Non-contact Culture Research Articles

Attenuation of Surface CXCR4 Down-Regulation in Human Cord Blood HSC during Ex Vivo Expansion Using the HUBEC Co-Culture System.

Down-modulation of surface CXCR4, a G-protein-coupled receptor, in hematopoietic stem cells (HSCs) undergoing ex vivo expansion culturing is considered to be one of the major causes of marrow reconstitution failure, possibly due to an HSC homing defect. Recently, it has been reported that severe combined immunodeficiency (SCID)-repopulating cells (SRC) were expanded from the CD34-enriched human adult bone marrow (ABM) or cord blood (CB) hematopoietic stem cells (HSC) using a human brain endothelial cell (HUBEC) co-culture system. We found that primitive cord blood cells expressing surface CXCR4 (82+5%) lost this capability significantly during 7 days of ex vivo expansion in the HUBEC co-culture containing the cytokines stem cell factor (SCF), flt-3, interleukin (IL)-6, IL-3, and granulocyte macrophage colony stimulating factor (GM-CSF). Expression levels of other surface proteins relevant to HSC homing, such as CD49d, CD95, CD26, or CD11a, were not down-modulated. We hypothesized that CXCR4 down-regulation was caused by a receptor internalization and tested several methods to reverse CXCR4 internalization back to the surface, such as elimination of GM-CSF in the culture media, performing a non-contact culture using the transwell, or adding either 0.3Mor 0.4M sucrose, or 25μg/ml chlorpromazine (CPZ), 24 hours prior to the analysis. CPZ and sucrose are known inhibitors of the cytokine-induced endocytosis of CXCR4 in neutrophils (Bruhl H. et al. Eur J Immunol 2003). Interestingly, 0.4M sucrose showed approximately a 2-fold increase of surface CXCR4 expression on CB CD34+ cells by flow cytometry analysis. CPZ and 0.3M sucrose showed a moderate increase expression of CXCR4. Using a transwell HUBEC co-culture system, CXCR4 surface expression on CD34+ cells was down-regulated during the ex vivo culture. In vitro HSC migration test showed 3.1-fold increase in migration compared to the control after incubation of HSC with 0.1M sucrose for 16 hours prior to the in vitro migration study. Eliminating GM-CSF from the cytokine cocktail or adding MG132 increased migration 1.36- and 1.2-fold compared to the control. We are currently performing an in vivo homing assay using nonobese diabetic (NOD)-SCID mice. In conclusion, the HUBEC ex vivo culture system down-regulates surface CXCR4 in human cord blood HSC. The mechanism of CXCR4 surface down regulation may be receptor internalization by cytokines. Sucrose may be useful in attenuation of CXCR4 surface expression in CD34+ HSC by inhibition of receptor internalization via clathrin-coated pits.

A paradoxical role for IFN-γ in the immune properties of mesenchymal stem cells during viral challenge

The functional "plasticity" and immune-suppressive effects of human bone marrow (BM)-derived mesenchymal stem cells (MSC) provide them with the potential to be used across allogeneic barriers. The immunosuppressive properties of MSC may be detrimental in a clinical setting in which viral exposure is common. The study hypothesizes that MSC-derived IFN-gamma could offset the immune-suppressive functions of MSC and mediate partial CTL responses during viral infection. CTL responses were studied in bioassays with (51)Cr-P815 targets and PBMC (uninfected or infected) as effectors. Immunofluorescence studied the relative expression of CD8(+) cells. Cytokine analyses were performed with microarrays. Roles for IFN-gamma in CTL responses were studied with IFNgammaRI mAb or with MSC knockdown for IFN-gamma by siRNA (pPMSKH1-IFNgamma). MSC showed no significant effect on circulating CTL of healthy subjects. For virus-induced CTL, MSC demonstrated approximately 50% suppression. CD8(+) cell expansion could not explain the suppressive effects of MSC. Soluble factors produced by MSC were responsible for the retention of 50% CTL responses. Cytokine microarray analyses, noncontact cultures, and functional assays identified a role for IFN-gamma. MSC were identified as the relevant source of IFN-gamma. The results show a facilitating role of IFN-gamma on CTL responses, although paradoxical in light of the veto properties of MSC. This report shows that in cases where MSC are used in transplantation for repair of damaged tissue, they can exert an additional role by protecting the host to viral challenges and thereby protect from its immunosuppressive properties.

