Pluripotent Stem Cells In Bone Marrow Research Articles

Delineating the effects of 5-fluorouracil and follicle-stimulating hormone on mouse bone marrow stem/progenitor cells.

BackgroundPluripotent, Lin–/CD45–/Sca-1+ very small embryonic-like stem cells (VSELs) in mouse bone marrow (BM) are resistant to total body radiation because of their quiescent nature, whereas Lin–/CD45+/Sca-1+ hematopoietic stem cells (HSCs) get eliminated. In the present study, we provide further evidence for the existence of VSELs in mouse BM and have also examined the effects of a chemotherapeutic agent (5-fluorouracil (5-FU)) and gonadotropin hormone (follicle-stimulating hormone (FSH)) on BM stem/progenitor cells.MethodsVSELs and HSCs were characterized in intact BM. Swiss mice were injected with 5-FU (150 mg/kg) and sacrificed on 2, 4, and 10 days (D2, D4, and D10) post treatment to examine changes in BM histology and effects on VSELs and HSCs by a multiparametric approach. The effect of FSH (5 IU) administered 48 h after 5-FU treatment was also studied. Bromodeoxyuridine (BrdU) incorporation, cell cycle analysis, and colony-forming unit (CFU) assay were carried out to understand the functional potential of stem/progenitor cells towards regeneration of chemoablated marrow.ResultsNuclear OCT-4, SCA-1, and SSEA-1 coexpressing LIN–/CD45– VSELs and slightly larger LIN–/CD45+ HSCs expressing cytoplasmic OCT-4 were identified and comprised 0.022 ± 0.002 % and 0.081 ± 0.004 % respectively of the total cells in BM. 5-FU treatment resulted in depletion of cells with a 7-fold reduction by D4 and normal hematopoiesis was re-established by D10. Nuclear OCT-4 and PCNA-positive VSELs were detected in chemoablated bone sections near the endosteal region. VSELs remained unaffected by 5-FU on D2 and increased on D4, whereas HSCs showed a marked reduction in numbers on D2 and later increased along with the corresponding increase in BrdU uptake and upregulation of specific transcripts (Oct-4A, Oct-4, Sca-1, Nanog, Stella, Fragilis, Pcna). Cells that survived 5-FU formed colonies in vitro. Both VSELs and HSCs expressed FSH receptors and FSH treatment enhanced hematopoietic recovery by 72 h.ConclusionBoth VSELs and HSCs were activated in response to the stress created by 5-FU and FSH enhanced hematopoietic recovery by at least 72 h in 5-FU-treated mice. VSELs are the most primitive pluripotent stem cells in BM that self-renew and give rise to HSCs under stress, and HSCs further divide rapidly and differentiate to maintain homeostasis. The study provides a novel insight into basic hematopoiesis and has clinical relevance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0311-6) contains supplementary material, which is available to authorized users.

Effect of autologous mesenchymal pluripotent stem cells transplantation on liver microcirculation in rats with experimental liver cirrhosis

Aim. To study the effect of autologous bone marrow pluripotent stem cells transplantation on liver microcirculation in experimental model of liver cirrhosis.Methods. 43 white Wistar male rats with body weight of 150-180 g aged at least 3 months were used, in which autologous pluripotent mesenchymal stromal cells transplantation was performed. Considering the animals mortality at the cirrhosis modeling stage, which was 9.3% (4 out of 43 rats), the first group included 19 rats in which stromal cells were transplanted into the portal vein; in the second group (20 rats) the cells were injected into the common hepatic artery. Liver microcirculation was studied using laser Doppler flowmetry and wavelet analysis. Examinations were performed during the operation prior to autologous pluripotent mesenchymal stromal cells transplantation in rats with experimental liver cirrhosis, as well as on the 8th week of treatment.Results.In modeled liver cirrhosis, the microcirculation index was decreased by 24.5% (pConclusion. The repeated studies of microcirculation based on laser Doppler during the treatment of animals with experimental liver cirrhosis indicates the advantages of intra-arterial autologous multipotent mesenchymal stromal cells transplantation.

