The study was aimed to establish a protocol of isolating and culturing adult mesenchymal stem cells (MSC) from human bone marrow aspirate and identify them by surface antigen analysis and committed differentiation in order to provide an experimental foundation for achieving a therapeutic benefit in applying MSC in hematopoietic stem cell transplantation. MSCs were obtained from fresh human bone marrow aspirate by gradient centrifugation with Percoll (1.073 g/ml) and anchoring culture in L-DMEM with 10% fetal bovine serum by a full medium exchange every 3 days. The MSC surface antigens, including CD34, CD45, CD73, CD105, CD166, were analyzed on FACScan flow cytometer. Under culture in conditioned medium for osteogenesis (the hormone cocktail containing 0.1 micromol/L dexamethasone, 10 mmol/L glycerol-2-phosphate and 50 micromol/L ascorbic acid) and adipogenesis (the cocktail containing 1 micromol/L dexamethasone, 5 mg/L insulin, 0.5 mmol/L 1-methyl-3-isobutylxanthine and 60 micromol/L indomethacin), MSCs committedly differentiated into osteoblasts and adipocytes. The differentiated mesenchymal stem cells were identified by morphological observation and immunohistochemical staining. The results showed that by gradient centrifugation and adhesion culture, MSCs could be isolated and culture-expanded from human bone marrow aspirate. These cells were uniformly negative for CD34, CD45 and positive for CD73, CD105 and CD166. The osteogenic differentiated cells were positive for alkaline phosphatase (ALP) and the adipogenic differentiated cells displayed accumulation of lipid vacuoles, as detected by oil red O. It is concluded that MSC can be isolated and expand-cultured from adult human bone marrow aspirate and committedly differentiate into osteoblasts and adipocytes. MSC primary identification can be accomplished by flow cytometry and induced differentiation. The set of methods in current experiment shows somewhat practical value for basic research and clinical application.
5'-Nucleotidase ; metabolism ; Antigens, CD ; metabolism ; Bone Marrow Cells ; cytology ; Cell Adhesion Molecules, Neuronal ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Separation ; methods ; Endoglin ; Fetal Proteins ; metabolism ; Humans ; Mesenchymal Stromal Cells ; cytology ; Receptors, Cell Surface ; metabolism

The objective of this research was to explore whether the number of CD34(+)CD38(+) cells infused affects hematopoietic reconstitution after cord blood transplantation. The number of CD34(+)CD38(+) cells in cord blood was analysed with flow cytometry after freezethawing. The body weight and time for neutrophil and platelet recovery were measured in 20 children with acute leukemia. The results showed that the median number of CD34(+)CD38(+) cells infused was 29.47 (9.85 - 325.71) x 10(4)/kg. A median time for neutrophil recovery (> 5 x 10(8)/L) in 20 patients was 18.5 (11 - 32) days, and time for platlet recovery (> 2 x 10(10)/L) in 19 of 20 patients was 45 (12 - 118) days. The number of CD34(+)CD38(+) cells infused correlated with time to neutrophil and platelet recovery (r = -0.577, P < 0.01 and r = 0.503, P < 0.05, respectively). In conclusion, the number of CD34(+)CD38(+) cells infused is correlated with the time for hematologic recovery.
ADP-ribosyl Cyclase ; analysis ; ADP-ribosyl Cyclase 1 ; Adolescent ; Antigens, CD ; analysis ; Antigens, CD34 ; analysis ; Child ; Child, Preschool ; Fetal Blood ; cytology ; transplantation ; Hematopoiesis ; Hematopoietic Stem Cell Transplantation ; methods ; Humans ; Leukemia, Myeloid, Acute ; blood ; therapy ; Membrane Glycoproteins ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; blood ; therapy

OBJECTIVESeveral studies have shown that L-selectin on CD34-positive cells play a role in hematopoietic reconstitution after peripheral blood stem cell transplantation and allograft bone marrow transplantation. This study sought to investigate whether the numbers of CD(34)(+)CD(62L)(+) cells infused affect the engraftment of hematopoietic stem cells (HSC) and the time to neutrophil and platelet recovery after unrelated umbilical cord blood transplantation for the treatment of childhood acute leukemia.

METHODSTwenty-three children with acute leukemia who received unrelated umbilical cord blood transplantation of mostly mismatched HLA locus were included in this study. Flow cytometry was used to count the numbers of CD(34)(+)CD(62L)(+) cells after freezing-thawing by labelling the cells with anti-CD(34) and anti-CD62L. The patients' clinical data including body weight, engraftment of the HSC, times to neutrophil and platelet recovery were evaluated.

