Long-term Culture-initiating Cell Assays Research Articles

Primitive and committed human hematopoietic progenitor cells interact with primary murine neural cells and are induced to undergo self-renewing cell divisions

Studies in animal models have indicated that hematopoietic progenitor cells (HPC) migrate and home to the central nervous system and might acquire neural features under specific circumstances. The interaction between HPC and the neural environment and the functional effect on hematopoiesis have not yet been defined. CD34(+)133(+) cells from mobilized peripheral blood were cocultured with primary murine neurons or astrocytes. Chemotaxis and adhesive interactions were studied by applying beta(1)- and beta(2)-integrin function-blocking anibodies. The impact of neural feeder layers on integrin expression of HPC and the presence of appropriate adhesion ligands on neural cells were determined by immunostaining and flow cytometry. The hematopoietic long-term fate was monitored by time-lapse microscopy of individual cell-division history followed by long-term culture-initiating cell (LTC-IC) and colony-forming cell (CFC) assays. Neural differentiation was assessed by immunostaining against specific neuronal and glial antigens. The 23.0% +/- 4.9% of HPC showed stromal cell-derived factor-1-induced migration toward neural cells, and 20.2% +/- 1.6% displayed firm beta(1)-integrin-mediated adhesion to astrocytes. The latter expressed appropriate adhesion ligands, stabilized beta(1)-integrin expression, and increased beta(2)-integrin expression of HPC. Neural differentiation of HPC could not be identified but astrocytes were able to induce limited self-renewing cell divisions of HPC and thus maintain 25.8% +/- 3.4% of the initial LTC-IC and 80.7% +/- 1.9% of the initial CFC. Human HPC are able to interact with neural cells and interaction maintains, albeit to a limited extent, the self-renewal capability of HPC.

Stromal Derived Growth Factor-1α: Another Mediator in Neural-Emerging Immune System through Tac1 Expression in Bone Marrow Stromal Cells

Stromal cell-derived growth factor-1alpha (SDF-1alpha) is a member of the CXC chemokines and interacts with the G protein, seven-transmembrane CXCR4 receptor. SDF-1alpha acts as a chemoattractant for immune and hemopoietic cells. The Tac1 gene encodes peptides belonging to the tachykinin family with substance P being the predominant member. Both SDF-1alpha and Tac1 peptides are relevant hemopoietic regulators. This study investigated the effects of SDF-1alpha on Tac1 expression in the major hemopoietic supporting cells, the bone marrow stroma, and addresses the consequence to hemopoiesis. Reporter gene assays with the 5' flanking region of Tac1 showed a bell-shaped effect of SDF-1alpha on luciferase activity with 20 ng/ml SDF-1alpha acting as stimulator, whereas 50 and 100 ng/ml SDF-1alpha acted as inhibitors. Gel shift assays and transfection with wild-type and mutant IkappaB indicate NF-kappaB as a mediator in the repressive effects at 50 and 100 ng/ml SDF-1alpha. Northern analyses and ELISA showed correlations among reporter gene activities, mRNA (beta-preprotachykinin I), and protein levels for substance P. Of relevance is the novel finding by long-term culture-initiating cell assays that showed an indirect effect of SDF-1alpha on hemopoiesis through substance P production. The results also showed neurokinin 1 and not neurokinin 2 as the relevant receptor. Another crucial finding is that substance P does not regulate the production of SDF-1alpha in stroma. The studies indicate that SDF-1alpha levels above baseline production in bone marrow stroma induce the production of substance P to stimulate hemopoiesis. Substance P, however, does not act as autocrine stimulator to induce the production of SDF-1alpha. This study adds SDF-1alpha as a mediator within the neural-immune-hemopoietic axis.

Mesenchymal Stem Cells from the Wharton's Jelly of Umbilical Cord Segments Support the Maintenance of Long Term Culture-Initiating Cells from Cord Blood.