Ptprg and BCR/ABL1 Expression In CML Patients At Diagnosis and Upon TKI Treatment: Preliminary Results

BackgroundChronic Myeloid Leukemia (CML), is a biologically and clinically heterogeneous disease that originates in a pluripotent bone marrow stem cell. The hallmark of CML is the presence of an aberrant gene, BCR-ABL1, which encodes for a constitutively activated tyrosine kinase. Protein tyrosine phosphatases can negatively regulate ABL1 mediated signaling by dephosphorylating the kinase and/or its substrates and impaired phosphatase activity has been linked to several solid cancers and leukemias. We have previously reported the role of the receptor-type phosphatase PTPRG as tumor suppressor gene and demonstrated a significant decrease in the PTPRGexpression level in CML.Here we have investigated the expression of PTPRG gene in 32 CML patients aiming at the evaluation of the clinical relevance of the PTPRGdown regulation. MethodsAll 32 CML patients included in this study were diagnosed in chronic phase. Thirteen untreated patients diagnosed with philadelphia-negative myelod disorders were included in the study as control group. The study was approved by the Local Ethics Committee, and informed consent in accordance with declaration of Helsinki was obtained from each patient. The expression level of PTPRG gene was evaluated by a sybr green real-time RT-PCR assay in 2 samples from each patient, taken at diagnosis and following TKI treatment. The beta-Actin (ACTB) housekeeping gene was used for normalization. Results were validated using predesigned TaqMan quantitative RT-PCR assays for PTPRG (intracellular domain) and ABL1 genes. Quantitative RT-PCR was used to quantify BCR-ABL1 gene fusion transcripts according to the European Leukemia Net guidelines. Statistical analysis and comparisons were performed by Mann-Whitney's and Student paired T tests using the SPSS software. ResultsThe mean levels of PTPRG transcript, expressed as PTPRG/ACTB ratio, were significantly lower in CML samples at diagnosis compared to the non-CML control group (0,44%, range 0-0,37 vs 6,29%, range 0,09-52; p=0.020). Recovery of PTPRG mRNA expression at variable levels, ranging from 0.17 to 30%, was detected in 29/32 follow up samples, taken at different time points of treatment (from 3 months to 10 years). Differences in PTPRG gene expression levels between CML samples at diagnosis and after treatment were statistically significant (p=0,027). No statistically significant correlation was observed between PTPRG/ACTB and BCR-ABL/ABL1 ratios nor with deepness of molecular response or SOKAL and EUTOS scores, even though 2 of the 3 patients showing higher level of PTPRGmRNA at diagnosis than in the follow up sample showed resistance to TKI treatment. ConclusionsIn this study we confirm a down regulation of PTPRG in a high percentage of CML patients and show that its expression is restored upon treatment with TKIs. Deregulated expression of PTPRG phosphatase underline its role as a tumor suppressor gene in CML and highlights its potential use as a new bio-marker of disease potentially usable in association with BCR/ABL1, allowing for a better understanding of the molecular mechanism underlying the development of CML, and propose possible new avenues for therapeutic treatment. Disclosures:Yassin:qatar national research fund: Patents & Royalties, Research Funding.

Changes in the frequencies of human hematopoietic stem and progenitor cells with age and site

This study enumerated CD45(hi)/CD34(+) and CD45(hi)/CD133(+) human hematopoietic stem cells (HSCs) and progenitor granulocyte-macrophage colony forming cells (GM-CFCs) in blood and trochanteric and femoral bone marrow in 233 individuals. Stem cell frequencies were determined with multiparameter flow cytometry and using an internal control to determine the intrinsic variance of the assays. Progenitor cell frequency was determined using a standard colony assay technique. The frequency of outliers from undetermined methodological causes was highest for blood, but less than 5% for all values. The frequency of CD45(hi)/CD133(+) cells correlated highly with the frequency of CD45(hi)/CD34(+) cells in trochanteric and femoral bone marrow. The frequency of these HSC populations in trochanteric and femoral bone marrow rose significantly with age. In contrast, there was no significant trend of either of these cell populations with age in the blood. Trochanteric marrow progenitor GM-CFCs showed no significant trends with age, but femoral marrow GM-CFCs trended downward with age, potentially because of the reported conversion of red marrow at this site to fat with age. Hematopoietic stem and progenitor cells exhibited changes in frequencies with age that differed between blood and bone marrow. We previously reported that side population (SP) multipotential HSC, which includes the precursors of CD45(hi)/CD133(+) and CD45(hi)/CD34(+), decline with age. Potentially the increases in stem cell frequencies in the intermediate compartment between SP and GM progenitor cells observed in this study represent a compensatory increase for the loss of more potent members of the HSC hierarchy.