RESULTSTwenty-one patients who received CD(34)(+)CD(62L)(+) cell infusion at a number ranging from 1.37 x 10(5)/kg to 2.68 x 10(6)/kg (median, 3.567 x 10(5)/kg) had successful engraftment of the unrelated umbilical HSC. The numbers of CD(34)(+)CD(62L)(+) cells infused were statistically different between patients who had successful engraftment of the umbilical HSC and those who had not (P < 0.05). The engraftment occurred more commonly in patients who received > 1.3 x 10(5) CD(34)(+)CD(62L)(+) cells/kg. The time of neutrophil recovery (> 500/ microl) ranged from 11 days to 32 days (median, 17.5 days). The data of the time to platelet recovery (> 2 x 10(5)/ microl) were obtained in 18 patients, and it ranged from 12 days to 118 days (median, 14 days). There seemed to be a tendency of correlation between the numbers of CD(34)(+)CD(62L)(+) cells infused and time to platelet recovery (gamma = -0.324, 0.05 < P < 0.1), whereas the numbers of CD(34)(+)CD(62L)(+) cells infused correlated with the time to platelet recovery (gamma = -0.470, P < 0.05).

CONCLUSIONThis study suggests that the numbers of CD(34)(+)CD(62L)(+) cells infused might be involved in the engraftment of HSC and hematologic reconstitution after umbilical cord blood transplantation.

Acute Disease ; Adolescent ; Antigens, CD34 ; blood ; Blood Platelets ; metabolism ; Child ; Child, Preschool ; Cord Blood Stem Cell Transplantation ; methods ; Female ; Humans ; Infant ; Infusions, Intravenous ; L-Selectin ; blood ; Leukemia ; immunology ; therapy ; Male ; Neutrophils ; metabolism ; Treatment Outcome

OBJECTIVETo summarize the clinical study of modified total aortic arch replacement and stent elephant trunk technique treatment to patients with DeBakey I thoracic aortic dissection.

METHODSFrom January 2006 to October 2010, 101 cases of DeBakey I aortic dissection were treated by modified total arch replacement and stent elephant trunk technique, in which emergency surgery for 73 cases. There were 76 male and 25 female patients, aged from 21 to 77 years with a mean of (49 ± 8) years. Intraoperative ascending aortic replacement in 31 cases, Bentall procedure in 29 cases, Wheat procedure in 7 cases, David procedure in 34 cases. At the same time stent elephant trunk in the left subclavian artery corresponding position was windowed to rebuild the blood supply. Deep hypothermic circulatory arrest cerebral protection was completed by bilateral antegrade cerebral perfusion.

RESULTSThe mean cardiopulmonary bypass time was (212 ± 40) min, mean myocardial occlusion time was (95 ± 16) min, mean circulatory arrest time was (42 ± 8) min. Operative mortality was 1 case and hospital mortality was 5 case, which died of septicemia, acute renal failure and hemiplegia complicated with multiple organ failure. Compared with selective cerebral perfusion, the incidence of postoperative cerebral vascular accident and transient neurological dysfunction decreased. Seventy-six cases received aorta CTA before discharged, the closure rate of descending thoracic aortic dissection false lumen was 78.9%. Seventy-one patients were followed up for 5 to 49 months, 50 cases was reviewed by CTA, of which closure rate of descending thoracic aortic dissection false lumen was 88.0%, no late death and re-surgery.

CONCLUSIONSThe modified total aortic arch replacement and stent elephant trunk technique treatment for patients with DeBakey I thoracic aortic dissection was safe and effective, with less postoperative complications.

Adult ; Aged ; Aneurysm, Dissecting ; surgery ; Aortic Aneurysm ; surgery ; Blood Vessel Prosthesis Implantation ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Stents ; Young Adult

Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells for transplantation with success being associated with the total nucleated cell (TNC) count, CD34(+) cells and colony-forming unit-granulocyte-macrophage (CFU-GM) content infused. This study was purposed to clarify the impact of maternal and neonatal factors on hematopoietic potential of UCB product. UCB samples were screened, processed, tested and cryopreserved according to the Standard Operation Procedure (SOP) of Guangzhou cord blood bank (GZCBB). Relationship of hematopoietic cell parameters with maternal and neonatal characteristics for 4615 UCB units was analyzed retrospectively. The results showed that both collected volume (Mean ± SD: 95.23 ± 22.42 ml; Median: 91.85 ml) and initial TNC [Mean ± SD: (1.34 ± 0.49) × 10(9); Median: 1.25 × 10(9)] correlated well with postprocessed TNC [Mean ± SD: (1.21 ± 0.42) × 10(9); Median: 1.14 × 10(9); p < 0.001], CD34(+)count [Mean ± SD: (5.14 ± 4.55) × 10(6); Median: 4.08 × 10(6); p < 0.001] and CFU-GM content [Mean ± SD: (9.72 ± 8.66) × 10(5); Median: 7.53 × 10(5); p < 0.001]. As for donor factors, only infant birth weight correlated strongly with volume collected and all hematopoietic cell parameters (p < 0.001). UCB samples from bigger babies had higher collected volume, TNC, CD34(+) count and CFU-GM content (p < 0.001). Mother's age had no correlation with all the above parameters. Gestational age correlated positively with initial/postprocessed TNC (p < 0.001) and negatively with CD34(+) count (p = 0.04), but no relation with collected volume and CFU-GM content. Cesarean section produced superior volume (Mean ± SD: 97.05 ± 22.23 ml vs 92.53 ± 22.43 ml; Median: 94.08 ml vs 88.82 ml; p < 0.001), but inferior cell count than vaginal delivery (p < 0.001). Male infants had more initial volume and CD34(+) count (Mean ± SD: 96.41 ± 22.31 ml vs 93.95 ± 22.47 ml; Median: 93.27 ml vs 90.14 ml; p < 0.001); [Mean ± SD: (5.28 ± 5.04) × 10(6) vs (5.00 ± 3.94) × 10(6); Median: 4.18 × 10(6) vs 3.94 × 10(6); p < = 0.042], but lower initial and postprocessed TNC than female ones [Mean ± SD: (1.31 ± 0.50) × 10(9) vs (1.37 ± 0.47) × 10(9); Median: 1.22 × 10(9) vs 1.28 × 10(9); p < 0.001]; [Mean ± SD: (1.18 ± 0.42) × 10(9) vs (1.24 ± 0.41) × 10(9); Median: 1.10 × 10(9) vs 1.17 × 10(9); p < 0.001], while no significant difference of CFU-GM were found between male and female infants. It is concluded that these data may be helpful to optimize the UCB donor selection and improve cost efficiency of UCB bank resource. The heavier infants after vaginal delivery should be selected and large-volume units with higher TNC should be chosen at first.
Adult ; Birth Weight ; Blood Banks ; methods ; Cord Blood Stem Cell Transplantation ; methods ; Delivery, Obstetric ; Donor Selection ; Female ; Fetal Blood ; cytology ; immunology ; Gestational Age ; Hematopoietic Stem Cells ; Humans ; Infant, Newborn ; Male ; Maternal Age ; Pregnancy ; Young Adult

To study the program of evaluating mothers and infants after 6 months of cord blood donation, from June 1998 to February 2004, all mothers after 6 months of cord blood donation were followed-up by phone calls or letters to report on the health condition. The results showed that when 3 195 mothers were visited by phone calls, 18 mothers declined to answer. 392 letter were send to those who could not be found by phone, 15 of whom wrote back. The average time to talk with each mother was approximately 12 minutes. Follow-up on the baby donors showed two cases with chromosome abnormality, one with hypothyroidism, one with neutropenia, one with albinism and 5 dead with unclear reasons. The cord blood components from all these abnormal donors found were discarded. In conclusion, the programs to evaluate mother and baby after 6 months of cord blood donation seems important in quality control of the components stored in cord blood bank.
Adult ; Blood Banks ; Blood Donors ; China ; Female ; Fetal Blood ; Follow-Up Studies ; Humans ; Infant ; Infant, Newborn ; Quality Assurance, Health Care ; methods ; statistics & numerical data ; Surveys and Questionnaires ; Time Factors