Hematopoietic Stem Cells (HSC) are routinely obtained from bone marrow, mobilized peripheral blood, and umbilical Cord Blood. Traditionally, adult bone marrow has been utilized as a source of Mesenchymal Stem Cells (MSC). Bone marrow derived MSC (BM-MSC) have previously been shown to maintain the growth of HSC obtained from cord blood and have been utilized for cord blood expansion purposes. However, the use of a mismatched BM-MSC feeder stromal layer to support the long term culture of cord blood HSC is not ideal for transplant purposes. The isolation of MSC from a novel source, the Wharton's Jelly of Umbilical Cord segments, was recently reported (Romanov Y, et al. Stem Cells. 2003; 21: 105–110) (Lee O, et al. Blood. 2004; 103: 1669–1675). We therefore hypothesized that Umbilical Cord derived MSC (UC-MSC) have the ability to support the long term growth of cord blood derived HSC similar to that previously reported for BM-MSC. To test this hypothesis, MSC were isolated from the Wharton's Jelly of Umbilical Cord segments and defined morphologically and by cell surface markers. UC-MSC were then tested for their ability to support the growth of pooled CD34+ cord blood cells in long term culture - initiating cell (LTC-IC) assays as compared to BM-MSC. We observed that like BM-MSC, CB-MSC express a defined set of cell surface markers. By flow cytometry we determined that that both UC-MSC and BM-MSC are positive for CD29, CD44, CD73, CD90, CD105, CD166, HLA-A and negative for CD45, CD34, CD38, CD117, HLA-DR expression. Utilizing Mitomycin C treated (200 μM, 15 min.) UC-MSC from multiple donors as a feeder layer we observed that UC-MSC have the ability to support the maintenance of long term hematopoiesis during the LTC-IC assay. Specifically, UC-MSC isolated from separate umbilical cord donors support the growth of 69.6±11.9 (1A), 31.7±3.9 (2B), 67.0±13.5 (3A), and 38.5±13.7 (3B) colony forming cells (CFC) per 1×104 CD34+ cord blood cells as compared to 64.0±4.2 CFC per 1×104 CD34+ cord blood cells supported by BM-MSC (Mean±SEM, N=4 separate segments from three different donors). Thus, Umbilical Cord derived Mesenchymal Stem Cells, a recently described novel source of MSC, have the ability to support long term maintenance of Hematopoietic Stem Cells, as defined by the LTC-IC assay. These results may have potential therapeutic application with respect to ex vivo stem cell expansion of Cord Blood Hematopoietic Stem Cells utilizing a Mesenchymal Stem Cell stromal layer. In addition, these data suggest the possibility of co-transplantation of matched Mesenchymal and Hematopoietic Stem Cells from the same umbilical cord and cord blood donor respectively. Lastly, these results describe a novel model system for the future study of the interaction between Cord Blood Hematopoietic Stem Cells and the appropriate supportive microenvironment represented by the Umbilical Cord - Mesenchymal Stem Cells.

Peripheral Blood, Cord Blood and Bone Marrow Cells Sorted into SP+, CD34+ and SP− Cells Are Distinct in Their ABCG2 and MDR1 Expression Levels, Which Are Most Increased in SP+ Cells and Substantially Decline after In Vitro Culture and Terminal Cell Differentiation.

Primitive hematopoietic stem cells (HSC) can be selected based on the rapid efflux of the DNA-binding dye Hoechst 33342 and are identified as side-population (SP+) cells. We and others have demonstrated that SP+ cells are negative for most lineage markers including CD34, are increased in long-term culture-initiating cell assays, reconstitute the bone marrow (BM) of lethally irradiated NOD/SCID mice and have been determined as early HSC (Engelhardt et al. Leukemia 2002; 16:1603, Guo et al. JHSCR 2003; 12:71). With further characterization of the SP phenotype, different members of the ABC-superfamily have been ascertained as an SP feature, of which ABCG2 and MDR1 are major efflux proteins. We analyzed the expression of ABCG2 and MDR1 (by RT- and Q-PCR) in an inducible system using different cell lines and in SP+ cells as compared to CD34+ and SP− cells from various cell sources (PB, CB and BM) before (d0) and after (d7) in vitro culture. The hypothesis was that 1. SP+ cells as compared to CD34+ and SP− cells show highest ABCG2 and MDR1 values before expansion and 2. expression levels substantially decrease with expansion and terminal cell differentiation. The tested cell lines CMK, AML 3.3 and AML 6.2 were strongly positive for ABCG2, less positive in HL60 and K562, and KG-1a was negative for both target genes. With use of PB, CB and BM SP+ cells before and after culture, ABCG2 expression was highest in SP+ cells, less in CD34+ and the least positive in SP− cells. This difference in ABCG2 was most prominent in CB cells, that showed a 7-fold higher expression in SP+ as compared to CD34+ cells, whereas this was similar for PB and BM SP+ and CD34+ cells. Of note, the differences in ABCG2 via matched pair analysis of SP+, CD34+ and SP− cells did not reach significance. After 7-days of expansion, ABCG2 expression was substantially reduced in all three hematopoietic cell subsets as compared to d0 values, reaching statistical significance with matched pair analysis for 34+ cells (p=0.001). Also for MDR1, the d0 expression was highest in SP+ as compared to CD34+ and SP− cells. Significant differences between SP+ and SP− cells and between CD34+ and SP− cells were observed (p<0.01), however not between SP+ and CD34+ values. MDR1 expression was also decreased on d7 as compared to d0 values, which reached significance for SP+ and CD34+ cells (p<0.05). Our results suggest that via ABCG2 and MDR1 expression, PB, CB and BM cells sorted into SP+, CD34+ and SP− cells show substantial differences in pump protein expression. Albeit ABCG2 and MDR1 expression was highest in SP+ as compared to CD34+ and SP− cells, which declined with cell differentiation in culture, the difference between SP+ and CD34+ cells was less distinct than expected. This suggests that ABCG2 and MDR1 serve as markers for primitive HSC, but also that 1. “contamination” of other cells within the SP+ cell population (e.g. early lymphocyte subsets) dilute the expression levels of ABCG2 and MDR1 in SP+ cells, and/or 2. transporter expression determination does not allowed to detect small differences in ontogeny and stem cells' primitiveness or 3. SP+ cells may not reflect - as much as initially postulated - very early HSC or are similar in its primitiveness as CD34+ cells.