Adult Rat Bone Marrow-Derived Stem Cells Promote Late Fetal Type II Cell Differentiation in a Co-Culture Model

Bronchopulmonary dysplasia develops in preterm infants due to a combination of lung immaturity and lung injury. Cultured pluripotent bone marrow stem cells (BMSC) are known to reduce injury and induce repair in adult and in immature lungs, possibly through paracrine secretion of soluble factors. The paracrine relationship between BMSC and primary fetal lung epithelial type II cells is unknown. We determined the effects of BMSC on type II cell and fibroblast behavior using an in vitro co-culture model. Rat BMSC were isolated and co-cultured with primary fetal E21 rat type II cells or lung fibroblasts in a Transwell® system without direct cell contact. Effects of BMSC conditioned media (CM) on type II cell and fibroblast proliferation and on type II cell surfactant phospholipid (DSPC) synthesis and mRNA expression of surfactant proteins B and C (sftpb and sftpc) were studied. We also determined the effect of fibroblast and type II cell CM on BMSC proliferation and surface marker expression. Co-culture with BMSC significantly decreased type II cell and fibroblast proliferation to 72.5% and 83.7% of controls, respectively. Type II cell DSPC synthesis was significantly increased by 21% and sftpb and sftpc mRNA expressions were significantly induced (2.1 fold and 2.4 fold, respectively). BMSC proliferation was significantly reduced during the co-culture. Flow cytometry confirmed that BMSC retained the expression of undifferentiated stem cell markers despite their exposure to fetal lung cell CM. We conclude that BMSC induce fetal type II cell differentiation through paracrine release of soluble factors. These studies provide clues for how BMSC may act in promoting alveolar repair following injury.

Acute Kidney Injury Presenting a Feature of Leukemic Infiltration during Therapy for Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a myeloproliferative disease that originates in abnormal pluripotent bone marrow stem cells and it is consistently associated with the Philadelphia chromosome and/or BCR/ ABL fusion gene. Renal infiltration of leukemic cells is relatively rare in CML and is associated with renal impairment. We describe a patient who developed acute renal failure by tubulointerstitial nephropathy during treatment with imatinib mesylate for CML. The acute kidney injury was subsequently found to be due to direct leukemic infiltration. Treatment with hydroxycarbamide and prednisolone resulted in stabilization of the renal function for approximately 4 months. Leukemic infiltration into the kidney should always be considered when a patient with CML presents with renal impairment, regardless of the clinical stage, as the renal failure often responds well to chemotherapy.

Does the Number or Quality of Pluripotent Bone Marrow Stem Cells Decrease with Age?

In the next 25 years, as the 37 million baby boomers age, we can expect a 400% increase in total joint arthroplasties that will challenge surgeons, the healthcare system, and the scientific community. The burden may be eased if we are able to manipulate side population stem cells and enhance peri-prosthetic bone remodeling thereby reducing the incidence of revisions. Therefore, as a preliminary question, we asked if the number and quality of side population stem cells, with the ability to proliferate into multiple cell lineages for long periods, correlates with age and can be evaluated in peripheral blood. Using flow cytometry we analyzed the quantity and quality of side population stem cells from bone marrow and peripheral blood in 54 patients (20 under 60 years of age, 34 over) undergoing THA. The total side population of stem cells decreased with age, but their long-term repopulating ability (quality) remained constant. The total count of side population stem cells in marrow correlated with the number found in peripheral blood. If these populations can be manipulated, periprosthetic remodeling may be beneficially enhanced.

