[A Case of Two Stage taTME for Perforated Rectal Cancer during Chemotherapy].

The patient was a 57-year-old male. He was diagnosed with locally advanced rectal cancer infiltrating the left levator ani muscle. Chemotherapy(S-1 plus L-OHP plus bevacizumab regimen)was started for the purpose of obtaining a negative circumferential radial margin. After the second course, he presented with perforation of the sigmoid colon for which an emergency operation was performed. The perforation was located 5 centimeters above the tumor in the sigmoid colon. We performed partial resection of the sigmoid colon to repair the perforation and create a sigmoid colostomy. CT, after the initial S-1 plus L-OHP plus bevacizumab chemotherapy regimen, revealed tumor shrinkage. Following 2 more courses of chemotherapy( S-1 plus L-OHP regimen), we performed transanal total mesenteric excision(taTME)as curative surgery. R0 resection was achieved. The combined transanal and laparoscopic approach was highly effective for a patient with pan-peritonitis.

[A Case of Urachal Cancer at the Inserted Site of Cystostomy after 23 Years].

A 66-year-old man had bilateral lower limb paralysis 30 years ago owing to traumatic injury of the thoracic spinal cord, and surgery(cystostomy)was performed 23 years ago. He was transferred to our hospital followingtreatment of sepsis owingto a worseningdecubitus. There was a 4 cm sized mucin-producingtumor at the inserted site of cystostomy. We performed tumor resection. Histological examination revealed the tumor to be a mucin-producingwell -differentiated adenocarcinoma. There was no tumor in any other organ. There was a residual tumor at the inserted site, and it was located at the dome of the bladder, which we considered to urachal cancer. Therefore, we performed partial resection of the bladder. Histological examination revealed a well-differentiated adenocarcinoma extendingfrom the urachal epithelium, and thus, it was diagnosed as urachal cancer. This is an extremely rare disease and is the first report from Japan.

[Postoperative Bile Leakage Following Liver Resection Due to Stenosis of a Choledochojejunostomy Anastomosis after Pancreaticoduodenectomy - A Case Report].

We report a rare case of intractable bile leakage after liver resection due to stenosis of the anastomosis of a choledochojejunostomy after pancreaticoduodenectomy. A 65-year-old woman was diagnosed with pancreatic and right breast cancer, and underwent pancreaticoduodenectomy and right mastectomy with simultaneous axillary lymph node dissection. Adjuvant chemotherapy and follow-up were performed in our department. After 18 months, computed tomography revealed a liver metastasis of 2.5 cm in segment 8. Because the primary nest of liver metastasis was unknown and performing a biopsy was difficult due to the location, partial resection of the liver was performed. Pathological examination confirmed liver metastasis from the breast cancer. She was rehospitalized due to a right subdiaphragmatic abscess 33 days post-surgery. Abscess drainage revealed bile leakage, and the cause was believed to be stenosis of the anastomosis created by the choledochojejunostomy. Percutaneous transhepatic cholangiographic drainage was performed, and the bile leakage disappeared immediately. However, it was difficult to release the anastomotic stenosis by choledochoscopy; therefore, a retrograde drainage tube was placed in the hepatic duct using enteroscopy, and it formed an internal fistula. The patient has continued to undergo chemotherapy for recurrence in the remnant liver that was observed 16 months after the hepatectomy. In conclusion, when hepatic resection is performed after pancreaticoduodenectomy, attention should be paid to the possible occurrence of bile leakage.

Advanced human T-cell leukemia virus type 1 carriers and early-stage indolent adult T-cell leukemia-lymphoma are indistinguishable based on CADM1 positivity in flow cytometry.

