Assessment of Benzene-Induced Hematotoxicity Using a Human-Like Hematopoietic Lineage in NOD/Shi-scid/IL-2R��null Mice

Despite recent advancements, it is still difficult to evaluate in vivo responses to toxicants in humans. Development of a system that can mimic the in vivo responses of human cells will enable more accurate health risk assessments. A surrogate human hematopoietic lineage can be established in NOD/Shi-scid/IL-2Rγ^null (NOG) mice by transplanting human hematopoietic stem/progenitor cells (Hu-NOG mice). Here, we first evaluated the toxic response of human-like hematopoietic lineage in NOG mice to a representative toxic agent, benzene. Flow cytometric analysis showed that benzene caused a significant decrease in the number of human hematopoietic stem/progenitor cells in the bone marrow and the number of human leukocytes in the peripheral blood and hematopoietic organs. Next, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice (Mo-NOG mice). A comparison of the degree of benzene-induced hematotoxicity in donor-derived hematopoietic lineage cells within Mo-NOG mice indicated that the toxic response of Hu-NOG mice reflected interspecies differences in susceptibilities to benzene. Responses to the toxic effects of benzene were greater in lymphoid cells than in myeloid cells in Mo-NOG and Hu-NOG mice. These findings suggested that Hu-NOG mice may be a powerful in vivo tool for assessing hematotoxicity in humans, while accounting for interspecies differences.     

Magnetically empowered bone marrow cells as a micro-living motor can improve early hematopoietic reconstitution

Background aimsBone marrow-derived hematopoietic stem cell transplantation/hematopoietic progenitor cell transplantation (HSCT/HPCT) is widely used and one of the most useful treatments in clinical practice. However, the homing rate of hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs) by routine cell transfusion is quite low, influencing hematopoietic reconstitution after HSCT/HPCT.MethodsThe authors developed a micro-living motor (MLM) strategy to increase the number of magnetically empowered bone marrow cells (ME-BMCs) homing to the bone marrow of recipient mice.ResultsIn the in vitro study, migration and retention of ME-BMCs were greatly improved in comparison with non-magnetized bone marrow cells, and the biological characteristics of ME-BMCs were well maintained. Differentially expressed gene analysis indicated that ME-BMCs might function through gene regulation. In the in vivo study, faster hematopoietic reconstitution was observed in ME-BMC mice, which demonstrated a better survival rate and milder symptoms of acute graft-versus-host disease after transplantation of allogeneic ME-BMCs.ConclusionsThis study demonstrated that ME-BMCs serving as MLMs facilitated the homing of HSCs/HPCs and eventually contributed to earlier hematopoietic reconstitution in recipients. These data might provide useful information for other kinds of cell therapies.     

Knockdown of HPRT for Selection of Genetically Modified Human Hematopoietic Progenitor Cells

The inability to obtain sufficient numbers of transduced cells remains a limitation in gene therapy. One strategy to address this limitation is in vivo pharmacologic selection of transduced cells. We have previously shown that knockdown of HPRT using lentiviral delivered shRNA facilitates efficient selection of transduced murine hematopoietic progenitor cells (HPC) using 6-thioguanine (6TG). Herein, we now extend these studies to human HPC. We tested multiple shRNA constructs in human derived cell lines and identified the optimal shRNA sequence for knockdown of HPRT and 6TG resistance. We then tested this vector in human umbilical cord blood derived HPC in vitro and in NOD/SCID recipients. Knockdown of HPRT effectively provided resistance to 6TG in vitro. 6TG treatment of mice resulted in increased percentages of transduced human CD45⁺ cells in the peripheral blood and in the spleen in particular, in both myeloid and lymphoid compartments. 6TG treatment of secondary recipients resulted in higher percentages of transduced human cells in the bone marrow, confirming selection from the progeny of long-term repopulating HPCs. However, the extent of selection of cells in the bone marrow at the doses of 6TG tested and the toxicity of higher doses, suggest that this strategy may be limited to selection of more committed progenitor cells. Together, these data suggest that human HPC can be programmed to be resistant to purine analogs, but that HPRT knockdown/6TG-based selection may not be robust enough for in vivo selection.     

