Isolation, culture and validation of CD34+ vascular wall-resident stem cells from mice / 生理学报

Vascular wall-resident stem cells (VW-SCs) play a critical role in maintaining normal vascular function and regulating vascular repair. Understanding the basic functional characteristics of the VW-SCs will facilitate the study of their regulation and potential therapeutic applications. The aim of this study was to establish a stable method for the isolation, culture, and validation of the CD34+ VW-SCs from mice, and to provide abundant and reliable cell sources for further study of the mechanisms involved in proliferation, migration and differentiation of the VW-SCs under various physiological and pathological conditions. The vascular wall cells of mouse aortic adventitia and mesenteric artery were obtained by the method of tissue block attachment and purified by magnetic microbead sorting and flow cytometry to obtain the CD34+ VW-SCs. Cell immunofluorescence staining was performed to detect the stem cell markers (CD34, Flk-1, c-kit, Sca-1), smooth muscle markers (SM22, SM MHC), endothelial marker (CD31), and intranuclear division proliferation-related protein (Ki-67). To verify the multipotency of the isolated CD34+ VW-SCs, endothelial differentiation medium EBM-2 and fibroblast differentiation medium FM-2 were used. After culture for 7 days and 3 days respectively, endothelial cell markers and fibroblast markers of the differentiated cells were evaluated by immunofluorescence staining and q-PCR. Furthermore, the intracellular Ca2+ release and extracellular Ca2+ entry signaling were evaluated by TILLvisION system in Fura-2/AM loaded cells. The results showed that: (1) High purity (more than 90%) CD34+ VW-SCs from aortic adventitia and mesenteric artery of mice were harvested by means of tissue block attachment method and magnetic microbead sorting; (2) CD34+ VW-SCs were able to differentiate into endothelial cells and fibroblasts in vitro; (3) Caffeine and ATP significantly activated intracellular Ca2+ release from endoplasmic reticulum of CD34+ VW-SCs. Store-operated Ca2+ entry (SOCE) was activated by using thapsigargin (TG) applied in Ca2+-free/Ca2+ reintroduction protocol. This study successfully established a stable and efficient method for isolation, culture and validation of the CD34+ VW-SCs from mice, which provides an ideal VW-SCs sources for the further study of cardiovascular diseases.

Effect of Hypoxia-Supported Umbilical Cord Mesenchymal Stem Cells on the Expansion of Cord Blood Mononuclear Cells in vitro / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of hypoxia-supported umbilical cord mesenchymal stem cell (UC-MSC) on the expansion of cord blood mononuclear cell （MNC） in vitro.@*METHODS@#The isolated cord blood mononuclear cells were inoculated on the preestablished umbilical cord mesenchymal stem cell layer and cultured under hypoxic conditions (3％ O2) and the experimental groups were normoxia (MNCs were cultured under normoxic conditions), hypoxia (MNCs were cultured under hypoxic conditions), UC-MSC (MNCs were cultured with UC-MSC under normoxic conditions), and UC-MSC+hypoxia (MNCs were cultured with UC-MSC under hypoxic conditions). To further investigate the combinational effect of 3 factors of SCF+FL+TPO (SFT) on expansion of cord blood MNCs in vitro in hypoxia-supported UC-MSC culture system, the experiments were further divided into group A (MNCs were cultured with UC-MSC and SFT under normoxic conditions), group B (MNCs were cultured with UC-MSC under hypoxic conditions), group C (MNCs were cultured with UC-MSC and SFT under hypoxic conditions). The number of nucleated cells (TNC), CD34+ cell, CFU and CD34+CXCR4+, CD34+CD49d+, CD34+CD62L+ cells of each groups were detected at 0, 7, 10 and 14 days, respectively.@*RESULTS@#Compared with group hypoxia and UC-MSC, group UC-MSC+hypoxia effectively promoted the expansion of TNC, CD34+ cell and CFU, and upregulated the expression level of adhesion molecule and CxCR4 of the cord blood CD34+ cell（P<0.05）. After culturing for 14 days, compared with group A and group B, group C effectively promoted the expansion of cord blood MNC at different time points（P<0.05）, and the effect of group A was better than that of group B at 7 and 10 days（P<0.05）.@*CONCLUSION@#Hypoxia-supported UC-MSC efficiently promoted the expansion and expression of adhesion molecule and CXCR4 of cord blood CD34+ cell, and the effect of expansion could be enhanced when SFT 3 factors were added.