We previously reported that the cell adhesion molecule 1 (CADM1) versus CD7 plot in flow cytometry reflects disease progression in human T-cell leukemia virus type 1 (HTLV-1) infection. In CD4(+) cells from peripheral blood, CADM1(-) CD7(+) (P), CADM1(+) CD7(dim) (D) and CADM1(+) CD7(-) (N) subpopulations are observed. The D and N subpopulations increase as asymptomatic HTLV-1 carriers (AC) progress to indolent adult T-cell leukemia-lymphoma (ATL) and the N subpopulation then expands in aggressive ATL. In the present study we examined whether the analysis can estimate the risk of developing ATL in advanced AC. Peripheral blood samples from AC (N = 41) and indolent ATL patients (N = 19) were analyzed by flow cytometry using the CADM1 versus CD7 plot for CD4(+) cells and inverse long PCR (clonality analysis) of FACS-sorted subpopulations. Almost all AC with a high HTLV-1 proviral load (>4 copies/100 cells) had a CADM1(+) (D + N) frequency of >10%. AC with 25% < CADM1(+) ≤ 50% contained expanded clones similar to smoldering-type ATL. In many patients in the 25% < CADM1(+) ≤ 50% group, the proportion of abnormal lymphocytes was distributed around the 5% line, which divides AC and smoldering-type ATL in Shimoyama's classification. In conclusion, the CADM1 versus CD7 plot is useful for selection of putative high-risk AC. The characteristics of some AC and smoldering ATL are said to be similar; however, long-term follow up is required and the clinical outcome (e.g. rate of transformation) of these cases should be used to determine whether to include them in the same clinical category.

Clinical outcomes of a novel therapeutic vaccine with Tax peptide-pulsed dendritic cells for adult T cell leukaemia/lymphoma in a pilot study.

Adult T cell leukaemia/lymphoma (ATL) is a human T cell leukaemia virus type-I (HTLV-I)-infected T cell malignancy with poor prognosis. We herein developed a novel therapeutic vaccine designed to augment an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL) response that has been implicated in anti-ATL effects, and conducted a pilot study to investigate its safety and efficacy. Three previously treated ATL patients, classified as intermediate- to high-risk, were subcutaneously administered with the vaccine, consisting of autologous dendritic cells (DCs) pulsed with Tax peptides corresponding to the CTL epitopes. In all patients, the performance status improved after vaccination without severe adverse events, and Tax-specific CTL responses were observed with peaks at 16-20 weeks. Two patients achieved partial remission in the first 8 weeks, one of whom later achieved complete remission, maintaining their remission status without any additional chemotherapy 24 and 19 months after vaccination, respectively. The third patient, whose tumour cells lacked the ability to express Tax at biopsy, obtained stable disease in the first 8 weeks and later developed slowly progressive disease although additional therapy was not required for 14 months. The clinical outcomes of this pilot study indicate that the Tax peptide-pulsed DC vaccine is a safe and promising immunotherapy for ATL.

Osteocytes up-regulate the terminal differentiation of pre-osteoblasts via gap junctions.

We examined cell-to-cell interaction between pre-osteoblasts and osteocytes using MC3T3-E1 and MLO-Y4, respectively. First, GFP expressing MC3T3-E1 (E1-GFP) cells were generated to isolate the cells from co-culture with MLO-Y4. No changes were observed in the expression of osteogenic transcription factors Runx2, Osterix, Dlx5 and Msx2, but expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP) in E1-GFP co-cultured with MLO-Y4 was 300-400-fold greater than that in mono-cultured E1-GFP. In addition, mineralized nodule formation was drastically increased in co-cultured E1-GFP cells compared to mono-cultured cells. Patch clamp assay showed the presence of gap junctions between E1-GFP and MLO-Y4. Furthermore, when the gap junction inhibitor carbenoxolone (CBX) was added to the culture, increased expression of ALP and BSP in E1-GFP co-cultured with MLO-Y4 was suppressed. These results suggest that gap junction detected between pre-osteoblasts and osteocytes plays an important role on the terminal differentiation of pre-osteoblasts.

Quantification of adult T-cell leukemia/lymphoma cells using simple four-color flow cytometry.