Long‐Term Maintenance of Hematopoietic Stem Cells Does Not Require Contact with Embryo‐Derived Stromal Cells in Cocultures

We recently established that two midgestation-derived stromal clones--UG26-1B6, urogenital ridge-derived, and EL08-1D2, embryonic liver-derived--support the maintenance of murine adult bone marrow and human cord blood hematopoietic repopulating stem cells (HSCs). In this study, we investigate whether direct HSC-stroma contact is required for this stem cell maintenance. Adult bone marrow ckit+ Ly-6C- side population (K6-SP) cells and stromal cells were cocultured under contact or noncontact conditions. These experiments showed that HSCs were maintained for at least 4 weeks in culture and that direct contact between HSCs and stromal cells was not required. To find out which factors might be involved in HSC maintenance, we compared the gene expression profile of EL08-1D2 and UG26-1B6 with four HSC-nonsupportive clones. We found that EL08-1D2 and UG26-1B6 both expressed 21 genes at a higher level, including the putative secreted factors fibroblast growth factor-7, insulin-like growth factor-binding proteins 3 and 4, pleiotrophin, pentaxin-related, and thrombospondin 2, whereas 11 genes, including GPX-3 and HSP27, were expressed at a lower level. In summary, we show for the first time long-term maintenance of adult bone marrow HSCs in stroma noncontact cultures and identify some secreted molecules that may be involved in this support.

Soluble factors elaborated by human brain endothelial cells induce the concomitant expansion of purified human BM CD34+CD38– cells and SCID-repopulating cells

AbstractThe CD34+CD38– phenotype identifies a population in the bone marrow that is enriched in the steady state for hematopoietic stem cells (HSCs). Following ex vivo culture of CD34+ cells, HSC content is difficult to measure since committed CD34+CD38+ progenitors down-regulate CD38 surface expression during culture. In this study, we sought to define the phenotype of human HSCs following ex vivo culture under conditions that support the expansion of human cells capable of repopulating non-obese diabetic/severe combined immunodeficiency (SCID)–repopulating cells (SRCs). Contact coculture of fluorescence-activated cell sorter (FACS)–sorted bone marrow (BM) CD34+CD38– cells with human brain endothelial cells (HUBECs) supported a 4.4-fold increase in CD34+CD38– cells with a concordant 3.6-fold increase in SRCs over 7 days. Noncontact HUBEC cultures and the addition of thrombopoietin, stem cell factor (SCF), and macrophage colony stimulating factor I receptor (Fms)–like tyrosine kinase 3 (Flt-3) ligand supported further increases in CD34+CD38– cells (6.4-fold and 13.1-fold), which correlated with significant increases in SRC activity. Moreover, cell-sorting studies performed on HUBEC-cultured populations demonstrated that SRCs were significantly enriched within the CD34+CD38– subset compared with the CD34–CD38– population after culture. These results indicate that human HSCs can be identified and characterized by phenotype following expansion culture. These studies also demonstrate that HUBEC-elaborated soluble factors mediate a unique and potent expansion of human HSCs.

Endothelial Cells Mediate the Repair of Human Bone Marrow Stem and Progenitor Cells Following Radiation Injury.