Long-Term Quality Control Program Plan for Cord Blood Banks in Korea: A Pilot Study for Cryopreservation Stability

BACKGROUND: Maintaining the quality of cryopreserved cord blood is crucial. In this pilot study, we describe the results of the internal quality control program for a cord blood bank thus far. METHODS: Donated cord blood units unsuitable for transplantation were selected for internal quality control once a month. One unit of cord blood, aliquoted into 21 capillaries, was cryopreserved and thawed annually to analyze the total nucleated cell count, CD34⁺ cell count, cell viability test, and colony-forming units assay. RESULTS: No significant differences in the variables (total nucleated cell count, cell viability, CD34⁺ cell count) were observed between samples cryopreserved for one and two years. Upon comparing the variables before cryopreservation and post thawing with the capillaries of one year of storage, cell viability and CD34⁺ cell counts decreased significantly. The use of cord blood samples in capillaries, which can be easily stored for a long period, was similar to the methods used for testing segments attached to the cord blood unit. CONCLUSIONS: The results of this study may be useful for determining the period during which the quality of cryopreserved cord blood units used for transplantation is maintained.

In ovo administration of human recombinant leptin shows dose dependent angiogenic effect on chicken chorioallantoic membrane

BACKGROUND: Leptin, the cytokine produced by white adipose tissue is known to regulate food energy homeostasis through its hypothalamic receptor. In vitro studies have demonstrated that leptin plays a major role in angiogenesis through binding to the receptor Ob-R present on ECs by stimulating and initiating new capillary like structures from ECs. Various in vivo studies indicate that leptin has diverse effect on angiogenesis. A few reports have showed that leptin exerts pro angiogenic effects while some suggested that it has antiangiogenic potential. It is theoretically highly important to understand the effect of leptin on angiogenesis to use as a therapeutic molecule in various angiogenesis related pathological conditions. Chicken chorio allantoic membrane (CAM) on 9th day of incubation was incubated with 1, 3 and 5 µg concentration of HRL for 72 h using gelatin sponge. Images where taken after every 24 h of incubation and analysed with Angioguant software. The treated area was observed under microscope and histological evaluation was performed for the same. Tissue thickness was calculated morphometrically from haematoxylin and eosin stained cross sections. Reverse transcriptase PCR and immunohistochemistry were also performed to study the gene and protein level expression of angiogenic molecules. RESULTS: HRL has the ability to induce new vessel formation at the treated area and growth of the newly formed vessels and cellular morphological changes occur in a dose dependent manner. Increase in the tissue thickness at the treated area is suggestive of initiation of new capillary like structures. Elevated mRNA and protein level expression of VEGF165 and MMP2 along with the activation of ECs as demonstrated by the presence of CD34 expression supports the neovascularization potential of HRL. CONCLUSION: Angiogenic potential of HRL depends on the concentration and time of incubation and is involved in the activation of ECs along with the major interaction of VEGF 165 and MMP2. It is also observed that 3 µg of HRL exhibits maximum angiogenic potential at 72 h of incubation. Thus our data suggest that dose dependent angiogenic potential HRL could provide a novel role in angiogenic dependent therapeutics such as ischemia and wound healing conditions.