BACKGROUND: The absolute number of adult T-cell leukemia/lymphoma (ATL) cells in peripheral blood is an essential indicator to evaluate disease status. However, microscopically counting ATL cells based on morphology requires experience and tends to be inaccurate due to the rarity of ATL. METHODS: Based on our research showing that acute-type ATL cells are specifically enriched in the CD4+/CD7- (CD7N) fraction, a new analytical method to accurately quantify ATL cells was established using an internal bead standard and simple four-color flow cytometry. This method was verified by comparison with microscopic examination of 49 peripheral blood samples and used to follow up patients. RESULTS: A strong correlation was observed between the number of CD7N cells measured by flow cytometry and the number of abnormal lymphocytes measured microscopically by experienced technicians [Pearson's R, 0.963; Spearman's rho, 0.921; intercorrelation coefficient, 0.962]. The linear regression coefficient was close to 1 (ß=1.013). Our method could detect 1 cell/µL, and the limit of quantitation was between 2.9 and 9.8 cells/µL. The frequency of CD7N cells among CD4+ cells changed during chemotherapy, which reflected differences between chemosensitive and chemoresistant cases. Kaplan-Meier analysis with a log-rank test showed that patients with decreased CD7N proportion after chemotherapy had significantly longer disease-specific survival (p=0.003). CONCLUSIONS: Our newly established method quantified tumor cells in patients with acute-type ATL. Furthermore, this method was useful for assessing the efficacy of chemotherapy, and the change of the CD7N proportion could be more important to predict prognosis.

The P75 neurotrophin receptor regulates proliferation of the human MG63 osteoblast cell line.

The 75 kDa transmembrane protein, p75(NTR), is a marker of mesenchymal stem cells (MSCs). Isolated MSCs are capable of differentiating into osteoblasts, but the molecular function of p75(NTR) in MSCs and osteoblasts is poorly understood. The aim of this study was to examine the function of p75(NTR) in the human MG63 osteoblast cell line compared to the murine MC3T3E-1 pre-osteoblast cell line. MG63 cells and MC3T3-E1 cells expressing exogenous p75(NTR) protein (denoted as p75-MG63 and p75GFP-E1, respectively) were generated to compare osteogenic differentiation and cell proliferation abilities. Overexpression of p75(NTR) induced alkaline phosphatase activity and the mRNA expression of osteoblast-related genes such as osterix and bone sialoprotein in both p75-MG63 and p75GFP-E1. Interestingly, exogenous p75(NTR) stimulated cell proliferation and cell cycle progression in p75GFP-E1, but not in p75-MG63. To elucidate any different effects of p75(NTR) expression on osteogenic differentiation and cell proliferation, we examined the mRNA expression of tropomyosin receptor kinase (trk) genes (trkA, trkB, trkC) and Nogo receptor (NgR), which are binding partners of p75(NTR). Although trkA, trkB, and trkC were detected in both p75-MG63 and p75GFP-E1, only NgR was detected in p75-MG63. We then used the K252a inhibitor of the trks to identify the signaling pathway for osteogenic differentiation and cell proliferation. Inhibition of trks by K252a suppressed p75(NTR)-mediated osteogenic differentiation of p75GFP-E1, whereas deletion of the GDI domain in P75(NTR) from the p75-MG63 produced enhanced cell proliferation compared to p75-MG63. These results suggest that p75(NTR) signaling associated with trk receptors promotes both cell proliferation and osteoblast differentiation, but that p75(NTR)-mediated proliferation may be suppressed by signaling from the p75(NTR)/NgR complex.

MRD detection of leukemia relapse using HLA typing by FACS in combination with FISH after mismatched allogeneic stem cell transplantation.

Loss of mismatched HLA is a cause of relapse following HLA-mismatched allo-SCT. We directly detected the loss of mismatched HLA alleles in relapsed leukemic cells at a MRD level using HLA typing by multicolor FACS (HLA-Flow) in combination with FISH in the BM of two patients with MLL-AF9-positive AML, at 6 and 10 months after mismatched allo-SCT. HLA-Flow with FISH analysis detected relapsed leukemic cells not expressing a mismatched HLA allele and harboring the MLL rearrangement. Simultaneously, real-time quantitative RT-PCR detected a low copy number of MLL-AF9 transcripts, consistent with MRD detection. HLA-Flow with FISH is a powerful method for detecting molecular relapse after mismatched allo-SCT and provides important information on the HLA expression status of the relapsed leukemic cells to help determine the next intervention.

