visible stem cells was rather low and decreased quickly. LESWT could recruit endogenous stem cells to the cavernous body and improve the microenvironment in diabetic cavernous tissue. Thus, we deduced that LESWT might benefit transplanted stem cell survival and improve the effects of stem cell transplantation. In this research, 42 streptozotocin-induced diabetic rats were randomized into four groups: the diabetic group (n = 6), the LESWT group (n = 6), the bone marrow-derived mesenchymal stem cell (BMSC) transplantation group (n = 15), and the combination of LESWT and BMSC transplantation group (n = 15). One and three days after BMSC transplantation, three rats were randomly chosen to observe the survival numbers of BMSCs in the cavernous body. Four weeks after BMSC transplantation, the following parameters were assessed: the surviving number of transplanted BMSCs in the cavernous tissue, erectile function, real-time polymerase chain reaction, and penile immunohistochemical assessment. Our research found that LESWT favored the survival of transplanted BMSCs in the cavernous body, which might be related to increased stromal cell-derived factor-1 expression and the enhancement of angiogenesis in the diabetic cavernous tissue. The combination of LESWT and BMSC transplantation could improve the erectile function of diabetic erectile function rats more effectively than LESWT or BMSC transplantation performed alone.

Clinical analysis of 60 relapsed pediatric patients with acute lymphoblastic leukemia

Objective To explore the factors for the relapse, prognosis and the treatment strategy for relapsed pediatric patients with acute lymphoblastic leukemia(ALL). Methods From January 2007 to January 2015, 60 pedia-tric relapsed ALL patients were recruited in Beijing Children's Hospital, Capital Medical University.The association between prognosis and different sites of relapse, and the duration of first complete remission and the treatment strategy were analyzed. Results The median age of the 60 patients was 70.5 months, 51 patients out of 60 cases were B-cell immunophenotype, with the median diagnosis-relapse interval 30.5 months.Forty-five patients belonged to marrow relapse and 15 had isolated extramedullary relapsed.Fifty-six patients received treatment after relapse.Thirty-two out of 56 patients survived without disease, the median follow-up was 19 months (3-78 months). The remission rates induced by Cyclophosphamide, Prednisone, Doxorubicin, L-Asparaginase, Vindecine (CODLP) induction, and Dexa-methasone, Doxorubicin, Pegaspargase, Vindecine (VDLD) regimens were 85.7%(12/14 cases) and 92.9%(39/42 cases), respectively.The overall survival (OS) of bone marrow relapsed patients and the isolate extramedullary relapsed patients were 57.14%(24/42 cases) and 78.57%(11/14 cases), respectively.However, there was no statistical difference(χ2=0.073, P=0.787; χ2=2.979, P=0.084). The OS of the early, intermediate and late relapsed patients were 43.75%(7/16 cases), 46.67%(7/15 cases) and 80.00%(20/25 cases). The differences between early, and late relapsed patients, intermediate and late relapsed patients were statistically significant, respectively(χ2=10.122, P=0.001; χ2=6.179, P=0.013). The OS of chemotherapy group versus hematopoietic stem cell transplantation group(HSCT) in bone marrow relapsed patients were 26.92% (7/26 cases) and 100.00%(16/16 cases). Statistically, the difference between the 2 groups was significant(χ2=18.481, P<0.001). Conclusions The accurate gradation treatment regimen on the original diagnosis could reduce relapse rate effectively, VDLD protocol was still an useful induction regimen for relapsed ALL patients, and allo-genetic HSCT was the good choice for relapsed patients at high risk, che-motherapy with local irradiation treatment strategy could assist longer survival for the relapsed patients with isolated testicular leukemia. Key words: Acute lymphoblastic leukemia; Relapse; Child