Adverse Prognostic Impact of Bone Marrow Microvessel Density in Multiple Myeloma

BACKGROUND: Angiogenesis is important for the proliferation and survival of multiple myeloma (MM) cells. Bone marrow (BM) microvessel density (MVD) is a useful marker of angiogenesis and is determined by immunohistochemical staining with anti-CD34 antibody. This study investigated the prognostic impact of MVD and demonstrated the relationship between MVD and previously mentioned prognostic factors in patients with MM. METHODS: The study included 107 patients with MM. MVD was assessed at initial diagnosis in a blinded manner by two hematopathologists who examined three CD34-positive hot spots per patient and counted the number of vessels in BM samples. Patients were divided into three groups according to MVD tertiles. Cumulative progression-free survival (PFS) and overall survival (OS) curves, calculated by using Kaplan-Meier method, were compared among the three groups. Prognostic impact of MVD was assessed by calculating Cox proportional hazard ratio (HR). RESULTS: Median MVDs in the three groups were 16.8, 33.9, and 54.7. MVDs were correlated with other prognostic factors, including beta2-microglobulin concentration, plasma cell percentage in the BM, and cancer stage according to the International Staging System. Multivariate Cox regression analysis showed that high MVD was an independent predictor of PFS (HR=2.57; 95% confidence interval, 1.22-5.42; P=0.013). PFS was significantly lower in the high MVD group than in the low MVD group (P=0.025). However, no difference was observed in the OS (P=0.428). CONCLUSIONS: Increased BM MVD is a marker of poor prognosis in patients newly diagnosed with MM. BM MVD should be assessed at the initial diagnosis of MM.

The New Sysmex XN-2000 Automated Blood Cell Analyzer More Accurately Measures the Absolute Number and the Proportion of Hematopoietic Stem and Progenitor Cells Than XE-2100 When Compared to Flow Cytometric Enumeration of CD34+ Cells

No abstract available.

Inflammatory Hepatic Adenoma / 대한소화기학회지

No abstract available.

CD34 and p53 Immunohistochemical Stains Differentiate Hypocellular Myelodysplastic Syndrome (hMDS) from Aplastic Anemia and a CD34 Immunohistochemical Stain Provides Useful Survival Information for hMDS

BACKGROUND: The presence of significant dysplasia in bone marrow (BM) aspirates helps to distinguish between hypocellular myelodysplastic syndrome (hMDS) and aplastic anemia (AA). Occasionally, diluted BM aspirates make it difficult to recognize dysplastic changes and can also negatively affect the detection of cytogenetic abnormalities in hMDS. We evaluated the usefulness of CD34 and p53 immunoreactivity for discriminating between hMDS and AA and for estimating survival outcomes in hMDS patients. METHODS: BM clot section (BMC) or BM biopsy (BMB) specimens were obtained from 64 hMDS/AA patients (33 with hMDS and 31 with AA) and seven controls. Immunohistochemical (IHC) staining for CD34 and p53 was performed by using the EnVision detection system (Dako, Denmark). We compared the results of IHC staining, BM findings, and chromosomal analyses, and determined overall survival outcomes. RESULTS: The number of CD34- and p53-positive BM cells was higher among the patients with hMDS than among the patients with AA (P<0.001 and P=0.001, respectively). hMDS patients with increased CD34-positive cells had significantly poorer survival outcomes compared with those with normal number of CD34-positive cells (P=0.013). CONCLUSIONS: CD34 and p53 IHC stains of BMC or BMB provide useful information for differentiating between hMDS and AA. CD34 IHC staining of BMC or BMB also provides useful information for estimating survival outcomes in hMDS patients.

Optimal Time to Start Peripheral Blood Stem Cell Collection in Children with High-Risk Solid Tumors

In order to clarify the optimal timing for peripheral blood stem cell (PBSC) collection, PBSC collection records of 323 children who were scheduled to undergo autologous stem cell transplantation from two study periods differing in the timing of PBSC collection were analyzed. In the early study period (March 1998 to August 2007, n=198), PBSC collection was initiated when the peripheral WBC count exceeded 1,000/microL during recovery from chemotherapy. Findings in this study period indicated that initiation of PBSC collection at a higher WBC count might result in a greater CD34+ cell yield. Therefore, during the late study period (September 2007 to December 2012, n=125), PBSC collection was initiated when the WBC count exceeded 4,000/microL. Results in the late study period validated our conclusion from the early study period. Collection of a higher number of CD34+ cells was associated with a faster hematologic recovery after transplant in the late study period. Initiation of PBSC collection at WBC count > 4,000/microL was an independent factor for a greater CD34+ cell yield. In conclusion, PBSC collection at a higher WBC count is associated with a greater CD34+ cell yield, and consequently a faster hematologic recovery after transplant.

