Pooled Umbilical Cord Blood Plasma for Culturing UCMSC and Ex Vivo Expanding Umbilical Cord Blood CD34⁺ Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of umbilical cord blood plasma (UCP) as a replacement for fetal bovine serum (FBS) for culturing mesenchymal stem cells (MSC) derived from umbilical cord, and to observe the supporting effects of these cells (served as a feeder layer) on ex vivo expanding of human umbilical cord blood CD34(+) cells.</p><p><b>METHODS</b>Umbilical cord blood (UCB) units were suitable if the Guangzhou cord blood bank donor selection criteria strictly were fulfilled. UCP were ready to use after the collection from the plasma depletion/reduction during the processing and pooling of suitable UCB units (at least 30 units were screened for pathogens and microorganisms, and qualified). Umbilical cord mesenchymal stem cells (UCMSC) were harvested from the umbilical cord tissue of health full-term newborns after delivery by enzyme digestion and divided into 3 groups: group 1 and 2 were cultured in the presence of DMEM/F12 containing either FBS or UCP; and group 3 was cultured in serum-free medium (StemPro® MSC SFM CTS™). Morphology, proliferation and surface marker expression were examined by flow cytometry, and the differentiation toward adipogenic and osteogenic lineages was used for investigating the effect of media on UCMSC after 3-5 passages. Next, the cells cultured in the three different media were cryopreserved and thawed, then prepared as feeder layers with the name of UCMSC(FBS), UCMSC(UCP), and UCMSC(SFM), respectively. The CD34⁺ cells were separated from UCB by magnetic activated cell sorting (MACS) and divided into 4 groups cultured in StemPro(-34) SFM medium added with hematopoietic cytokine combination (StemSpan® CC100). The control group included only CD34⁺ cells as group A (blank control) and experimental groups included UCMSC(FBS) + CD34⁺ cells as group B, UCMSC(UCP) + CD34⁺ cells as group C, UCMSC(SFM) + CD34⁺ cells as group D, and cells in all groups were cultured ex vivo for 7 days. The nucleated cell (NC) number was counted by cell counter, CD34⁺ cells were measured by flow cytometry, and clonogenic assay was conducted at day 0 and 7 of culture. The expansion efficiency was assessed.</p><p><b>RESULTS</b>The morphology (spindle-shaped and plastic-adherent), the immunophenotype (high positive percentage of CD73, CD90, CD105 and CD166) and the differentiation potential (osteogenic and adipogenic) were almost indistinguishable among the cells cultured in any of these three media except for the expression of CD105 in group 3 (serum-free medium) was lower than that in other 2 groups (P < 0.05). UCMSC grown in UCP medium demonstrated significantly higher proliferation rates than that in media containing FBS or commercial serum-free supplement (P < 0.05). After co-culture for 7 days, the CD34⁺ cell percentage decreased in all the groups, while NC were amplified effectively and the CD34⁺ cell number increased with the same order as group C or D group B or A (control group) (P < 0.05). As compared with the colony-forming unit (CFU) number at day 0, there was no significant difference in the expansion multiple between group C and D, while the expansion of CFU in group C were higher than that in group B and A.</p><p><b>CONCLUSION</b>The UCP can be used as a better animal-free serum supplement for growth, maintenance and differentiation of UCMSC, thus would be a safe choice for clinical-scale production of human MSC.</p>

Analysis of post-thaw infused cell dose for predicting engraftment after unrelated cord blood transplantation / 中国实验血液学杂志

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.

Analysis of maternal and neonatal factors associated with hematopoietic reconstruction potential in umbilical cord blood units / 中国实验血液学杂志

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.

Correlative analysis between HLA high resolution typing and GVHD in 48 cases received unrelated umbilical cord blood transplantation / 中国实验血液学杂志

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.

Clinical application of HLA sequencing in unrelated umbilical cord blood transplantation / 中国实验血液学杂志

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.

Results of unrelated umbilical cord blood stem cell transplantation for 65 patients in China / 中华儿科杂志

<p><b>OBJECTIVE</b>From 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.</p><p><b>METHODS</b>ALL (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.</p><p><b>RESULTS</b>Out 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%.</p><p><b>CONCLUSIONS</b>Unrelated allogeneic umbilical cord blood transplantation may be a good substitution for unrelated allogeneic bone marrow transplantation with a good prospect.</p>

A modified method to isolate and identify the adult mesenchymal stem cells from human bone marrow / 中国实验血液学杂志

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.

HLA-B gene polymorphism detected by high-resolution sequence-based typing in Guangdong Han populations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the genetic polymorphism of HLA-B locus in Guangdong Han population and compare the characteristic of the allele frequency distribution with that in other populations.</p><p><b>METHODS</b>A total of 562 cord blood samples from Guangzhou Cord Blood Bank were analyzed by sequence-based typing. Then the sequences encompassing exons 2, 3, and 4 for HLA-B gene were analyzed by direct sequencing of PCR products. The allele frequency distribution of HLA-B in this population was compared with that in other populations.</p><p><b>RESULTS</b>A total of 59 different HLA-B alleles were detected, and among them were 6 HLA-B alleles with frequencies higher than 5%: HLA-B*4601 (14.5%), HLA-B*400101 (14.4%), HLA-B*1502 (11.5%), HLA-B*1301 (8.6%), HLA-B*5801 (8.1%) and HLA-B*380201 (6.4%); the total frequency of these six alleles was 63.5%. At the same time, there were 30 kinds of HLA-B allele with frequencies lower than 0.5%; the total frequency of these alleles was 4.9%. Maximum variation at HLA-B was seen in the HLA-B*15 allele family (nine alleles). Comparison of the HLA-B frequencies in different populations showed a close relationship of Guandong Han population with the Chinese populations from Hong Kong and Singapore, respectively.</p><p><b>CONCLUSION</b>The results have shown the characteristic of HLA-B distribution and provided more accurate genotypic data that may serve as normal reference value for the Han population in Guangdong, China.</p>