Systematic investigation of oxygen and growth factors in clinically valid ex vivo expansion of cord blood CD34(+) hematopoietic progenitor cells

Background aimsCord blood is considered to be a superior source of hematopoietic stem and progenitor cells for transplantation, but clinical use is limited primarily because of the low numbers of cells harvested. Ex vivo expansion has the potential to provide a safe, effective means of increasing cell numbers. However, an absence of consensus regarding optimum expansion conditions prevents standard implementation. Many studies lack clinical applicability, or have failed to investigate the combinational effects of different parameters.MethodsThis is the first study to characterize systematically the effect of growth factor combinations across multiple oxygen levels on the ex vivo expansion of cord blood CD34⁺ hematopoietic cells utilizing clinically approvable reagents and methodologies throughout.ResultsOptimal fold expansion, as assessed both phenotypically and functionally, was greatest with thrombopoietin, stem cell factor, Flt-3 ligand and interleukin-6 at an oxygen level of 10%. With these conditions, serial expansion showed continual target population expansion and consistently higher expression levels of self-renewal associated genes.ConclusionsThis study has identified optimized fold expansion conditions, with the potential for direct clinical translation to increase transplantable cell dose and as a baseline methodology against which future factors can be tested.     

The effects of Arctigenin-Valine ester on chemotherapy-induced myelosuppression in mice

ObjectiveTo explore whether Arctigenin-Valine ester (ARG-V) can treat myelosuppression caused by chemotherapy.MethodsThe number of peripheral blood cells of the mice was measured by an automatic blood analyzer, and the hematopoietic progenitor colonies CFU-GM, CFU-E, BFU-E, and CFU-Meg were cultured in vitro. Hematopoietic progenitor colonies and BMNCs were counted under an inverted microscope. The expressions of cytokines GM-CSF, EPO and TPO were detected by ELISA. The cell cycle was measured by flow cytometry. The expressions of related proteins MEK and p-ERK were quantitated by western blots, and the thymus index and spleen index were quantitated.ResultsAfter taking ARG-V, the peripheral blood cells of the mice gradually returned to normal, the number of nucleated cells in the bone marrow increased, the thymus index increased, the spleen index decreased, the number of hematopoietic progenitor cells increased, and the hematopoietic cytokines decreased. And ARG-V promoted the transformation of myelosuppression cells from G0/G1 to S and from S to G2/M. ARG-V could up-regulate the expression of MEK and p-ERK, and low dose ARG-V is not as effective in all aspects as high dose ARG-V.ConclusionARG-V can effectively alleviate the myelosuppression that caused by intraperitoneal injection of CTX in 100mg/kg, and ARG-V can promote the proliferation and differentiation of hematopoietic progenitor cells and improve immunity, and the effect of high-dose Arctigenin-Valine ester is more significant to some extent.     

Proteinase-Activated Receptor 1 (PAR1) Regulates Leukemic Stem Cell Functions

External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients'' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34⁺ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1^−/− hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1^−/− leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.     

The dynamics of murine mammary stem/progenitor cells

The stem/progenitor cells in the murine mammary gland are a highly dynamic population of cells that are responsible for ductal elongation in puberty, homeostasis maintenance in adult, and lobulo-alveolar genesis during pregnancy. In recent years understanding the epithelial cell hierarchy within the mammary gland is becoming particularly important as these different stem/progenitor cells were perceived to be the cells of origin for various subtypes of breast cancer. Although significant advances have been made in enrichment and isolation of stem/progenitor cells by combinations of antibodies against cell surface proteins together with flow cytometry, and in identification of stem/progenitor cells with multi-lineage differentiation and self-renewal using mammary fat pad reconstitution assay and in vivo genetic labeling technique, a clear understanding of how these different stem/progenitors are orchestrated in the mammary gland is still lacking. Here we discuss the different in vivo and in vitro methods currently available for stem/progenitor identification, their associated caveats, and a possible new hierarchy model to reconcile various putative stem/progenitor cell populations identified by different research groups.     

Increased hematopoietic toxicity following administration of interferon-å with combination dideoxynucleoside therapy (zidovudine plus DDI) administered in normal mice