Human Mesenchymal Stromal Cells Regulate Initial Self-Renewing Divisions of Hematopoietic Progenitor Cells by a β1-Integrin-Dependent Mechanism

In previous reports, we have demonstrated that only direct cell-cell contact with stromal cells, such as the murine stromal cell line AFT024, was able to alter the cell division kinetics and self-renewing capacity of hematopoietic progenitor cells (HPC). Because beta(1)-integrins were shown to be crucial for the interaction of HPC with the bone marrow microenvironment, we have studied the role of beta(1)-integrins in the regulation of self-renewing cell divisions. For this purpose, we used primary human mesenchymal stromal (MS) cells as in vitro surrogate niche and monitored the division history and subsequent functional fate of individually plated CD34(+)133(+) cells in the absence or presence of an anti-beta(1)-integrin blocking antibody by time-lapse microscopy and subsequent long-term culture-initiating cell (LTC-IC) assays. beta(1)-Integrin-mediated contact with MS cells significantly increased the proportion of asymmetrically dividing cells and led to a substantial increase of LTC-IC. Provided that beta(1)-integrin-mediated contact was available within the first 72 hours, human MS cells were able to recruit HPC into cell cycle and accelerate their division kinetics without loss of stem cell function. Activation of beta(1)-integrins by ligands alone (e.g., fibronectin and vascular cell adhesion molecule-1) was not sufficient to alter the cell division symmetry and promote self-renewal of HPC, thus indicating an indirect effect. These results have provided evidence that primary human MS cells are able to induce self-renewing divisions of HPC by a beta(1)-integrin-dependent mechanism.

Leukemia Targeting Via Cytotoxic T Cells Expressing a CD33 Specific Chimeric Receptor.

CD33 is a surface protein expressed on the malignant blast cells of most acute myeloid leukemia (AML) and is a potential tumor-associated target antigen. Immunotoxin-based therapy against CD33+ myeloid leukemia has shown promising results with no long-term side effects on CD33+ progenitor cells, but is associated with liver toxicity. We describe a cell therapy approach targeting CD33 for AML, which should be more effective than the immunotoxin and have lower non-specific toxicity. The genetic modification of human T cells to express tumor specific chimeric immune receptors (TCR) can produce large numbers of tumor-specific T cells. Unfortunately, these modified T cells have only a limited response to tumor cells in vivo. By contrast, EBV-specific cytotoxic T cells (EBV-CTLs) expand and have long-term activity in vivo. We exploited this phenomenon and redirected EBV-CTLs against CD33. EBV-CTLs were generated from six EBV-seropositive healthy donors and transduced using a retroviral vector encoding the chimeric CD33 specific TCR. The cRCD33-EBV-CTL maintained their pre-transduction immunophenotype and specifically lyzed EBV-LCL and CD33+ targets (specific lysis of 30% and 35% at E:T ratio 25:1). They also produced Th1 and Th2-type cytokines on exposure to CD33+ targets. Addition of the CD28 intracellular domain did not significantly increase cytotoxicity to CD33+ targets, but resulted in a 5–7 and 2–3 fold increase in IFN-γand IL-5 production. We confirmed the specificity of the TCR interactions with CD33 by inhibiting them with MHC class I, class II and CD33 blocking monoclonal antibodies (MoAb). Preincubation of CD33+ target cells with the CD33-blocking MoAb resulted in 40% inhibition of lysis and 60% inhibition of cytokine release by cRCD33-EBV-CTLs, whereas preincubation of CD33+ targets with class I and II blocking MoAb had no significant effect. Incubation of autologous LCLs with MHC Class I and Class II MoAb showed MHC class I was the major restriction element for the native antigen receptor of both nontransduced and modified EBV-CTLS. Of note, the cRCD33-EBV-CTLs proliferated only in response to autologous LCLs and not to CD33+ targets. Failure to proliferate was not overcome by incorporating the CD28 endodomain into the CAR. By contrast, cRCD33-EBV-CTLs can be expanded and maintain their specific activities toward CD33+ targets by co-culture with LCLs, and these effects are sustained for over 30 days in culture. Despite their activity against CD33+ blast cells, long-term culture-initiating cell assay demonstrated that neither cRCD33-EBV-CTLs nor cRCD33-CD28-EBV-CTLs had measurable toxicity against CD33+ progenitor cells isolated from bone marrow samples. To assess activity in vivo, we injected NOD-SCID mice bearing a human CD33+ AML tumor with EBV-CTLs x4 weekly, starting 5 days after tumor inoculation. Whereas the administration of PBS or untransduced EBV-CTLs had no effect on tumor progression, cRCD33-EBV-CTLs showed a trend to reduced tumor growth, the statistical significance of which is being confirmed on a larger series of animals. Incorporation of the CD28 endodomain had no additive effect on anti-tumor activity. Importantly, no adverse effects wee seen on normal CD33+ progenitor cells in vivo. We conclude that EBV-CTL expressing the CD33 chimeric receptor may be appropriate as a therapeutic modality for the treatment of myeloid leukemia.