Progress of study on placenta mesenchymal stem cells--review / 中国实验血液学杂志

Recently mesenchymal stem cells have been successfully obtained from various sources of human body, including bone marrow, compact bone, peripheral blood, adipose tissue, cord blood, amniotic fluid and other fetal tissues. Placenta, as a temporary organ keeping substance exchange between mother and fetus, consisted of decidua basalis and chorion frondosum, which derived from cytotrophoblast and extraembryonic mesoderm, thus involved both primary embryonic cells and adult stem cells. As a castoff after parturition, along with the ease of accessibility, lack of ethical concerns, placenta may be an attractive source of mesenchymal stem/progenitor cells for basic and clinical application. Therefore, the origin, isolation, characteristics and potential uses in future therapy are mainly reviewed in this paper.

A retrospective analysis on multiple cord blood transplantation in 13 cases / 中国实验血液学杂志

In order to study the feasibility of multi-umbilical cord blood transplantation (multi-UCBT) in adult, 13 cases of unrelated allogeneic multi-UCBT performed from June 1998 to July 2003 were analyzed retrospectively. All cord blood units were obtained from full term normal vaginal and cesarean deliveries in Guangzhou Maternity and Neonatal Hospital. The fractionation, cryopreservation and thawing of cord blood were done according to the regulation of New York Umbilical Cord Blood Bank and pertinent literatures. Donors of HLA 1/6-2/6 mismatch were accepted at registry search. The results showed that from June 1998 to July 2003, 28 umbilical cord blood units were selected by 7 transplantation centers for 13 cases. The median age of recipients was 22 (8 - 41) years, and the median weight was 50 (21 - 75) kg, the median infused dose of total nuclear cells was 2.91 x 10(7)/kg. Six out of thirteen cases were engrafted after cord blood infusion with absolute neutrophil count of > 5.0 x 10(8)/L at 19 days post-infusion. Only one case suffered from graft versus host disease, the total survival of multi-UCBT was 46.2% (6/13). It is concluded that good prospects in the field of multi-umbilical cord blood transplantation is likely to be realized.

Multiplex PCR for detecting genotypes of deletional alpha-thalassemia / 中国实验血液学杂志

To investigate the clinical application of multiplex PCR in detecting genotypes of deletional alpha-thalassemia in South China and observe the distribution frequency of alpha-globin gene deletion, 145 patients with silent carrier, alpha thalassemia trait or HbH were identified by M-PCR and 1.2% agarose gel electrophoresis. There are 1.3, 1.6, 1.8 and 2.0 kb bands which indicate --(SEA), -alpha(4.2), alphaalpha and -alpha(3.7), respectively. The results showed that among 145 patients, 100 patients with --(SEA)/alphaalpha (68.9%), 15 with -alpha(3.7)/alphaalpha (10.3%), 8 with -alpha(4.2)/alphaalpha (5.52%), 2 with -alpha(3.7)/-alpha(4.2) (1.38%), 1 with -alpha(3.7)/-alpha(3.7) (0.69%), 1 with -alpha(4.2)/-alpha(4.2) (0.69%), 14 with --(SEA)/-alpha(3.7) (9.65%), 2 with --(SEA)/-alpha(4.2) (1.38%) were found. Two patients prenatal diagnosed were confirmed with Bart's hydrops fetuses. In conclusion, M-PCR analysis is a simple, rapid and accurate method for detection of alpha-thalassemia gene deletion. This technique is helpful in screening, carrier identification and prenatal diagnosis of deletional alpha-thalassemia.

Analysis of HLA matching probability in Guangzhou Cord Blood Bank / 中国实验血液学杂志

The HLA matching results for 1 060 patients searching donors within 3 000 units of umbilical cord blood in Guangzhou Cord Blood Bank from 1998 to 2002 were analyzed. There were 119 (11.23%) and 992 (93.58%) patients found 6/6 and more than 4/6 of HLA matched loci of unrelated cord blood donors respectively. 61.29% of probability in all patients could find one or more cord blood with 4/6 or more matched loci and total nucleated cell (TNC) dose of >or= 3.7 x 10(7)/kg. The highest mean body weight in these supplied patients was 79 kg. The probability was 89.79% for those patients with TNC dose of >or= 2.0 x 10(7)/kg and >or= 4/6 of HLA loci matched. In these patients, the highest weight was 175 kg. In conclusion, a cord blood bank with 3 000 units or more of cord blood in stock shows a high probability of HLA matching and can meet the requirement of TNC >or= 3.7 x 10(7)/kg dose in child and part of adult patients. The umbilical cord blood is a good alternative stem cell source for all patients including adults.