The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation.

While the role of p75(NTR) signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75(NTR) on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75(NTR)-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75(NTR) in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75(NTR), as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75(NTR)-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75(NTR) signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.

Development of donor cell leukemia mimicking hematogones after unrelated cord blood transplantation for relapsed acute lymphoblastic leukemia.

A 26-year-old woman, who developed ALL when she was eighteen years old, achieved remission after chemotherapy. Her ALL relapsed when she was twenty-two years old. After re-induction therapy, she underwent cord blood transplantation. Her bone marrow examination on the 42nd day revealed a lymphoblast count of 16%. She was observed without any therapy, but her bone marrow blast count continued to be around 6% for three years without any symptoms. The bone marrow blast fraction originated from the cord blood. Surface marker analysis of the blast fraction initially revealed a pattern of hematogones that was CD10 and CD19 positive, but then showed a myeloblast pattern that was CD13 and CD33 positive. AML developed as donor cell leukemia. When blasts appear in the early phase after transplantation and persist, an observation period is necessary with molecular chimerism, morphology, and surface marker analysis of the blast fraction to consider relapse, hematogones, or donor cell leukemia.

Leukemic T cells are specifically enriched in a unique CD3(dim) CD7(low) subpopulation of CD4(+) T cells in acute-type adult T-cell leukemia.

The morphological discrimination of leukemic from non-leukemic T cells is often difficult in adult T-cell leukemia (ATL) as ATL cells show morphological diversity, with the exception of typical "flower cells." Because defects in the expression of CD3 as well as CD7 are common in ATL cells, we applied multi-color flow cytometry to detect a putative leukemia-specific cell population in the peripheral blood from ATL patients. CD4(+) CD14(-) cells subjected to two-color analysis based on a CD3 vs CD7 plot clearly demonstrated the presence of a CD3(dim) CD7(low) subpopulation in each of nine patients with acute-type ATL. The majority of sorted cells from this fraction showed a flower cell-like morphology and carried a high proviral load for the human T-cell leukemia virus type 1 (HTLV-I). Genomic integration site analysis (inverse long-range PCR) and analysis of the T cell receptor Vß repertoire by flow cytometry indicated that the majority of leukemia cells were included in the CD3(dim) CD7(low) subpopulation. These results suggest that leukemic T cells are specifically enriched in a unique CD3(dim) CD7(low) subpopulation of CD4(+) T cells in acute-type ATL.

Novel approach for transient protein expression in primary cultures of human dental pulp-derived cells.

Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7-3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7-3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7-3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis.

Inhibitory effects of a tryptamine derivative on ultraviolet radiation-induced apoptosis in MC3T3-E1 mouse osteoblasts.

MS-IPA1 is a new synthetic compound that is synthesized from tryptamine. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to MS-IPA1, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of MS-IPA1 on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In this study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of MS-IPA1 on apoptosis by analyzing caspase3/7 activity, translocation of phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. In addition, it was investigated whether MS-IPA1 affects cell proliferation and cell cycle progression. We found that MS-IPA1 had no effect on cell proliferation or cell cycle progression. However, MS-IPA1 suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of caspase activation and translocation of PS. Furthermore, after UV exposure, Bcl-2 expression was increased in the MS-IPA1-treated cells as compared to that in the vehicle-treated cells. In contrast, Bax expression was decreased in the MS-IPA1-treated cell as compared to that in the vehicle-treated cells. These results suggest that MS-IPA1 has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway.

Inhibitory Effects of a Tryptamine Derivative on Ultraviolet Radiation-Induced Apoptosis in MC3T3-E1 Mouse Osteoblasts.