Clinical and Pathologic Analysis of 16 Cases of Relapsed Chronic Myeloid Leukemia After Stem Cell Transplantation

Chronic myeloid leukemia (CML) is a myeloproliferative disease that originates in an abnormal pluripotent bone marrow stem cell and is characteristically associated with the Philadelphia chromosome and/or the bcr/abl fusion gene. Despite the exciting success of the bcr/abl tyrosine kinase-specific inhibitor imatinib for CML treatment, hematopoietic stem cell (bone marrow or peripheral blood stem cell) transplantation (HCT) remains the only "curative" approach for the majority of patients. Although HCT outcomes for patients with CML have improved considerably during the past 2 decades, relapse after HCT may occur. We analyzed the clinical and pathologic features of 16 cases of hematologically relapsed CML after HCT during a 5-year period at City of Hope National Medical Center, Duarte, CA. The results of our analysis showed that relapsed CML after HCT frequently manifested with advanced disease with a more aggressive clinical course and was often refractory to therapy. The frequency of acute leukemic transformation at time of relapse was largely associated with pre-HCT disease status and acquired secondary cytogenetic abnormalities. Disease mortality in patients with relapsed CML after HCT was closely associated with advanced disease and HCT-related complications.

Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl 4-injured rats

The autologous transplantation of bone marrow cells is a promising treatment for liver disease. Pluripotent bone marrow stem cells can differentiate into hepatocytes, but few reports address the therapeutic effect of transplanting these stem cells into damaged liver in vivo. Here, we transplanted bone marrow-derived mesenchymal cells (BMMCs) to test their effect in liver-injured rats. Rat bone marrow cells were cultivated for 2 weeks in the presence or absence of hepatocyte growth factor (HGF), labeled with a fluorescent marker, and transplanted by injection into CCl(4)-injured rats. Blood samples collected 4 weeks later were analyzed for albumin production and transaminase levels. The amount of fibrosis was determined by histology. RT-PCR analysis detected alpha-fetoprotein and albumin mRNAs in BMMCs cultured with HGF for 2 weeks. Albumin protein was also produced in the BMMC cultures by a subpopulation of cells. Transplantation of the BMMCs into liver-injured rats restored their serum albumin level and significantly suppressed transaminase activity and liver fibrosis. These effects were not seen when the BMMCs were cultured without HGF. The transplantation of BMMCs cultured with HGF effectively treats liver injury in rats. This is a promising technique for autologous transplantation in humans with liver injury.

Induction of functional neovascularization by combined VEGF and angiopoietin-1 gene transfer using AAV vectors

Vectors based on the adeno-associated virus (AAV) deliver therapeutic genes to muscle and heart at high efficiency and maintain transgene expression for long periods of time. Here we report about the synergistic effect on blood vessel formation of AAV vectors expressing the 165 aa isoform of vascular endothelial growth factor (VEGF165), a powerful activator of endothelial cells, and of angiopoietin-1 (Ang-1), which is required for vessel maturation. High titer AAV-VEGF165 and AAV-Ang-1 vector preparations were injected either alone or in combination in the normoperfused tibialis anterior muscle of rats. Long term expression of VEGF165 determined massive cellular infiltration of the muscle tissues over time, with the formation of a large set of new vessels. Strikingly, some of the cells infiltrating the treated muscles were found positive for markers of activated endothelial precursors (VEGFR-2/KDR and Tie-2) and for c-kit, an antigen expressed by pluripotent bone marrow stem cells. Expression of VEGF165 eventually resulted in the formation of structured vessels surrounded by a layer of smooth muscle cells. Presence of these arteriolae correlated with significantly increased blood perfusion in the injected areas. Co-expression of VEGF165 with angiopoietin-1-which did not display angiogenic effect per se-remarkably reduced leakage of vessels produced by VEGF165 alone.

Control of hematopoietic differentiation: lack of specificity in signaling by cytokine receptors.