Primitive hematopoietic cell populations reside in the spleen: Studies in the pig, baboon, and human

We previously observed high levels (>40%) of multilineage hematopoietic cell chimerism following spleen transplantation across full MHC barriers in immunosuppressed miniature swine. We therefore investigated the spleen as a source of hematopoietic progenitor cells (HPCs). Specific cell-surface markers were used to identify HPCs in the spleen and bone marrow (BM) of young adult (n = 15) and fetal (n = 9) miniature swine by flow cytometry. Hoechst dye-effluxing side population (SP) cells were analyzed in adult spleen, BM, and blood for their expression of c-kit. Functional HPC activity of varying repopulation potential in vitro was investigated by the ability of spleens and BM to give rise to colony-forming units (CFUs) and cobblestone area-forming cells (CAFCs) in long-term stromal cultures. Studies were also carried out on baboon and human spleens and BM. Spleen c-kit+ cells co-expressed more lymphoid markers, but equal myeloid markers, when compared with BM c-kit+ cells. BM and spleen both contained significant percentages of c-kit+ SP cells. Although the frequency of early-forming CFUs in the spleen was only 0.1 to 1.3% of that in the BM, the frequency of CAFCs developing after 8 weeks in culture was comparable to that of BM. Secondary CFUs in long-term culture-initiating cell assays confirmed the presence of long-term repopulating cells at comparable frequencies in spleen and BM. Similar findings were found with regard to baboon and human spleen cells. The adult spleen is a relatively rich source of very primitive HPCs, possibly hematopoietic stem cells (HSCs), and may be of therapeutic value.

Comparative analysis of proliferative potential and clonogenicity of MACS‐immunomagnetic isolated CD34+ and CD133+ blood stem cells derived from a single donor

A novel stem cell marker prominin-1 (CD133) has been shown to be expressed on a subpopulation of CD34(+) haematopoietic stem and progenitor cells. The aim of this study was to compare in parallel commercially available CD34(+) and CD133(+) isolation methods based on paramagnetic bead-coupled antibodies using clinical-grade samples of mobilized peripheral blood from 10 individual healthy donors under identical conditions. The CD133 negative fraction from the first selection was used for CD34(+) enrichment to obtain an additional CD34(+)/CD133(-) population. Although no significant difference in total cell expansion between cells isolated from the three procedures was observed in a 7-day cytokine-driven suspension culture, the long-term culture-initiating cell assay demonstrated that cells derived by CD34(+) isolation contain less primitive progenitors than those isolated based on CD133(+) selection. Interestingly, CD34(+)-enriched progenitors, especially the CD34(+)/CD133(-) fraction, contained a significantly higher proportion of erythroid colony-forming cells, whereas the highest content of myeloid colony-forming cells was concentrated in the CD133(+) selected cells. These subtle differences between CD34(+) and CD133(+) immunomagnetic selection will have to be explored for their potential clinical relevance.

Multilevel Regulation of IL‐6R by IL‐6–sIL‐6R Fusion Protein According to the Primitiveness of Peripheral Blood‐Derived CD133+Cells

Interleukin-6 (IL-6) and its soluble receptor (sIL-6R) are major factors for maintenance and expansion of hematopoietic stem cells (HSCs). Sensitivity of HSCs to IL-6 has been previously studied, in part by measuring the expression of IL-6R on the membrane (mIL-6R). Several studies have described the regulation of cell surface expression of IL-6R by several cytokines, but the role of glycoprotein 130 activation has not yet been investigated. In this study, CD133(+) cells were purified from adult peripheral blood and were precultured in the absence or presence of 5-fluorouracil (5-FU) for selection of quiescent HSCs. Cells were cultured with continuous or pulsed stimulations of an IL-6-sIL-6R fusion protein (hyperinterleukin-6 [HIL-6]) to 1) detect mIL-6R by flow cytometry, 2) assess mIL-6R and sIL-6R RNAs by reverse transcription-polymerase chain reaction, 3) measure sIL-6R in supernatants by enzyme-linked immunosorbent assay, 4) analyze cell-cycle status, and 5) perform long-term culture-initiating cell assays. The level of mIL-6R(-) cells was preserved by 5-FU incubation. HIL-6 increased steady-state mIL-6R RNA and expression rate on HSCs, independently of treatment with 5-FU. Enhanced production of sIL-6R was observed with short pulses of HIL-6 on CD133(+) 5-FU-pretreated cells. This overproduction of sIL-6R was abrogated by tumor necrosis factor-alpha protease inhibitor-1, an inhibitor of a disintegrin and metalloprotease proteases, suggesting the shedding of mIL-6R. This phenomenon was mediated through the phosphatidylinositol-3'-kinase pathway and was involved in the maintenance of primitive HSCs. In conclusion, expression and production of IL-6R are tightly regulated and stage specific. We assume that sIL-6R produced by shedding should be involved in autocrine and paracrine loops in the HSC microenvironment.