Because of the urgency to develop drugs which will effectively combat HIV infection, many combination therapies which have proved effective against HIV in vitro have undergone, or are undergoing clinical trial. Unfortunately many of drugs are being used without rigorous and exhaustive preclinical evaluation to assess their potential to develope hematopoeitic toxicity. We report here the results of two in vivo studies performed to analyze the effect of combined zidovudine (AZT) plus didanosine (ddl) therapy, either with or without interferon-å (IFN-å), on murine hematopoiesis. Normal C57BL/6 female mice were administered AZT (1.0 mg/ml) plus dose-escalation ddI (0.1, 1.0 and 2.5 mg/ml) placed in their drinking water. Control mice received IFN-å (100 units/ml) alone. Mice were serially bled and sacrified over a six-week period for assessment of hematopoietic toxicity measured by peripheral blood indices and assays of hematopoietic progenitors, i.e., erythroid (BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) cultured from bone marrow and spleen. AZT plus dose-escalation ddI decreased the hematocrit and white blood cell count when administered to normal mice compared to untreated controls during the six-week examination period. Marrow derived BFU-E, CFU-GM, and CFU-Meg were all reduced, however an increase was observed from the spleen for all three progenitor cell types. Use of IFN-å, in addition to combination AZT plus ddI further decreased the hematocrit, white blood cell and platelets. Marrow derived CFU-GM and CFU-Meg were increased slightly and only marginally for BFU-E with a similar response observed from the spleen. These results demonstrate that combination AZT plus ddI when used in vivo may produce synergistic hematopoietic toxicity, and that the addition of IFN-å to this treatment regimen increases this toxicity. These data indicate caution when this therapeutic approach is suggested for patients infected with HIV. If used, these patients wil require careful monitoring for blood cel toxicity.     

Mesenchymal stromal/stem cells markers in the human bone marrow

Background aimsMesenchymal stromal/stem cells (MSCs) can be isolated from human bone marrow (BM), expanded ex vivo and identified via numerous surface antigens. Despite the importance of these cells in regenerative therapy programs, it is unclear whether the cell membrane signature defining MSC preparations ex vivo is determined during culture or may reflect an in vivo counterpart. BM-MSC phenotype in vivo requires further investigation.MethodsTo characterize cells in their natural BM environment, we performed multi-parametric immunohistochemistry on trabecular bone biopsy specimens from multiple donors and described cells by different morphology and micro-anatomic localization in relationship to a precise pattern of MSC antigen expression.ResultsMicroscopically examined high-power field marrow sections revealed an overlapping in vivo expression of antigens characterizing ex vivo expanded BM-MSCs, including CD10, CD73, CD140b, CD146, GD2 and CD271. Expanding this panel to proteins associated with pluripotency, such as Oct4, Nanog and SSEA-4, we were able to identify different cellular populations in the human trabecular bone and BM expressing different progenitor cell markers.ConclusionsTargeting several multipotency and pluripotency markers, we found that the BM contains identifiable and distinct progenitor cells further justifying their introduction for a wide range of applications in regenerative medicine.     

Active RHOA favors retention of human hematopoietic stem/progenitor cells in their niche

Background

Hematopoietic stem/progenitor cells (HSPCs) maintain the hematopoietic system by balancing their self-renewal and differentiation events. Hematopoietic stem cells also migrate to various sites and interact with their specific microenvironment to maintain the integrity of the system. Rho GTPases have been found to control the migration of hematopoietic cells and other cell types. Although the role of RAC1, RAC2 and CDC42 has been studied, the role of RHOA in human hematopoietic stem cells is unclear.

Results

By utilizing constitutively active and dominant negative RHOA, we show that RHOA negatively regulates both in vitro and in vivo migration and dominant negative RHOA significantly increased the migration potential of human HSC/HPCs. Active RHOA expression favors the retention of hematopoietic stem/progenitor cells in the niche rather than migration and was found to lock the cells in the G0 cell cycle phase thereby affecting their long-term self-renewal potential.

Conclusion

The current study demonstrates that down-regulation of RHOA might be used to facilitate the migration and homing of hematopoietic stem cells without affecting their long-term repopulating ability. This might be of interest especially for increasing the homing of ex vivo expanded HSPC.     

Mitochondrial Reactive Oxygen Species Regulate Adipocyte Differentiation of Mesenchymal Stem Cells in Hematopoietic Stress Induced by Arabinosylcytosine

Objective

The increase in adipocytes induced by chemotherapeutic drugs may play a negative role in hematopoietic recovery. However, the mechanism underlying adipocyte differentiation of mesenchymal stem cells (MSCs) in hematopoietic stress is still unknown. Hence, the involvement of reactive oxygen species (ROS) in adipocyte differentiation under hematopoietic stress was investigated in vitro and in vivo.

Methods

The roles of cellular ROS in adipogenesis were investigated in vivo through an adipocyte hyperplasia marrow model under hematopoietic stress induced by arabinosylcytosine (Ara-C) and in vitro via adipocyte differentiation of human MSCs. ROS levels were detected using the CM-H₂DCFDA probe and Mito-SOX dye. Adipogenesis was evaluated by histopathology and oil red O staining, whereas detection of mRNA levels of antioxidant enzymes and adipogenesis markers was performed using quantitative real-time polymerase chain reaction analysis.