Blood consists of eight principal cell types of diverse function. They have in common a limited life span and an inability to replicate; they therefore must be replaced continuously throughout life. A small pool of pluripotent hemopoietic stem cells that reside in the bone marrow gives rise to all blood cell types through a process of simultaneous lineage commitment, cell proliferation, and differentiation (Fig. 1). In the last two decades, numerous cytokines have been identified as essential extracellular factors in this process (1–4). Some, like stem cell factor, interleukin 3, or granulocyte–macrophage colony-stimulating factor (GM-CSF), contribute to the progeny of many blood cell types. Others, like Epo, G-CSF, or thrombopoietin, exert their principal action on progenitors of a single lineage. Mice in which the genes for lineage- specific cytokines or their receptors are disrupted show a severe deficiency in the blood cells that constitute the progeny of their physiological targets (5, 6). For example, disruption of the EpoR gene results in embryonic lethality caused by the absence of red blood cells; similarly, disruption of the interleukin 7 receptor leads to severe lymphocyte deficiency. Therefore, lineage-restricted cytokine receptors each play an essential and specific role in differentiation. What is it that makes their signaling essential? The report in this issue of Proceedings by Goldsmith et al. (7) builds on a series of reports in the last year (8–11) that is fundamentally altering our view on the role of cytokine receptors in hematopoietic differentiation. Figure 1 Structure of the hematopoietic compartment. Bone marrow pluripotent stem cells may either self-renew or give rise to eight different hematopoietic lineages through a gradual process of commitment and differentiation. Some of the cytokines involoved in supporting this process are illustrated. M-CSF, Macrophage colony stimulating factor; SCF, stem cell factor; Epo, erythropoietin; Tpo, thrombopoietin; …

Global vascular expression of murine CD34, a sialomucin-like endothelial ligand for L-selectin

Extravasation of leukocytes into organized lymphoid tissues and into sites of inflammation is critical to immune surveillance. Leukocyte migration to peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) depends on L-selectin, which recognizes carbohydrate-bearing, sialomucin-like endothelial cell surface glycoproteins. Two of these ligands have been identified at the molecular level. One is the potentially soluble mucin, GlyCAM 1, which is almost exclusively produced by high endothelial venules (HEV) of PLN and MLN. The second HEV ligand for L-selectin is the membrane-bound sialomucin CD34. Historically, this molecule has been successfully used to purify human pluripotent bone marrow stem cells, and limited data suggest that human CD34 is present on the vascular endothelium of several organs. Here we describe a comprehensive analysis of the vascular expression of CD34 in murine tissues using a highly specific antimurine CD34 polyclonal antibody. CD34 was detected on vessels in all organs examined and was expressed during pancreatic and skin inflammatory episodes. A subset of HEV-like vessels in the inflamed pancreas of nonobese diabetic (NOD) mice are positive for both CD34 and GlyCAM 1, and bind to an L-selectin/immunoglobulin G (IgG) chimeric probe. Finally, we found that CD34 is present on vessels of deafferentiated PLN, despite the fact that these vessels are no longer able to interact with L-selectin or support lymphocyte binding in vitro or trafficking in vivo. Our data suggest that the regulation of posttranslational carbohydrate modifications of CD34 is critical in determining its capability to act as an L-selectin ligand. Based on its ubiquitous expression, we propose that an appropriately glycosylated form of vascular CD34 may act as a ligand for L-selectin-mediated leukocyte trafficking to both lymphoid and nonlymphoid sites.

Global Vascular Expression of Murine CD34, a Sialomucin-Like Endothelial Ligand for L-Selectin

Extravasation of leukocytes into organized lymphoid tissues and into sites of inflammation is critical to immune surveillance. Leukocyte migration to peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) depends on L-selectin, which recognizes carbohydrate-bearing, sialomucin-like endothelial cell surface glycoproteins. Two of these ligands have been identified at the molecular level. One is the potentially soluble mucin, GlyCAM 1, which is almost exclusively produced by high endothelial venules (HEV) of PLN and MLN. The second HEV ligand for L-selectin is the membrane-bound sialomucin CD34. Historically, this molecule has been successfully used to purify human pluripotent bone marrow stem cells, and limited data suggest that human CD34 is present on the vascular endothelium of several organs. Here we describe a comprehensive analysis of the vascular expression of CD34 in murine tissues using a highly specific antimurine CD34 polyclonal antibody. CD34 was detected on vessels in all organs examined and was expressed during pancreatic and skin inflammatory episodes. A subset of HEV-like vessels in the inflamed pancreas of nonobese diabetic (NOD) mice are positive for both CD34 and GlyCAM 1, and bind to an L-selectin/immunoglobulin G (IgG) chimeric probe. Finally, we found that CD34 is present on vessels of deafferentiated PLN, despite the fact that these vessels are no longer able to interact with L-selectin or support lymphocyte binding in vitro or trafficking in vivo. Our data suggest that the regulation of posttranslational carbohydrate modifications of CD34 is critical in determining its capability to act as an L-selectin ligand. Based on its ubiquitous expression, we propose that an appropriately glycosylated form of vascular CD34 may act as a ligand for L-selectin-mediated leukocyte trafficking to both lymphoid and nonlymphoid sites.