The transcription factor MEF/ELF4 regulates the quiescence of primitive hematopoietic cells

The transcriptional circuitry that regulates the quiescence of hematopoietic stem cells is largely unknown. We report that the transcription factor known as MEF (or ELF4), which is targeted by the t(X;21)(q26;q22) in acute myelogenous leukemia, regulates the proliferation of primitive hematopoietic progenitor cells at steady state, controlling their quiescence. Mef null HSCs display increased residence in G0 with reduced 5-bromodeoxyuridine incorporation in vivo and impaired cytokine-driven proliferation in vitro. Due to their increased HSC quiescence, Mef null mice are relatively resistant to the myelosuppressive effects of chemotherapy and radiation. Thus, MEF plays an important role in the decision of stem/primitive progenitor cells to divide or remain quiescent by regulating their entry to the cell cycle.

Idiopathic CD4+ Lymphocytopenia May Be due to Decreased Bone Marrow Clonogenic Capability

Background: Idiopathic CD4+ lymphocytopenia is defined by a stable decrease of CD4+ T cells in the absence of any known cause of immune deficiency. The mechanisms responsible for the immunological impairment are still unknown, but a regenerative failure of hematopoietic stem/progenitor cells has been hypothesized. Methods: We evaluated in the bone marrow (BM) of 5 patients with idiopathic CD4+ lymphocytopenia the phenotype of BM progenitor cells, their differentiation capacity with colony-forming cells and long-term culture-initiating cell assays, in parallel with the spontaneous IL-7 production in the patient sera. Results: Compared with controls, a regenerative failure of hematopoietic stem cells has been observed, both in ‘committed’ and in ‘uncommitted’ progenitor cells, despite high IL-7 serum levels. The percentage of phenotypically primitive CD34+CD38–DR+ cells (this includes the lymphoid precursor cells) was decreased, suggesting an involvement of the more primitive BM compartment in the de novo T cell generation. Conclusions: Despite the low number of patients, due to the low incidence of the disease, the decrease of primitive precursors sustains the possibility that diminished stem cell precursors might contribute to the development of CD4+ T cell depletion.

HIV Type 1 Protease Inhibitors Enhance Bone Marrow Progenitor Cell Activity in Normal Subjects and in HIV Type 1-Infected Patients

HIV-1 protease inhibitors (PIs) may improve hematopoietic functions owing to their direct effects on bone marrow (BM) progenitor cells. In this study we investigated this hypothesis evaluating the effect of adding ritonavir (RTV) and indinavir (IND) on hematopoietic colony formation assays by colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, on apoptosis, on cytokine production and stromal cells, in subjects with HIV-1 infection, and in seronegative controls. After PI addition, CFC and LTC-IC assays in HIV-1-infected patients showed levels of colony growth significantly higher than those observed at baseline; the same PI activity on colony formation was observed in healthy subjects. No significant modifications on Fas, the membrane form of Fas (mFas) and Fas-ligand (FasL) expression, and on cytokine production were observed at BM level after the addition of PIs. At baseline, in HIV-1-infected patients, the majority of the stromal cells appeared as large and rounded, whereas after the addition of RTV or IND the stromal cells exhibited a "fibroblast-like" morphology and produced higher stem cell factor (SCF) and lower MIP-1alpha levels when compared with the stromal production without the addition of IND. RTV and IND increased colony growth of BM obtained either from HIV-1-infected patients or from normal individuals, in parallel with the normalization of functional and morphological characteristics of stromal cells.

Forced Overexpression of Ahi-1 Confers a Proliferative Advantage on Primitive Murine Hematopoietic Cells and Enhances the Effects of BCR-ABL.