MS-IPA1 is a new synthetic compound that is synthesized from tryptamine. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to MS-IPA1, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of MS-IPA1 on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In this study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of MS-IPA1 on apoptosis by analyzing caspase3/7 activity, translocation of phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. In addition, it was investigated whether MS-IPA1 affects cell proliferation and cell cycle progression. We found that MS-IPA1 had no effect on cell proliferation or cell cycle progression. However, MS-IPA1 suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of caspase activation and translocation of PS. Furthermore, after UV exposure, Bcl-2 expression was increased in the MS-IPA1-treated cells as compared to that in the vehicle-treated cells. In contrast, Bax expression was decreased in the MS-IPA1-treated cell as compared to that in the vehicle-treated cells. These results suggest that MS-IPA1 has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway.

Limited but heterogeneous osteogenic response of human bone marrow mesenchymal stem cells to bone morphogenetic protein-2 and serum.

Although there are numerous reports describing the in vivo bone forming capability of recombinant human bone morphogenetic proteins-2 (rhBMP-2), studies have reported limited effects on human mesenchymal stem cells (hMSCs). However, the reasons for these discrepancies are not well understood. The aim of this study was to investigate the responsiveness of hMSCs to osteoinductive signals, focusing on rhBMP-2 and the effect of serum on that responsiveness. Human MSCs from six donors were analysed. When those cells were treated with osteoinduction medium including dexamethasone (Dex), alkaline phosphatase (ALP) activities increased in all cell lines. On the other hand, rhBMP-2-containing medium failed to increase ALP activity. When five different sera were used for cultivation and induction with rhBMP-2, ALP activities increased in two of them, but not in the others. The expression of BMP-2 antagonist noggin was induced in almost all combinations regardless of the responsiveness to rhBMP-2. On the other hand, the expression of follistatin showed significant variations depending on the serum and cell line. However, the expression did not correlate with the responsiveness to rhBMP-2. The results from this study showed limited but heterogeneous osteogenic response of hMSCs to rhBMP-2 and that the results are affected by the choice of serum. This fact should be concerned for the successful and effective clinical application of rhBMP-2.

Mapping of BrdU label-retaining dental pulp cells in growing teeth and their regenerative capacity after injuries.

Recent studies have demonstrated that human dental pulp contains adult stem cells. A pulse of the thymidine analog BrdU given to young animals at the optimal time could clarify where slow-cycling long-term label-retaining cells (LRCs), putative adult stem cells, reside in the pulp tissue. This study focuses on the mapping of LRCs in growing teeth and their regenerative capacity after tooth injuries. Two to seven peritoneal injections of BrdU into pregnant Wistar rats revealed slow-cycling long-term dense LRCs in the mature tissues of born animals. Numerous dense LRCs were postnatally decreased in number and reached a plateau at 4 weeks after birth when they mainly resided in the center of the dental pulp, associating with blood vessels. Mature dental pulp cells were stained with Hoechst 33342 and sorted into (<0.76%) side population cells using FACS, which included dense LRCs. Some dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 or CD146. Tooth injuries caused degeneration of the odontoblast layer, and newly differentiated odontoblast-like cells contained LRCs. Thus, dense LRCs in mature pulp tissues were supposed to be dental pulp stem cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells. The present study proposes the new hypothesis that both granular and dense LRCs are equipped in the dental pulp and that the dense LRCs with proliferative capacity play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with the granular LRCs.

Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions.

Current standard techniques for bone tissue engineering utilize ex vivo expanded osteogenic cells. However, ex vivo expansion requires serum, which may hinder clinical applications. Here, we report the feasibility and efficacy of bone tissue engineering with human bone marrow stromal cells (BMSCs) expanded in serum-free conditions. Bone marrow was aspirated from 4 healthy donors and adherent cells were cultured in either serum-free medium (STEMPRO((R)) MSC SFM) or conventional serum-containing medium (alpha-MEM supplemented with 10% serum). Efficacy of expansion was greater in serum-free medium. Phenotypically, serum-free expanded BMSCs were smaller in cell-size and showed expression of CD105(++) and CD146(dim). After osteogenic induction, serum-free expanded BMSCs showed lower alkaline phosphatase activity. However, they showed higher responsiveness to induction. In vivo bone-forming ability was also confirmed. In conclusion, bone tissue engineering with serum-free expanded BMSCs is feasible and as efficient as that obtained with BMSCs expanded in conventional serum-containing medium.