The purposeful misuse of ionizing radiation has been recognized as a major bioterrorism threat in the United States. Although the myeloablative effects of ionizing radiation exposure have been well established, definitive treatments for potential victims of radiation injury are lacking. We have recently demonstrated that BM stem cells can be harvested from mice following exposure to lethal dose total body irradiation and can subsequently recover multilineage repopulating potential via ex vivo culture with microvascular endothelial cells (EC). However, it remains to be determined whether human hematopoietic stem cells (HSC) can be recovered in a similar manner following high dose radiation injury. In this study, we examined the capacity for ex vivo culture with and without primary human endothelial cells to support the cellular repair and recovery of primary human BM CD34+ cells following exposure to high dose irradiation. Exposure of primary BM CD34+ cells to 400 cGy resulted in a 40% decline in total viable cells and an 89% decline in CD34+CD38− cells compared to input despite 10 day culture with optimal concentrations of thrombopoietin, stem cell factor, and flt-3 ligand alone. Conversely, co-culture of 400 cGy irradiated BM CD34+ cells with primary human brain endothelial cells (LS-01) resulted in a 6-fold and 22-fold increase in total cells and CD34+CD38− cells compared to input, respectively. Non-contact cultures with LS-01 resulted in comparable recovery of total cells and the CD34+CD38− subset by 10 days. Concordantly, TSF-cultured progeny contained 5-fold greater numbers of early apoptotic and necrotic cells within the total and CD34+CD38− fractions as compared to the progeny of contact and non-contact endothelial cultures. Colony forming cell (CFC) assays demonstrated that 400 cGy exposure caused an 8-fold reduction in CFU-total compared to normal BM CD34+ cells and no recovery of CFU-GM, BFU-E, or CFU-Mix was observed following TSF culture. Co-culture with LS-01 resulted in the recovery of 50% of CFU-total compared to normal BM CD34+ cells. In order to assess HSC content post-irradiation, NOD/SCID mice were transplanted with normal BM CD34+ cells, 400 cGy irradiated BM CD34+ cells, and the progeny of 400 cGy irradiated cells following ex vivo culture. 100% of mice transplanted with normal BM CD34+ cells (7.5 x 105) demonstrated human hematopoietic engraftment at 6 weeks, whereas 0% of mice transplanted with 400 cGy irradiated BM CD34+ cells showed detectable human repopulation. Twenty-five percent of mice transplanted with 400 cGy irradiated/LS-01 cultured cells demonstrated human repopulation, whereas 0% of mice transplanted with 400 cGy irradiated/TSF-cultured cells showed human engraftment. These data demonstrate that ionizing radiation has a profoundly toxic effect on both human HSC and committed progenitor cells. Endothelial cells and endothelial cell-derived soluble factors appear to provide anti-apoptotic signals to BM progenitor cells following radiation damage, allowing cellular repair of committed progenitors as well as cells with in vivo repopulating capacity. Cytokine combinations alone, such as TSF, appear ineffective toward rescuing human BM stem/progenitor cells from radiation damage. Studies are ongoing to optimize the application of endothelial cells and endothelial cell-derived growth factors to stimulate HSC repair following radiation injury.

Multipotent neural precursors express neural and hematopoietic factors, and enhance ex vivo expansion of cord blood CD34+ cells, colony forming units and NOD/SCID-repopulating cells in contact and noncontact cultures

In view of the possible crosstalks between hematopoiesis and neuropoiesis, we evaluated two microenvironments, murine neonatal neural cell line C17.2 and primary embryonic aorta-gonad-mesonephros (AGM) stromal cells, on the ex vivo expansion of CD34+ cells from human cord blood. In a contact culture system, C17.2 or AGM cells significantly enhanced the expansion of CD34+ cells to a panel of early and committed hematopoietic progenitor cells. In a noncontact transwell system, pre-established C17.2 cells significantly increased the expansion of total nucleated cells, CD34+ cells and multilineage colony forming cells (P<0.01). Expanded cells were infused into nonobese diabetic/severe-combined immunodeficient mice. The engraftment of human (hu)CD45+ cells in the bone marrow of these mice was consistently higher in all the 10 experiments conducted with the support of C17.2 cells when compared with those in respective control groups (11.9 vs 2.43%, P=0.03). Using RT-PCR and Southern blot analysis, we showed that AGM and C17.2 cells expressed a panel of hematopoietic, bone morphogenetic and neurotrophic factors. Our data provided the first evidence on the promoting effects of a neural progenitor cell line on hematopoiesis at a noncontact condition. The mechanism could be mediated by the expression of multilineage regulatory factors.

Quantitative analysis demonstrates expansion of SCID-repopulating cells and increased engraftment capacity in human cord blood following ex vivo culture with human brain endothelial cells.

Initial clinical trials examining the transplantation of ex vivo expanded cord blood (CB) cells have failed to demonstrate an impact on hematopoietic recovery compared with historical unmanipulated CB controls. In this study, we tested whether coculture with primary human brain endothelial cells (HUBECs) could increase the engraftment capacity and repopulating cell frequency within CB CD34+ cells. Quantitative analysis demonstrated that HUBEC coculture for 7 days supported a 19-fold greater number of CD34+ cells and 3.4-fold and 2.6-fold greater severe combined immunodeficient (SCID)-repopulating cell (SRC) frequencies than fresh CB CD34+ cells and liquid suspension-cultured cells. Mice transplanted with day-14 HUBEC-cultured cells showed 4.2-fold higher levels of human engraftment than mice transplanted with day-7 HUBEC-cultured cells, indicating that SRC enrichment continued to occur through day 14. Noncontact HUBEC cultures also maintained SRCs at levels comparable with contact HUBEC cultures, demonstrating that HUBEC-secreted soluble factors critically supported SRC self-renewal. Seeding efficiency studies demonstrated that HUBEC-cultured CB CD34+ cells engrafted nonobese diabetic/SCID marrow at significantly higher levels than either fresh CB CD34+ cells or liquid suspension-cultured CD34+ cells. These studies indicate that the application of HUBEC coculture or HUBEC-conditioned media can potentially improve upon current strategies for the clinical expansion of CB stem cells.