Ahi-1 (Abelson helper integration site-1) is a novel gene that is commonly activated by proviral insertional mutagenesis in v-abl or myc-induced murine leukemias and lymphomas. Ahi-1 encodes a unique protein with known signaling features including SH3 and WD40-repeat domains but its normal function is unknown. Involvement of Ahi-1 in leukemogenesis is suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias and by the gross perturbations seen in the expression of human AHI-1 and its isoforms in several human leukemic cell lines, as well as in the primary lin−CD34+CD38− leukemic stem cell-enriched population in patients with chronic phase CML. To further investigate the role of Ahi-1 as a potential co-operating oncogene relevant to BCR-ABL-mediated leukemogenesis, we compared the biological behavior of primitive murine hematopoietic cells from the bone marrow of 5-FU-treated adult C57BL/6 mice after their transduction with MSCV-Ahi-1-IRES-YFP, MSCV-BCR-ABL-IRES-GFP retroviruses, either alone or in combination. Quantitative real-time RT-PCR analysis of RNA from FACS-purified lin−YFP+ (Ahi-1+), lin−GFP+ (BCR-ABL+) and lin−YFP+GFP+ (Ahi-1+ + BCR-ABL+)-transduced bone marrow cells showed that Ahi-1 transcripts were present at 40-fold higher levels in the Ahi-1-transduced cells by comparison to the control cells transduced with the empty MIY vector. Immediately post-transduction, the Ahi-1-transduced cells produced a similar number of colonies as the MIY-transduced control cells in semi-solid cultures containing Steel factor (SF) + IL-3 + IL-6 + EPO, although a small proportion of the Ahi-1+ CFCs (~10%) were already growth factor-independent. In addition, the proliferative activity of the FACS-purified lin−YFP+ (Ahi-1+) cells (as indicated by the rate of expansion of viable cells in a week in liquid cultures containing SF, IL-3, and IL-6) was ~3-fold higher than in cultures initiated with control (lin−YFP+) cells. Moreover, after 4 weeks in longterm culture-initiating cell (LTC-IC) assays, the Ahi-1+ cells produced 2x more CFCs than the control cells. All of these endpoints (proliferative activity, growth factor-dependence and CFC output in LTC-IC assays) are also perturbed by BCR-ABL transduction. Interestingly, in cells that were co-transduced with Ahi-1 and BCR-ABL, all of these effects were further enhanced as compared to cells transduced with either BCR-ABL alone (2–4-fold) or Ahi-1 alone (3–6-fold). Thus, overexpression of Ahi-1 alone deregulates the proliferation control of primitive murine hematopoietic cells and this is additive with the effects of BCR-ABL, suggesting that Ahi-1 and BCR-ABL can cooperate to promote the progression of BCR-ABL-associated diseases like CML.

Imatinib Mesylate (STI571) Considerably Affects Normal Human Bone Marrow Stromal Cell Growth in Vitro.

Imatinib mesylate (STI571) is a selective Bcr-Abl tyrosine kinase inhibitor used for the treatment of Philapdelphia chromosome positive leukemias and other malignancies. Typical non-hematological side effects of imatinib are mostly moderate whereas hematological side effects include all three lineages and are dose-dependent (e.g. 35% grade 3/4 neutropenia at 800 mg daily). Recently, a significant dose-dependent inhibitory effect of imatinib on normal hematopoietic progenitor cells has been reported. However, since alterations of the hematopoietic system might also result (at least in part) from a possible drug-induced damage of the hematopoietic environment, we studied the effects of imatinib on normal human marrow microenvironmental cells. Marrow stromal progenitor cell growth was considerably inhibited by imatinib in a dose-dependent fashion: CFU-F were reduced to 75 ± 35.5%, 52 ± 29.2%, 32 ± 25.7%, 33.7 ± 23.4%, and 20.6 ± 20.6% of control at 0.04, 0.16, 0.62, 2.5, and 10.0 μM imatinib, respectively (IC50: 0.44 μM). Marrow stromal cells (MSC, culture-derived from normal bone marrow mononuclear cells) were also affected dose-dependently: After two weeks in culture, MSC were reduced to 20.0 ± 2.2% (0.62 μM), 14.6 ± 1.2% (1.25 μM), 10.1 ± 0.3% (2.5 μM), 5.8 ± 0.5% (5 μM), and 2.8 ± 0.3% (10 μM) of controls. After 4 weeks, corresponding data were 3.1 ± 0.5%, 2.2 ± 0.4%, 1.8 ± 0.3%, 1.1 ± 0.2%, and 0.5 ± 0.1%. Furthermore, following a 7-day culture period in imatinib-containing medium (0.62 – 10 μM) and subsequent washing and resuspension, the growth kinetics of imatinib-treated MSC were delayed for up to 2 weeks after imatinib withdrawal. Moreover, the total cellular expansion of imatinib-pretreated MSC after 4 weeks of culture was lower when compared with controls. To assess whether imatinib would impair functional MSC capacities, long-term culture initiating cell assays were set up using imatinib-treated MSC (0 μM, 1.25 μM, 5 μM) as feeder cells and normal CD34pos-PBPC for inoculation. After 5 weeks, numbers of clonogenic progenitors assayed per well were not different (control: 20.0 ± 7.1, imatinib 1.25 μM: 16.2 ± 6.5, imatinib 5 μM: 21.3 ± 6). Experiments addressing a possible role of PDGF- and SCF-receptor signaling revealed that the growth inhibitory effects of imatinib (10 μM) clearly exceeded those of receptor blocking antibodies, thereby indicating that imatinib acts - at least in part - independently of PDGFR- and c-kit-signaling. Cell cycle analysis showed that the fraction of imatinib-treated MSC (0.62 - 10 μM) in S- and G2/M-phase was slightly lower albeit not significantly different when compared with controls. Furthermore, no differences with regard to the fraction of apoptotic cells were observed. Nevertheless, MSC cultures in maximum growth phase showed a markedly higher susceptibility to imatinib compared to those in the steady-state. Taken together, imatinib severely affected marrow stromal cells in-vitro. This effect was at least partly independent of PDGFR- and c-kit signaling and appeared to be related to the proliferative status of the cells. This information might be relevant for the administration of imatinib in situations with increased MSC turnover, such as regeneration after intensive chemo/radiotherapy.