A Case of Splenic Hamartoma Diagnosed by Contrast-enhanced Ultrasonography and Magnetic Resonance Imaging / 대한소화기학회지

Splenic hamartoma is a very rare benign tumor, which is usually found incidentally after splenectomy or autopsy. Although percutaneous needle biopsy can be performed, it carries a high risk of bleeding after the procedure. Therefore, diagnosis is usually made by surgical resection. Herein, we report a case of splenic hamartoma diagnosed by magnetic resonance imaging and contrast-enhanced ultrasonography, which enables visualization of the unique signals of microbubbles in the vessels in real time. Relevant literature is also reviewed.

Primary extragastrointestinal stromal tumors: A clinicopathological and immunohistochemical study-A tertiary care center experience.

Background: Extra gastrointestinal stromal tumors (EGIST) are uncommon compared to their gastrointestinal counterparts. EGISTs involve omentum, mesentery, retroperitoneum, pancreas, and pelvis. Materials and Methods: Ten EGISTs were analyzed in this study from January 1995 to November 2011. They were analyzed with respect to clinical features, imageological, histopathological, and immunohistochemical findings. The immunohistochemical stains used were Smooth muscle actin (SMA), Desmin, S-100 protein, CD34 and CD-117. Results: There was slight female preponderance with wide age range. Four of the tumors were in retroperitoneum, three in mesentery, and two in omentum and one in pelvis. Histopathologically majority were spindle cell tumors. Immunohistochemically CD117 was consistently positive followed by CD34. Smooth muscle actin was positive in eight cases, S-100 protein and desmin were positive in two cases each. Conclusion: EGISTs are rare and should be considered in the differential diagnosis of the mesenchymal tumors and immunohistochemistry helps to confirm the diagnosis. Further study with better follow-up is desired to characterize these uncommon tumors.

Epithelioid hemangioendothelioma of the liver

No abstract available.

Hepatocellular carcinoma composed of two different histologic types: imaging features on gadoxetic acid-enhanced liver MRI

No abstract available.

Hematologic Recovery after Tandem High-Dose Chemotherapy and Autologous Stem Cell Transplantation in Children with High-Risk Solid Tumors

Although the number of studies using tandem high-dose chemotherapy and autologous stem cell transplantation (HDCT/autoSCT) for the treatment of high-risk pediatric solid tumors has been increasing, documentation of hematologic recovery after tandem HDCT/autoSCT is very limited. For this reason, we retrospectively analyzed the hematologic recovery of 236 children with high-risk solid tumors who underwent tandem HDCT/autoSCT. The median numbers of CD34+ cells transplanted during the first and second HDCT/autoSCT were 4.3 x 10(6)/kg (range 0.6-220.2) and 4.1 x 10(6)/kg (range 0.9-157.6), respectively (P = 0.664). While there was no difference in neutrophil recovery between the first and second HDCT/autoSCT, platelet and RBC recoveries were significantly delayed in the second HDCT/autoSCT (P < 0.001 and P < 0.001, respectively). Delayed recovery in the second HDCT/autoSCT was more prominent when the number of transplanted CD34+ cells was lower, especially if it was < 2 x 10(6)/kg. A lower CD34+ cell count was also associated with increased RBC transfusion requirements and a higher serum ferritin level after tandem HDCT/autoSCT. More CD34+ cells need to be transplanted during the second HDCT/autoSCT in order to achieve the same hematologic recovery as the first HDCT/autoSCT.

Prognostic Significance of Angiogenesis by Chalkley Counting in Node Negative Cancer of the Ampulla of Vater

Angiogenesis is essential for tumor growth and metastasis. Currently, the Chalkley assay with CD34 immunostaining is the proposed standard method for angiogenesis quantification in solid tumor sections. The purpose of this study was to evaluate the expression of CD34 and its prognostic significance using the Chalkley method in node negative carcinoma of the ampulla of Vater. Between January 1997 and December 2006, 56 node negative patients who had curative resection for carcinoma of the ampulla of Vater were retrospectively reviewed. The Chalkley count was expressed as the mean value of the three counts for each tumor and further divided into two groups according to the mean value of the Chalkley count: low or = 4. The mean Chalkley count value was 4.0 (+/- 3.1). In the low Chalkley group, the 1- and 3-yr recurrence rates were 18.3%, 47.6% respectively; in the high Chalkley group, the 1- and 3-yr recurrence rates were 26.5% and 60.6% respectively. Only high Chalkley count had statistical significance as a factor in recurrence of node negative ampulla of Vater carcinoma. Assessment of angiogenesis may have an important role in the prognostic evaluation of node negative cancer of the ampulla of Vater.