Territorial Behavior in Residence Halls

This research enhances the understanding of cultural differences in the degree of territoriality in residence halls and how territorial behavior differs based on gender, culture, and knowing one's roommate previously. The present study was conducted in two distinct cultures: American, representing a noncontact culture, and Turkish, representing a contact culture. The sample was representative of male and female freshmen living in double-occupancy rooms at single-sex residence halls. Factor analysis yielded two dimensions of territorial experience: firmness of boundaries and personalization and claiming. In both cultures, men tended to exhibit more nonsharing behavior and less personalization of space than women. Regardless of culture and gender, students who knew their roommates tended to share their personal belongings and other features of the room to a greater extent than students who did not know their roommates previously. American students experienced their rooms as more personal and expressive of the self than did Turkish students.

Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow

Adult human bone marrow (ABM) is an important source of hematopoietic stem cells for transplantation in the treatment of malignant and nonmalignant diseases. However, in contrast to the recent progress that has been achieved with umbilical cord blood, methods to expand ABM stem cells for therapeutic applications have been disappointing. In this study, we describe a novel culture method that uses human brain endothelial cells (HUBECs) and that supports the quantitative expansion of the most primitive measurable cell within the adult bone marrow compartment, the nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cell (SRC). Coculture of human ABM CD34(+) cells with brain endothelial cells for 7 days supported a 5.4-fold increase in CD34(+) cells, induced more than 95% of the CD34(+)CD38(-) subset to enter cell division, and produced progeny that engrafted NOD/SCID mice at significantly higher rates than fresh ABM CD34(+) cells. Using a limiting dilution analysis, we found the frequency of SRCs within fresh ABM CD34(+) cells to be 1 in 9.9 x 10(5) cells. Following HUBEC culture, the estimated frequency of SRCs increased to 1 in 2.4 x 10(5) cells. All mice that received transplants of HUBEC-cultured cells showed B-lymphoid and myeloid differentiation, indicating that a primitive hematopoietic cell was preserved during culture. Noncontact HUBEC cultures also maintained SRCs at a level comparable to contact HUBEC cultures, suggesting that cell-to-cell contact was not required. These data demonstrate that human brain endothelial cells possess a unique hematopoietic activity that increases the repopulating capacity of adult human bone marrow.

Dendritic cells support hematopoiesis of bone marrow cells.

We previously observed that vaccination of normal mice with bone marrow (BM) -derived dendritic cells (DCs) could increase the number of peripheral white blood cells (WBCs) and platelets. In the present study, we investigated the potential of DCs to support the hematopoiesis of BM cells in vitro and in vivo. In the absence of exogenous cytokines, the expansion of CD34+ stem cells was observed when cultured with DC-derived supernatant or contact cocultured with DC. After culture in supernatant of DCs or contact coculture with DCs for 3 days, CD34+ progenitor cells were cultured in the semisolid media to test their ability to generate the clonogeneic cells. Then, BM cells combined with DCs or not were transferred into lethally irradiated syngeneic recipients to determine the effects of DCs on hematopoietic recovery. After culture in the supernatant of DCs, especially in the supernatant of OVA-DCs (OVA-stimulated DC), the proliferation of CD34+ stem cells and generation of clonogeneic cells were augmented in correspondence with the concentration of DCs. After contact coculture with DCs, the proliferation of CD34+ stem cells and generation of clonogeneic cells were more significant than that in noncontact cultures. Moreover, when cultured with DCs or supernatant of DCs, CD34+ progenitor cells were preferentially differentiated to megakaryocytes. After coculture with OVA-DCs, markedly greater generation of colony forming units-granulocyte/macrophages (CFU-GM): colony forming units-megakaryocytes (CFU-MK) was found than that in coculture with unstimulated DCs. Pretreatment of DC with antibodies to thrombopoietin (TPO), interleukin (IL) -6, IL-12, or anti-mouse intercellular adhesion molecule-1 (ICAM-1) could inhibit the ability of DCs to support the generation of CFU-GM, CFU-MK. After transplant with BM cells and DCs, the number of peripheral platelets of the recipients increased significantly and, to a lesser extent, peripheral WBC counts increased. The survival periods were significantly prolonged when the lethally irradiated mice were transplanted with BM cells combined with DCs or OVA-DCs. High levels of TPO, IL-6, and IL-12 could be detectable in the supernatant of DCs, and TPO expression by DCs was further confirmed by reverse transcription-polymerase chain reaction analysis and intracellular staining with anti-TPO antibody. We first demonstrated that DCs, especially antigen-stimulated DCs, can promote the expansion of hematopoietic progenitors and support hematopoiesis, preferentially support megakaryopoiesis of BM cells, by expressing soluble factors, including TPO, IL-6, IL-12, and by direct cell-to-cell interaction with stem cells in vitro and in vivo.