A theoretical simulation of hematopoietic stem cells during oxygen fluctuations: prediction of bone marrow responses during hemorrhagic shock.

The bone marrow (BM) responds to various diseases, including infections and hemorrhagic shock, by generating immune and blood cells. These cells are derived from a finite number of lymphohematopoietic stem cells (LHSC) close to the endosteal region of the BM. This study presumes that studies on LHSC involving proteomics, computational biology, and genomics could be aided by mathematical models. A theoretical model is developed to predict the responses of proliferating (P) nonproliferating (N) BM cells during acute blood loss when the Po2 in the BM is decreased. Hematopoietic responses were simulated for otherwise healthy individuals who have been subjected to various degrees of blood loss, as represented by 3%, 5%, and 20% O2. The model is robust and could predict hematopoietic activity in the area close to the endosteum during low Po2 as for acute blood loss. Steady-state hematopoiesis at oxygen saturation (80%) in healthy individuals could not be simulated with the equations. Functional assays tested the model with an in vitro assay of the most primitive LHSC (modified long-term culture-initiating cell assay, LTC-IC). The LTC-IC assay showed that 1%, 3% - 5%, and 20% O2 mediate significant increases in the proliferation of the most primitive BM progenitors, as compared with 80% O2. Thus, the functional studies show that the theoretical model is robust and could be used to gain insights into the biology of LHSC during different degrees of blood loss. The utility of such a model in surgical trauma is discussed.

A systematic strategy to optimize ex vivo expansion medium for human hematopoietic stem cells derived from umbilical cord blood mononuclear cells

Objective In this study, a serum-free, stroma-free, and chemically defined medium for hematopoietic stem cell (HSC) expansion was systematically developed and optimized using factorial design and the steepest ascent method. Materials and methods Mononuclear cells (MNCs) were isolated from umbilical cord blood (UCB). HSCs were stimulated to proliferate ex vivo in the MNC culture system with variable serum substitutes, cytokines, and basal media according to experimental design. The expanded cells were assessed for cellular characteristics by surface antigen analysis, colony-forming cell assay (CFC assay), and long-term culture-initiating cell assay (LTC-IC assay). Results The optimal compositions of serum substitutes and the cytokine cocktail for HSC expansion in the MNC culture system were BIT (4 g/L BSA, 0.71 μg/mL insulin, and 27.81 μg/mL transferrin), and CC-9 (5.53 ng/mL TPO, 2.03 ng/mL IL-3, 16 ng/mL SCF, 4.43 ng/mL FL, 2.36 ng/mL IL-6, 1.91 ng/mL G-CSF, 1.56 ng/mL GM-CSF, 2.64 ng/mL SCGF, and 0.69 ng/mL IL-11) in the Iscove's modified Dulbecco's medium. After 6-day culture, the absolute fold expansions for white blood cells, CD34 + cells, CD34 +CD38 − cells, CFC, and LTC-IC were 1.4-, 30.4-, 63.9-, 10.7-, 2.8-fold, respectively. Conclusion Using the statistic methodology to develop HSC medium, our formula had lower cytokine concentrations comparing to other literatures and commercial media, but had superior or comparable expansion ability on HSC growth.

Quiescent (5-fluorouracil-resistant) aplastic anemia hematopoietic cells in vitro

Objective Bone marrow from aplastic anemia (AA) patients shows reduced numbers in long-term culture (LTC)-initiating cell (LTC-IC) assays. The LTC-IC assay is based on assumptions of the culture kinetics of normal hematopoietic stem cells (HSC), which are not necessarily justified in a disease state. We therefore undertook a detailed examination of the kinetics of quiescent HSC from AA patients in LTC. Methods Colony formation by quiescent HSC in LTC was tested by pretreating control (n = 6) and AA bone marrow (n = 7) with 5-fluorouracil. Secondly, we manipulated normal samples to inoculate cultures with proportions of CD34 + cells similar to those from AA samples. We obtained enough CD34 + cells to reconstitute one AA sample to “normal” levels. Results Patient cells showed altered kinetics with rapid proliferation and premature termination of LTC. In vivo, decreased numbers of HSC may induce rapid proliferation and differentiation; a similar phenomenon could explain the observations in culture. We therefore manipulated normal samples to contain a proportion of CD34 + HSC similar to that in AA samples. Although absolute numbers of secondary colonies in LTC were reduced, the kinetics of culture were not altered. However, when AA CD34 + HSC were reconstituted to “normal” levels, the cultures still demonstrated early termination. Conclusions The kinetics of LTC are not affected by CD34 + HSC number. However, quiescent HSC derived from patients with AA have qualitative differences from normal cells, as reflected by distinct kinetics in long-term culture. This has implications for the interpretation of the LTC-IC assay with AA samples.