Diagnosis of an Accessory Spleen Mimicking a Gastric Submucosal Tumor Using Endoscopic Ultrasonography-guided Fine-needle Aspiration / 대한소화기학회지

Accessory spleen can be mistaken as a gastric subepithelial mass, and may not be differentiated in CT or endoscopic ultrasonography (EUS). A gastric subepithelial mass was detected on routine endoscopy in a 39-year old woman with history of splenectomy. In subsequent CT and EUS, the subepithelial mass was located on the fourth layer of the stomach. To make a definite diagnosis, EUS-guided fine needle aspiration (FNA) was performed, and a splenic tissue was demonstrated in histologic examination. EUS-guided FNA can be beneficial in the diagnosis of accessory spleen which mimics a gastric subepithelial mass.

MR imaging of hepatic lymphangioma / 대한간학회지

No abstract available.

Iron Overload during Follow-up after Tandem High-Dose Chemotherapy and Autologous Stem Cell Transplantation in Patients with High-Risk Neuroblastoma

Multiple RBC transfusions inevitably lead to a state of iron overload before and after high-dose chemotherapy and autologous stem cell transplantation (HDCT/autoSCT). Nonetheless, iron status during post-SCT follow-up remains unknown. Therefore, we investigated post-SCT ferritin levels, factors contributing to its sustained levels, and organ functions affected by iron overload in 49 children with high-risk neuroblastoma who underwent tandem HDCT/autoSCT. Although serum ferritin levels gradually decreased during post-SCT follow-up, 47.7% of the patients maintained ferritin levels above 1,000 ng/mL at 1 yr after the second HDCT/autoSCT. These patients had higher serum creatinine (0.62 vs 0.47 mg/mL, P = 0.007) than their counterparts (< 1,000 ng/mL). Post-SCT transfusion amount corresponded to increased ferritin levels at 1 yr after the second HDCT/autoSCT (P < 0.001). A lower CD34+ cell count was associated with a greater need of RBC transfusion, which in turn led to a higher serum ferritin level at 1 yr after HDCT/autoSCT. The number of CD34+ cells transplanted was an independent factor for ferritin levels at 1 yr after the second HDCT/autoSCT (P = 0.019). Consequently, CD34+ cells should be transplanted as many as possible to prevent the sustained iron overload after tandem HDCT/autoSCT and consequent adverse effects.

Iron Overload during Follow-up after Tandem High-Dose Chemotherapy and Autologous Stem Cell Transplantation in Patients with High-Risk Neuroblastoma

Multiple RBC transfusions inevitably lead to a state of iron overload before and after high-dose chemotherapy and autologous stem cell transplantation (HDCT/autoSCT). Nonetheless, iron status during post-SCT follow-up remains unknown. Therefore, we investigated post-SCT ferritin levels, factors contributing to its sustained levels, and organ functions affected by iron overload in 49 children with high-risk neuroblastoma who underwent tandem HDCT/autoSCT. Although serum ferritin levels gradually decreased during post-SCT follow-up, 47.7% of the patients maintained ferritin levels above 1,000 ng/mL at 1 yr after the second HDCT/autoSCT. These patients had higher serum creatinine (0.62 vs 0.47 mg/mL, P = 0.007) than their counterparts (< 1,000 ng/mL). Post-SCT transfusion amount corresponded to increased ferritin levels at 1 yr after the second HDCT/autoSCT (P < 0.001). A lower CD34+ cell count was associated with a greater need of RBC transfusion, which in turn led to a higher serum ferritin level at 1 yr after HDCT/autoSCT. The number of CD34+ cells transplanted was an independent factor for ferritin levels at 1 yr after the second HDCT/autoSCT (P = 0.019). Consequently, CD34+ cells should be transplanted as many as possible to prevent the sustained iron overload after tandem HDCT/autoSCT and consequent adverse effects.

Exophytic combined hepatocellular carcinoma and cholangiocarcinoma

No abstract available.