Unrelated umbilical cord blood units for 54 cases in 21 transplant centers were provided by Guangzhou Cord Blood Bank in China from 1998 to 2003. This study was aimed to identify HLA-DRB1 alleles by means of PCR sequencing based typing methods (SBT) and to analyze the correlation between HLA-DRB1 alleles and GVHD in unrelated umbilical cord blood transplantation (UCBT). 48 out of 54 patients received UCBT were followed up. DNA were extracted from cryopreservation blood of recipients/donors with UCBT, HLA-DRB1 alleles typing were done by SBT. Compared with low resolution results of HLA-DRB1 alleles, high resolution results were analyzed to see any correlation between HLA-DRB1 alleles and GVHD. by double-blind statistically analysis of HLA-DRB1 high resolution in 48 donor/recipient typings in UCBT. The results showed that the incidence of GVHD (25%) in patients who has HLA-DRB1 alleles matched with donors significantly lower (65.6%) than that in the patients with HLA-DRB1 alleles mismatched (P = 0.008). It is concluded that HLA-DRB1 by high resolution typing method is important in clinical application in UCBT.
Alleles ; Blood Donors ; Cord Blood Stem Cell Transplantation ; methods ; Graft vs Host Disease ; genetics ; immunology ; HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Histocompatibility Testing ; methods ; Humans ; Retrospective Studies ; Sequence Analysis, DNA ; methods

From June 1998 to July 2004, Guangzhou umbilical cord blood bank provided unrelated umbilical cord blood for 54 patients to more than 21 transplantation centers. HLA sequencing-based typing (SBT) was used to re-analyze the results of HLA antigens and alleles so as to investigate the relationship between HLA alleles and GVHD. The information about 48 out of 54 patients was obtained after 6 months of follow up. SBT was used to identify HLA-A, B, DRB1 alleles in 48 patients received the unrelated umbilical cord blood units, and the obtained results were compared with the results of HLA-SSP Low Resolution Typing. The results showed that the difference of GVHD incidence between less than 2 mismatched HLA sites and less than 3 sites was statistically significant (P < 0.05). In the results from single factor analysis and high-resolution typing of HLA-A, B and DRB1 alleles, the mismatch between HLA-B and HLA-DRB1 alleles was found to be a significant factor for the occurence of GVHD. It is concluded that SBT plays an important role in umbilical cord blood transplantation, and the incidence of GVHD is higher in the transplantation with HLA-DRB1 alleles mismatching.
Adolescent ; Adult ; Aged ; Alleles ; Child ; Child, Preschool ; Cord Blood Stem Cell Transplantation ; adverse effects ; Female ; Fetal Blood ; cytology ; immunology ; Graft vs Host Disease ; prevention & control ; HLA-A Antigens ; genetics ; immunology ; HLA-B Antigens ; genetics ; immunology ; HLA-DR Antigens ; genetics ; immunology ; HLA-DRB1 Chains ; Histocompatibility Testing ; methods ; Humans ; Leukemia ; therapy ; Male ; Middle Aged ; Sequence Analysis

This study was purposed to investigate the role of post-thaw infused donor cells for predicting engraftment and hematopoietic reconstitution after unrelated cord blood transplantation (UCBT). The retrospective analysis was performed on clinical data of 97 children with malignant or non-malignant diseases received single unit UCBT from August 1999 to April 2010. The impact of pre-freezing and post-thaw cell dose of total nucleated cells (TNC), CD34(+) cells and colony-forming units-granulocyte/macrophage (CFU-GM) on engraftment and hematological recovery after UCBT was analyzed. Unrelated donors were from Guangzhou cord blood bank (GZCBB) entirely. The results indicated that the pre-freezing TNC (/kg) (mean ± SD: 7.65 × 10⁷ ± 4.26 × 10⁷; median: 6.34 × 10⁷), CD34(+)cells (/kg) (mean ± SD: 4.64 × 10(5) ± 4.47 × 10⁵; median: 3.03 × 10⁵) and CFU-GM (/kg) (mean ± SD: 0.79 × 10⁵ ± 1.09 × 10⁵; median: 0.57 × 10⁵) showed a good correlation with their post-thaw counterparts including TNC(/kg) (mean ± SD: 6.98 × 10⁷ ± 4.12 × 10⁷; median: 6.00 × 10⁷), CD34(+)cells (/kg)(Mean ± SD: 6.86 × 10⁵ ± 8.56 × 10⁵; Median: 4.17 × 10⁵), and CFU-GM (/kg) (mean ± SD: 0.52 × 10⁵ ± 0.52 × 10⁵; median: 0.39 × 10⁵) (r = 0.952, p < 0.001; r = 0.794, p < 0.001; r = 0.478, p < 0.001). Either the pre-freezing or post-thaw number of infused CFU-GM was significant higher in patients who achieved engraftment (n = 70) than those who suffered graft failure (n = 22) (p = 0.023 and 0.011, respectively), but no significant difference of TNC and CD34(+) cells dose (pre-freezing or post-thaw) were found between these two groups. Pre-freezing CFU-GM, TNC, CD34(+) cell dose negatively correlated with the time of neutrophil engraftment (r = -0.285, p = 0.018; r = -0.396, p = 0.002; r = -0.373, p = 0.002), as well as the post-thaw number of TNC and CD34(+) cells (r = -0.260, p = 0.031; r = -0.483, p < 0.001), whereas only pre-freezing CD34(+) cells showed a significant correlation with platelet engraftment time (r = -0.352, p = 0.013). It is concluded that the CFU-GM amount is useful for predicting engraftment of UCBT, while pre-freezing hematopoietic cell doses show superior correlation with the speed of engraftment and hematopoietic reconstitution than their post-thaw counterparts in pediatric recipients, suggesting that it is essential to perform hematopoietic potency assay on each cord blood unit prior to listing or release for administration.
Adolescent ; Antigens, CD34 ; blood ; Blood Banks ; Child ; Child, Preschool ; Cord Blood Stem Cell Transplantation ; methods ; Female ; Fetal Blood ; cytology ; Graft Survival ; Granulocyte-Macrophage Progenitor Cells ; Humans ; Infant ; Male ; Retrospective Studies ; Tissue Donors