Trauma inhibits erythroid burst-forming unit and granulocyte-monocyte colony-forming unit growth through the production of TGF-beta1 by bone marrow stroma.

To examine the effect of trauma plasma on clonogenic progenitor cultures. Severely injured trauma patients often experience altered hematopoietic functions, manifested by an increased susceptibility to infection and the development of a persistent anemia. Experimental and clinical data suggest that trauma results in the release of cytokines into the plasma that have hematopoietic regulatory function, but few studies have examined human bone marrow. Plasma was obtained from 42 severely injured patients admitted to the surgical intensive care unit from days 1 to 15 after injury. Bone marrow and normal plasma were obtained from volunteers. Bone marrow mononuclear cells were isolated and plated for granulocyte-monocyte colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) growth. Parallel cultures were incubated with 2% (v/v) trauma or normal plasma. Additional cultures were plated with neutralizing concentrations of antibodies to transforming growth factor (TGF)-beta1 and MIP-1alpha. Circulating plasma TGF-beta1 was determined by bioassay. mRNA from bone marrow stromal cultures was extracted and probed for TGF-beta1 and macrophage inflammatory protein (MIP)-1alpha. Trauma plasma suppressed CFU-GM and BFU-E colony growth by 40% to 60% at all time periods after injury compared with cultures incubated with normal plasma. Using a noncontact culture system, the authors showed that this inhibition of BFU-E and CFU-GM colony growth was mediated by bone marrow stroma. The inhibition appeared to be due to soluble plasma-induced bone marrow stromal products that did not require direct cell-cell contact. The addition of anti-TGF-beta1 antibodies reversed the suppressive effect of trauma plasma on CFU-GM and BFU-E colony growth during the early but not late time points after injury. Trauma but not normal plasma induced TGF-beta1 mRNA in bone marrow stroma. Trauma plasma inhibits bone marrow BFU-E and CFU-GM colony growth for up to 2 weeks after injury. This inhibition is mediated through the interaction of trauma plasma with bone marrow stroma. TGF-beta1 production by bone marrow stroma appears to plays an important role in the early but not late bone marrow suppression after injury.

Soluble stromal factors enhance gene transfer in primitive human hematopoietic cells in mobilized peripheral blood

Hematopoietic stem cells present mobilized peripheral blood (MPB) are attractive targets for retroviral gene therapy. Previously, we have found that in contrast to primitive hematopoietic cells in umbilical cord blood, primitive cells in G-CSF MPB are not efficiently transduced. Based on earlier studies demonstrating a positive effect of the fetal liver stromal cell line AFT024 on the maintenance of primitive hematopoietic cells ex vivo, we hypothesized that AFT024 may enhance the level of gene transfer (GT) into these cells. To test this hypothesis, CD34+ cells from G-CSF MBP were cultured in the presence or absence of irradiated AFT024 cells using trans-well (non-contact) cultures for 4 days with either G-CSF, SCF and TPO (GST; 100 ng/mL) or Flt3-L, SCF, IL-7 and TPO (FS7T; 10-20 ng/mL), followed by infection on two consecutive days with a GALV-pseudotyped MFG-EGFP retroviral vector on fibronectin fragment FN CH-296 (Retronectin®, Takara Shuzo). Cultured and transduced cells were evaluated for the level of expansion as well as for the level of GT in CFC and LTC-IC assays. AFT024 enhanced the expansion of CFC and LTC-IC (1.5 to 2-fold). The level of GT in CFC (ranging from 1 to 26% in BFU-E and CFU-GM; n=10) was higher in the groups pre-stimulated with GST as compared to FS7T. In contrast, the level of GT in LTC-IC (ranging from 1.5 to 47%) was higher with FS7T (n=6), but only in the presence of AFT024. Finally, the overall recovery of transduced LTC-IC in the presence of AFT024 with FS7T was 6-fold higher as compared to our previously used strategy using GST in the absence of stroma (p<0.001). These data demonstrate that the recovery of transduced primitive hematopoietic cells in G-CSF MPB can be enhanced using low doses of early acting cytokines and a soluble factor produced by AFT024.