Transcriptional regulation of T cell receptor genes.

The diverse lineages of the mammalian hematopoietic system including both B and T lymphocytes are derived from a single mesodermal progenitor, the pluripotent bone marrow stem cell. The coordinate transcriptional regulation of sets of lineage-specific genes is one of the important molecular mechanisms underlying hematopoietic lineage determination and differentiation. The immunoglobulin and T cell receptor (TCR) genes have been used as model systems to study lineage-specific transcriptional regulation during lymphoid development. This review summarizes our current understanding of the regulation of TCR gene expression during thymocyte ontogeny. Expression of each of the TCR genes is controlled by T cell-specific transcriptional enhancers that bind partially overlapping sets of ubiquitous and lymphoid-specific transcription factors. These include members of both the ATF/CREB family of basic-leucine zipper proteins and the Ets protooncogene family, as well as the T cell-specific zinc finger transcription factor, GATA-3, and the T cell-specific high mobility group proteins TCF-1 and TCF-1 alpha/LEF-1. The identification of binding sites for these same transcription factors in a number of additional T cell-specific genes suggests that they may play important roles in the coordinate regulation of gene expression that specifies the development of the T cell lineages. Recent studies of the TCR alpha and gamma genes have suggested that negative regulatory elements of transcriptional silencers may also play an important role in controlling the lineage-specific expression of these genes. On going studies are designed to clarify the role of each of the TCR enhancer binding proteins in regulating T cell development in vivo, to more precisely define the interactions between the TCR enhancer binding proteins, and to elucidate the molecular mechanisms underlying transcriptional silencer activity.

Dye-Mediated Photolysis Is Capable of Eliminating Drug-Resistant (MDR +) Tumor Cells

Abstract We evaluated the potential role of photoradiation therapy with a benzoporphyrin derivative, monoacid ring A (BPD-MA), and dihematoporphyrin ether (DHE), for the ex vivo purging of residual tumor cells from autologous bone marrow (BM) grafts. BPD-MA and DHE photosensitizing activity was tested against two human large-cell lymphoma cell lines and colony-forming unit-leukemia (CFU-L) derived from patients with acute myelogenous leukemia (AML). In mixing experiments, 4-log elimination of tumor cell lines was observed after 1 hour of incubation with 75 ng/mL of BPD-MA or 30 minutes of treatment with 12.5 micrograms/mL of DHE followed by white light exposure. By comparison, using the same concentration of BPD-MA, the mean recovery of normal BM progenitors was 4% +/- 0.8% (mean +/- SD) for granulocyte- macrophage colony-forming unit (CFU-GM) and 5% +/- 0.8% for burst- forming unit-erythroid (BFU-E). Similarly, DHE treatment resulted in the recovery of 5.2% +/- 2% and 9.8% +/- 3% of CFU-GM and BFU-E, respectively. Furthermore, equivalently cytotoxic concentrations of both DHE and BPD-MA and light were found not to kill normal pluripotent stem cells in BM, as demonstrated by their survival in two-step long- term marrow culture at levels equal to untreated controls. The T- lymphoblastic leukemia cell line CEM and its vinblastine (VBL)- resistant subline CEM/VBL, along with the acute promyelocyte leukemia cell line HL-60 and its vincristine (VCR)-resistant subline HL-60/VCR, were also tested. BPD-MA at 75 ng/mL was able to provide a greater than 4-log elimination of the drug-sensitive cell lines, but only a 34% and 55% decrease of the drug-resistant HL-60/VCR and CEM/VBL cell lines, respectively. On the contrary, 12.5 micrograms/mL of DHE reduced the clonogenic growth of all the cell lines by more than 4 logs. Further experiments demonstrated decreased uptake of both BPD-MA and DHE by the resistant cell lines. However, all the cell lines took up more DHE than BPD-MA under similar experimental conditions. Our results demonstrate the preferential cytotoxicity of BPD-MA and DHE toward neoplastic cell lines and CFU-L from AML patients. In addition, DHE was slightly more effective in purging tumor cells expressing the p-170 glycoprotein. These results suggest that photoradiation with DHE would be useful for in vitro purging of residual drug-resistant leukemia and lymphoma cells.