Results

ROS were found to play an important role in regulating adipocyte differentiation of MSCs by activating peroxisome proliferator-activated receptor gamma (PPARγ,) while the antioxidant N-acetyl-L-cysteine acts through ROS to inhibit adipocyte differentiation. The elevated ROS levels induced by Ara-C were caused by both over-generation of mitochondrial ROS and reduction of antioxidant enzymes (Cu/Zn Superoxide dismutase and catalase). Our findings suggest that a mitochondrial-targeted antioxidant could diminish adipocyte differentiation.     

Inhibition of histone deacetylases by Trichostatin A leads to a HoxB4-independent increase of hematopoietic progenitor/stem cell frequencies as a result of selective survival

BackgroundDNA and chromatin modifications are critical mediators in the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. One type of modification, the acetylation and deacetylation of histones, occurs reversibly on lysine ε-NH₃⁺ groups of core histones via histone acetyl transferases (HAT) and histone deacetylases (HDAC). Hyperacetylated histones are associated with active chromatin domains, whereas hypoacetylated histones are enriched in non-transcribed loci.MethodsWe analyzed global histone H4 acetylation and HDAC activity levels in mature lineage marker-positive (Lin⁺) and progenitor lineage marker-negative (Lin^?) hematopoietic cells from murine bone marrow (BM). In addition, we studied the effects of HDAC inhibition on hematopoietic progenitor/stem cell (HPSC) frequencies, cell survival, differentiation and HoxB4 dependence.ResultsWe observed that Lin^? and Lin⁺ cells do not differ in global histone H4 acetylation but in HDAC activity levels. Further, we saw that augmented histone acetylation achieved by transient Trichostatin A (TSA) treatment increased the frequency of cells with HPSC immunophenotype and function in the heterogeneous pool of BM cells. Induction of histone hyperacetylation in differentiated BM cells was detrimental, as evidenced by preferential death of mature BM cells upon HDAC inhibition. Finally, TSA treatment of BM cells from HoxB4^?/? mice revealed that the HDAC inhibitor-mediated increase in HPSC frequencies was independent of HoxB4.ConclusionsOverall, these data indicate the potential of chromatin modifications for the regulation of HPSC. Chromatin-modifying agents may provide potential strategies for ex vivo expansion of HPSC.     

Reconstitution of Mammary Epithelial Morphogenesis by Murine Embryonic Stem Cells Undergoing Hematopoietic Stem Cell Differentiation

Background

Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.

Methodology/Principal Findings

To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs) under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.

Conclusions/Significance

Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.     

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood^[1-2].We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.     

Identification of an Effective Early Signaling Signature during Neo-Vasculogenesis In Vivo by Ex Vivo Proteomic Profiling

Therapeutic neo-vasculogenesis in vivo can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the in vivo signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours in vivo, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation in vitro as well as modulating therapeutic vasculogenesis in vivo. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation in vitro and significantly decreased vasculogenesis in vivo. Proteomic profiling ex vivo thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis in vivo.     

Ex vivo immunological evaluation of stable mixed chimeric patients after matched related donor allogeneic transplantation in sickle cell disease

Background aimsAllogeneic hematopoietic stem cell transplantation is curative for sickle cell disease, and the use of matched related donors, non-myeloablative conditioning and sirolimus immunosuppression results in stable mixed chimerism without graft-versus-host disease (GVHD). However, the time to terminate sirolimus while maintaining mixed chimerism is unclear.MethodsIn this study, we developed a two-way mixed lymphocyte reaction (MLR) to evaluate ex vivo immunoreaction in mixed chimeric patients.ResultsIn co-culture of peripheral blood mononuclear cells (PBMCs) from two healthy controls (without irradiation), we detected proliferation at various ratios of PBMC mixtures (1:9 to 9:1) as well as various concentrations of sirolimus, suggesting that two-way MLR is applicable to patients (having >10% chimerism) undergoing sirolimus treatment. In two-way MLR using PBMCs (including donor and recipient cells) from mixed chimeric patients (n = 28), greater ex vivo proliferation was observed <6 months compared with >6 months post-transplant and healthy control PBMC monoculture. Robust ex vivo proliferation was observed in a patient with acute GVHD, and persistent ex vivo proliferation (until 2 years) was observed in a patient with decreasing donor chimerism.ConclusionsIn summary, we demonstrated that in two-way MLR, ex vivo immunoreaction decreases to low levels ~6 months post-transplant. These findings suggest a rationale to continue immunosuppression for 6 months.