OBJECTIVEFrom December 1998 to April 2004, 3960 umbilical cord blood units were stored in Guangzhou cord blood bank, which provided 100 umbilical cord blood units to 25 transplant center for 83 patients with malignant or non-malignant diseases. To study the related factors affecting unrelated umbilical cord blood stem cell transplantation, the authors analyzed retrospectively the results of transplantation of unrelated umbilical cord blood stem cells for 65 patients.

METHODSALL (acute lymphocytic leukemia) cord blood units were obtained from full term normal vaginal and cesarean deliveries in Guangzhou Women and Infants Hospital. The fractionation, cryopreservation and thawing of the cord blood were performed according to the regulations of New York umbilical cord blood bank and pertinent literature. The selection of cord blood was based on HLA typing and the number of nucleated cells. The sex and HLA antigens of donors were defined as the evidence of engraftment. Time to engraftment was recorded when the absolute number of neutrophil ANC (absolute neutrophil count) was higher than 5.0 x 10(8) for three days. Event-free survival and graft versus host disease (GVHD) were provided by transplant centers.

RESULTSOut of 65 patients who received unrelated cord blood stem cell transplant, 49 patients were diagnosed as having malignant diseases [including 23 with ALL, 16 with AML (acute myeloid leukemia), 7 with CML (chronic myelogenous leukemia), 3 with lymphoma and one with MDS (myelodysplastic syndrome)], 16 patients had non-malignant disease. The 65 transplanted patients (42 male, 23 female) had a median age of 10 years (range 1 - 33 years) and a median body weight of 27 kg (range 10 - 67 kg). The patients received cord blood stem cells from unrelated 0-locus (n = 9) or 1-locus (n = 43) or 2-locus (n = 13) HLA mismatched donor. The median dose of infused cells was: total neutrophil count (TNC) 5.7 x 10(7), CD(34)(+) 5.1 x 10(5), CFU-GM 3.8 x 10(4). Fifty of 65 (77%) patients had engraftment. GVHD occurred in 41 patients (63%), including acute grade I - II GVHD in 31 patients (76%), acute grade III - IV GVHD in 8 patients (20%) and chronic GVHD in 2 patients (5%). Fifty patients had engraftment (ANC > 5.0 x 10(8)) after a median time of 17 (range 7 to 44) days after transplant, while an autologous hematopoietic reconstitution was observed in 6 patients; 24 patients died of severe pneumonia (n = 8), acute GVHD (n = 4), or sepsis (n = 12) and the disease-free survival probability was 61%.

CONCLUSIONSUnrelated allogeneic umbilical cord blood transplantation may be a good substitution for unrelated allogeneic bone marrow transplantation with a good prospect.

Adolescent ; Adult ; Child ; Child, Preschool ; China ; Cord Blood Stem Cell Transplantation ; adverse effects ; methods ; Disease-Free Survival ; Female ; Graft vs Host Disease ; etiology ; mortality ; Humans ; Infant ; Leukemia ; mortality ; therapy ; Male ; Retrospective Studies ; Survival Rate ; Transplantation, Homologous ; Treatment Outcome ; Young Adult