Facilitating Role of Preprotachykinin-I Gene in the Integration of Breast Cancer Cells within the Stromal Compartment of the Bone Marrow

Despite early detection of breast cancer, patients' survival may be compromised if the breast cancer cells (BCCs) enter the bone marrow (BM). It is highly probable that BCCs enter the BM long before clinical detection. An in vitro coculture model with BM stroma and BCCs (cell lines; primary cells from stage III BC, n = 7, and stage M0, n = 3) mimicked early entry of BCCs into the BM. In coculture, BCCs exhibit contact inhibition and do not require otherwise needed growth supplements. Stromal growth rate was increased 2-fold in coculture. The inclusion of BCCs in stromal support of long-term culture-initiating cell assay frequencies show no difference (38 +/- 3 versus 36 +/- 6). Nontumorigenic breast cells (patients and cell lines) did not survive in coculture, suggesting that the model could select for malignant population in surgical breast tissues. Cocultures were able to select cells with 73 +/- 7% cloning efficiencies and with the ability to form cocultures with BM stroma. Preprotachykinin-I (PPT-I), a gene that is conserved by evolution, facilitates BCC integration as part of the stromal compartment. This was deduced as follows: (a) nontumorigenic breast cells (n = 4) genetically engineered to express PPT-I and led to anchorage-independent growth, foci formation, and formation of cocultures; and (b) suppression of PPT-I in BCCs (n = 5) with pPMSKH1-PPT-I small interfering RNA reverted the cells to nontumorigenic phenotypes and was undetectable in the BM nude mice. The evidence supports that the PPT-I gene facilitates the integration of BCCs in the stromal compartment during a period before clinical detection, without disrupting hematopoietic activity.

Stroma from different developmental stages of fetal liver has different effects on human mononuclear fraction of umbilical cord blood during ex vivo co-culture

Fetal liver (FL) has transient and intense hematopoietic activity during gestation. It has been shown that hematopoietic activity in FL begins at embryonic day (E)11, is maximal at E16 and rapidly ceases thereafter. This development of FL is simultaneous with transition of several hematopoietic features. Stromal cells (SCs) appear to regulate hematopoiesis in FL. To compare in vitro hematopoietic support ability of FL SCs from different developmental stages, we established three mouse primary FL SCs from different gestation days and compared their capacity in supporting the growth and differentiation of human umbilical cord blood, by flow cytometry and long-term culture-initiating cell (LTC-IC) assays. Results showed that expansion of hematopoietic progenitor in an E15.5 FL SC microenvironment was superior to that in E12.5 and E18.5 FL SC microenvironments. Co-culture on E15.5 FL SCs resulted in a 3.04-fold increase in early CD34+/CD38− progenitors and a 6.4-fold increase in LTC-ICs after 2 weeks. However, the comparison of CD2+/CD19− cell output and CD19+ cell output in different conditions did not attain statistical significance. These results show that murine E15.5 FL SCs are most effective in sustaining stem cell function along with greatest expansion of total nuclear cells of human umbilical cord blood (hUCB). We conclude that the change in SC function is responsible for the transition of hematopoietic function in FL.

CD34 +CDw90(Thy-1) + subset colocated with mesenchymal progenitors in human normal bone marrow hematon units is enriched in colony-forming unit megakaryocytes and long-term culture-initiating cells

Objective The progress made in the supportive care of allografts and the identification of mesenchymal stem cells in adult human bone marrow (BM) has prompted renewed interest in the use of BM as a form of cell therapy. With the aim of optimizing the collection of BM cells, we evaluated the hematopoietic and mesenchymal immature cell contents of BM hematon units (HUs), which usually are eliminated during graft processing. Materials and methods Hematopoietic CD34 + progenitors from HU and buffy coat (BC) compartments were characterized in short-term culture. The sorted CD34 +CDw90(Thy-1) + primitive subset was assessed in colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, then further characterized by the expression of additional antigens. In parallel, we evaluated the colony-forming unit fibroblast (CFU-F) number and phenotyped the fresh adherent (D1-3) cells. Results The plating efficiencies of CD34 + cells derived from HU and BC were identical. However, the HU CD34 +CDw90(Thy-1) + subset was enriched in colony-forming unit megakaryocyte (2.3×), LTC-IC (4.6×), and cells coexpressing CD105 (5×). We found a higher frequency of CFU-F (4.7×), considered to be the mesenchymal stem cell-containing population, correlated with an enrichment in fresh adherent (CD45/GPA) −CD14 − cells. Conclusions We show for the first time that functional properties of the CD34 +CDw90 + subset are related to its in vivo location in HU, which may represent the BM mesenchymal reserve compartment. The location in HU of 35.6%, 59.1%, and 58.7% of CD34 + cells, CD34 +CDw90 + LTC-IC, and CFU-F, respectively, justifies the development of a procedure to collect them in order to reduce the therapeutic BM volume.