Dye-mediated photolysis is capable of eliminating drug-resistant (MDR+) tumor cells

We evaluated the potential role of photoradiation therapy with a benzoporphyrin derivative, monoacid ring A (BPD-MA), and dihematoporphyrin ether (DHE), for the ex vivo purging of residual tumor cells from autologous bone marrow (BM) grafts. BPD-MA and DHE photosensitizing activity was tested against two human large-cell lymphoma cell lines and colony-forming unit-leukemia (CFU-L) derived from patients with acute myelogenous leukemia (AML). In mixing experiments, 4-log elimination of tumor cell lines was observed after 1 hour of incubation with 75 ng/mL of BPD-MA or 30 minutes of treatment with 12.5 micrograms/mL of DHE followed by white light exposure. By comparison, using the same concentration of BPD-MA, the mean recovery of normal BM progenitors was 4% +/- 0.8% (mean +/- SD) for granulocyte-macrophage colony-forming unit (CFU-GM) and 5% +/- 0.8% for burst-forming unit-erythroid (BFU-E). Similarly, DHE treatment resulted in the recovery of 5.2% +/- 2% and 9.8% +/- 3% of CFU-GM and BFU-E, respectively. Furthermore, equivalently cytotoxic concentrations of both DHE and BPD-MA and light were found not to kill normal pluripotent stem cells in BM, as demonstrated by their survival in two-step long-term marrow culture at levels equal to untreated controls. The T-lymphoblastic leukemia cell line CEM and its vinblastine (VBL)-resistant subline CEM/VBL, along with the acute promyelocyte leukemia cell line HL-60 and its vincristine (VCR)-resistant subline HL-60/VCR, were also tested. BPD-MA at 75 ng/mL was able to provide a greater than 4-log elimination of the drug-sensitive cell lines, but only a 34% and 55% decrease of the drug-resistant HL-60/VCR and CEM/VBL cell lines, respectively. On the contrary, 12.5 micrograms/mL of DHE reduced the clonogenic growth of all the cell lines by more than 4 logs. Further experiments demonstrated decreased uptake of both BPD-MA and DHE by the resistant cell lines. However, all the cell lines took up more DHE than BPD-MA under similar experimental conditions. Our results demonstrate the preferential cytotoxicity of BPD-MA and DHE toward neoplastic cell lines and CFU-L from AML patients. In addition, DHE was slightly more effective in purging tumor cells expressing the p-170 glycoprotein. These results suggest that photoradiation with DHE would be useful for in vitro purging of residual drug-resistant leukemia and lymphoma cells.

Hematological toxicity of tetrachloroethylene in mice.

Female C57/BL/6 x DBA/2 hybrid mice were exposed to two concentrations (270 and 135 ppm) of tetrachloroethylene (PER) in inhalation chambers for 6 h/day, 5 days per week, up to 11.5 weeks (270 ppm) and 7.5 weeks (135 ppm), respectively, followed by a 3-week exposure-free period. In the peripheral blood a reduction of lymphocytes/monocytes and of neutrophils was observed with an almost complete regeneration in the exposure-free period. A reticulocytosis during and also after the exposure indicated a compensatory reaction in the erythroid cell system. Bone marrow pluripotent stem cells (CFU-S) were not affected up to 7.5 weeks. Erythroid committed cells (BFU-E and CFU-E) did react, with a reduction of CFU-E numbers at 7.5 and 11.5 weeks. A slight reduction of CFU-C numbers at 7.5 weeks indicated, in accordance with the peripheral blood data, a disturbance of the granulocytic cell series as well.

Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis.