The significance of MMP-1 and MMP-2 in peritoneal disseminated metastasis of gastric cancer.

The purpose of this study was to clarify the role of the matrix metalloproteinases (MMPs), collagenase (MMP-1), and gelatinase A (MMP-2), both of which are known to be involved in the development of gastric cancer, in peritoneal dissemination. The concentrations of MMP-1 and MMP-2 in the supernatant of mixed culture simulated peritoneal dissemination were measured in vitro with mesothelial cells and cancer cells using an enzyme-linked immunosorbent assay. The concentration of MMP-1 increased significantly after the contact culture was mixed with these two cells, in comparison with the non-contact mixed culture or the mesothelial cell culture alone. These results demonstrate that the production of MMP-1 derived from mesothelial cells was increased by contact with cancer cells. To clarify the effect of MMP-1 and MMP-2 on gastric cancer invasion, an invasion assay using matrigel was performed. After treatment with anti-MMP-1 monoclonal antibody (mAb) or anti-MMP-2 mAb, the number of matrigel-penetrating cancer cells was significantly reduced, indicating that MMP-1 and MMP-2 derived from mesothelial cells had a strong reaction to gastric cancer invasion. In conclusion, as MMP-1 showed a paracrine-like action responding to stimulus from cancer cells, it seemed to play an important role in the progression of peritoneal dissemination.

Highly efficient retroviral transduction of cord blood hematopoietic stem cells

Retroviral transduction of hematopoietic stem cells(HSC) requires that these cells divide in vitro while retaining their primitive phenotype. We have recently developed the Myeloid-Lymphoid Initiating Cell (ML-IC) assay (Punzel et al, Blood 93(11)), an in vitro culture system that allows for enumeration of single cells capable of generating secondary progenitors that can re-initiate long term myeloid and lymphoid cultures. The ML-IC is therefore believed to be closely related to HSC. CD34+CD38−CD33− cord blood cells were plated in a stroma noncontact culture in CH296 coated transwells above AFT024 stromal feeders in a Flt 3, SCF, IL7 and Tepo containing medium. After 48 hours, and again after 72 hours, they were exposed for 6 hours to GALV pseudotyped MFG-eGFP containing viral supernatant. After a total of 5 days, CD34+CD38−CD33− cells were single cell sorted to assess ML-IC frequency and transduction efficiency. On average there was a 2-fold expansion of total cell number. 14.6±11.3% retained the CD34+CD38−CD33− phenotype, and of these 80.0±14.3% were GFP+, whereas only 39.7±16.1% of the total cell population was GFP+ (p=0.016). Within the CD34+ CD38−CD33− fraction, the ML-IC frequency was 4.3±1.8%, and 73.9±15.7% of these were transduced. In 16% of the transduced ML-IC, GFP expression was silenced in the myeloid arm but present in the NK-IC. 5/69 ML-IC arising from cells in the GFP− population on day 5 proved transduced, i.e. GFP+, upon read-out. The generative potential of the ML-IC, defined as the fraction of ML-IC that generates 2 LTC-IC and 2 NK-IC, rather than just 1 of each, was similar for GFP+ and GFP− ML-IC (39.6±20.5% vs. 51.3±11.8%, p=0.24). In conclusion, we describe a transduction protocol that efficiently targets primitive progenitors, while retaining their generative potential. Selection of GFP+ cells shortly after transduction may lead to underestimation of transduction efficiency. Finally, the ML-IC assay proves to be useful in studying differential silencing in different lineages arising from a single cell.