Effect of ischemic culture conditions on the survival and differentiation of porcine dental pulp-derived cells.

Although differentiated and undifferentiated cells can be exposed to ischemic conditions in cases of injury or inflammation, the effects of ischemia on cell survival and differentiation have not been well characterized. Here, we characterize the response of porcine dental pulp-derived cells (pDPCs) to culture conditions that approximate ischemia. Dental pulp is often exposed to ischemia due to narrow vascular openings in the tooth, which may affect the differentiation status of pDPCs. In this study, we investigated the influence of various ischemic conditions on differentiation-induced and non-induced pDPCs. To understand the character of cells used in this study, reported cell surface markers for dental pulp stem cells were investigated. pDPCs were CD90(low), CD105(+), and alpha-smooth muscle actin positive and showed osteogenic/chondrogenic differentiation potential. Anoxia was the most detrimental factor to cell viability, whereas hypoxia did not significantly affect survival. Glucose concentrations had a significant, mechanism-dependent effect on cell death. The presence of glucose correlated with caspase-dependent cell death, whereas the absence of glucose was linked to caspase-independent cell death. In contrast, differentiation status (i.e., induced versus non-induced pDPCs) did not affect the degree or mechanism of cell death. Finding depletion of specific markers by reverse transcription-polymerase chain reaction in both induced and non-induced cells suggests that the cells are de-differentiating under anoxia. Non-induced pDPCs were susceptible to anoxic induction of Oct-4, Sox-2, and hypoxia inducible factor-2alpha, while these genes did not change in induced pDPCs. Re-differentiation analysis revealed that the surviving cells from non-induced pDPCs showed twofold higher alkaline phosphatase activity as compared with induced pDPCs, which suggest greater plasticity among the surviving fraction of non-induced pDPCs. These data showed that the ischemic conditions have similar detrimental influence on both undifferentiated and differentiated pDPCs, and affect differentiation status of pDPCs. Furthermore, ischemic conditions may influence the plasticity of undifferentiated pDPCs.

Recipient-derived cells after cord blood transplantation: dynamics elucidated by multicolor FACS, reflecting graft failure and relapse.

Although umbilical cord blood has been increasingly used as an alternative donor source to treat hematologic malignancies, cord blood transplantation (CBT) is frequently complicated by graft failure and relapse of primary diseases. Because persistence or increase of recipient-derived hematopoietic or malignant cells has pathogenic import under these conditions, analysis of recipient-derived cells should be useful to understand the pathogenesis of graft failure and relapse of primary disease. Because most CBT involves human leukocyte antigen (HLA)-mismatched transplantation, we developed a 9-color fluorescence activated cell sorter (FACS)-based method of mixed chimerism (MC) analysis using anti-HLA antibodies to detect mismatched antigens (HLA-Flow method). Among CD4(+) T cells, CD8(+) T cells, B cells, NK cells, monocytes, and granulocytes, donor- and recipient-derived cells alike could be individually analyzed simultaneously in a rapid, quantitative and highly sensitive manner, making the HLA-Flow method very valuable in monitoring the engraftment process. In addition, this method was also useful in monitoring recipient-derived cells with leukemia-specific phenotypes, both as minimal residual disease (MRD) and as early harbingers of relapse. Leukemia relapse can be definitively diagnosed by cytogenetic or PCR studies using recipient-derived cells sorted for leukemia markers. Multicolor HLA-fFlow analysis and cell sorting in early diagnosis of graft failure and relapse was confirmed as valuable in 14 patients who had received HLA-mismatched CBT.