Mechanisms triggering the commitment of pluripotent bone marrow stem cells to differentiated lineages such as mononuclear macrophages or multinucleated megakaryocytes are still unknown, although several lines of evidence suggested correlation between cholinergic signaling and hematopoietic differentiation. We now present cloning of a cDNA coding for CHED (cholinesterase-related cell division controller), a human homolog of the Schizosaccharomyces pombe cell division cycle 2 (cdc2)-like kinases, universal controllers of the mitotic cell cycle. Library screening, RNA blot hybridization, and direct PCR amplification of cDNA reverse-transcribed from cellular mRNA revealed that CHED mRNA is expressed in multiple tissues, including bone marrow. The CHED protein includes the consensus ATP binding and phosphorylation domains characteristic of kinases, displays 34-42% identically aligned amino acid residues with other cdc2-related kinases, and is considerably longer at its amino and carboxyl termini. An antisense oligodeoxynucleotide designed to interrupt CHED's expression (AS-CHED) significantly reduced the ratio between CHED mRNA and actin mRNA within 1 hr of its addition to cultures, a reduction that persisted for 4 days. AS-CHED treatment selectively inhibited megakaryocyte development in murine bone marrow cultures but did not prevent other hematopoietic pathways, as evidenced by increasing numbers of mononuclear cells. An oligodeoxynucleotide blocking production of the acetylcholine-hydrolyzing enzyme, butyrylcholinesterase, displayed a similar inhibition of megakaryocytopoiesis. In contrast, an oligodeoxynucleotide blocking production of the human 2Hs cdc2 homolog interfered with production of the human 2Hs cdc2 homolog interfered with cellular proliferation without altering the cell-type composition of these cultures. Therefore, these findings strengthen the link between cholinergic signaling and cell division control in hematopoiesis and implicate both CHED and cholinesterases in this differentiation process.

Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells.

It has been established that murine mast cells are derived from a pluripotent bone marrow stem cell. In humans, the corresponding pluripotent cell is included in the CD34+ bone marrow population. To determine whether human mast cells arise from CD34+ human progenitor cells, enriched CD34+ cells were cultured over agarose surfaces (interphase cultures) or cocultured with mouse 3T3 fibroblasts in the presence of recombinant human (rh) IL-3. The presence of both mast cells and basophils was determined using a variety of histochemical and immunohistologic techniques, including immunogold labeling for IgE receptors and mast cell tryptase. Mast cells and basophils continued to appear in cultures when T cell, B cell, macrophage, and eosinophil committed progenitor cells were removed, but were not seen in cultures from which CD34+ cells were removed. CD34+ cells layered over agarose in the presence of rhIL-3 were shown to give rise to cultures that contained mast cells (1 to 5%) and basophils (25 to 40%). Cultures supplemented with rhIL-4 showed no additional increase in mast cells or basophils. CD34+ cells cocultured with 3T3 fibroblasts in the presence of rhIL-3 gave rise to mast cells within the fibroblast monolayer, which by 6 wk comprised up to 46% of the monolayer. CD34-cells on 3T3 fibroblasts gave rise to few mast cells (2% of the monolayer). Mast cell granules from interphase cultures contained homogeneous electron-dense material. In contrast, mast cells within 3T3 monolayers at 6 wk contained a variety of granule morphologies, including scroll, mixed, reticular, dense core, or homogeneous patterns. We conclude that both human mast cells and basophils arise from CD34+ human progenitor cells.

Bone marrow granulopoietic response to scalds and wound infection in mice

Phagocytic cell function is often suppressed after thermal injury. It has been suggested that one of the mechanisms of suppression is a direct effect of thermal injury upon these cells. In the present study we examined whether bone marrow haemopoietic stem cells were suppressed by thermal injury similar to the effect seen on mature haemopoietic cells. Results of both in vitro bone marrow cell cultures and in vivo spleen colony assays showed that granulopoiesis was not suppressed but activated early on day 3 following scald injury or scald plus wound infection in mice. In the latter group, stimulation of granulopoiesis persisted throughout the entire observation period of 9 days. These studies indicate that bone marrow pluripotent stem cells are capable of modulating their direction of differentiation according to body demand and deviate toward the granulocyte series following thermal injury.