Regulation of megakaryocytopoiesis in an in vitro stroma model: Preferential adhesion of megakaryocytic progenitors and subsequent inhibition of maturation

Objective Studies of megakaryocytic progenitor cell interactions have focused on single receptor-ligand interactions using isolated components of the extracellular matrix. To approach a physiologic condition, we studied megakaryocytic development of human progenitor cells cultured on two stromal cell lines and on human bone marrow stroma. Materials and Methods Human CD34 + cells were cocultured with stromal layers in the presence of thrombopoietin. Megakaryocytes were quantified by monoclonal antibodies against glycoprotein (GP) IIb/IIIa (CD41) and GPIX (CD42a). Megakaryocytic clonogenic capacity (burst-forming unit-megakaryocyte and colony-forming unit-megakaryocyte) was determined using fibrin clot assays. Results After 6 days, a higher percentage of megakaryocytes and more megakaryocytic colonies were recovered from the adherent cell fraction compared to the nonadherent cell fraction. In contrast, significantly more granulocytic and erythroid colonies were recovered from the nonadherent cell fraction. Repeated replating of nonadherent cells onto fresh stroma showed a decline in megakaryocytic recovery of the remaining adherent cells, pointing toward selective adhesion of megakaryocytic progenitors. This was supported further by the finding that burst-forming unit and colony-forming unit megakaryocytes were preferentially recovered from the adherent cell fraction at 24 hours. No effect of blocking the β 1 integrins VLA-4 and VLA-5 on human progenitor cells was observed. A higher expression of CD42a antigen and a higher percentage of morphologically recognizable polyploid megakaryocytes were found when cells were grown in noncontact cultures compared to when grown adhered to stroma. Conclusion In contrast to granulocytic and erythroid progenitors, both very early and more mature megakaryocytic progenitors are preferentially located in the adherent fraction in an in vitro stromal model, leading to inhibition of maturation of megakaryocytes. This suggests that the presence of stroma components in ex vivo expansion cultures, aimed at preservation and expansion of megakaryocytic progenitors, might be a prerequisite.

Factor(s) secreted by AFT024 fetal liver cells following stimulation with human cytokines are important for human LTC-IC growth.

Soluble factors produced by human marrow stroma or the murine marrow derived M2-10B4 cell line support ex vivo maintenance for 5-8 weeks of 50% of human long-term culture initiating cells (LTC-IC). As the AFT024 cell line supports LTC-IC cultured in contact conditions better than M2-10B4 feeders, we evaluated LTC-IC support in non-contact conditions above AFT024 feeders. We show that only 15% of LTC-IC were maintained for 5 weeks in AFT024 non-contact cultures (n=6, P<0.05). As AFT024-conditioned media added to M2-10B4 non-contact cultures did not inhibit LTC-IC maintenance, AFT024 cells do not secrete factors that inhibit LTC-IC growth. We next characterized heparan sulfate glycosaminoglycans (HS-GAGs) and cytokines produced by AFT024 cells, which are both required for LTC-IC maintenance in M2-10B4 non-contact cultures. The size and extent of O-sulfation of HS-GAGs in AFT024 and M2-10B4 conditioned medium were similar, indicating that absence of hematopoietic specific HS-GAGs is not responsible for the lack of hematopoietic in AFT024 non-contact cultures. Levels of 13 different cytokines secreted in AFT024- and M2-10B4-conditioned medium were similar. However, addition of human SCF, G-CSF, GM-CSF, LIF, MIP-1alpha and IL-6 in concentrations found in human marrow stroma-conditioned medium to AFT024 non-contact cultures increased LTC-IC-maintenance to 72% at 5 weeks. These cytokines improved LTC-IC maintenance in part through interaction with the progenitors and in part, through interaction with the AFT024 feeder. Thus, although LTC-IC maintenance is poor in AFT024 non-contact cultures, addition of human cytokines enhances LTC-IC maintenance in part through indirect effects on the AFT024 feeder. Characterization of known or novel growth factors secreted by AFT024 cells before and after cytokine stimulation may lead to the identification of cytokines that support growth of human hematopoietic stem cells.

Megakaryocytic differentiation of human progenitor cells is negatively influenced by direct contact with stroma.

The recently defined ligand for the Mpl receptor, thrombopoietin (TPO), has been found to be the principal regulatory cytokine of megakaryocytopoiesis. Furthermore, it has been hypothesized that direct interaction between stroma or stroma-derived extracellular matrix (ECM) and human progenitor cells (HPC) may modulate megakaryocytopoiesis. For that purpose, we studied megakaryocytic development of HPC, obtained from patients with hematological malignancies in complete remission or solid tumors without bone marrow involvement, under the influence of megakaryocyte growth and development factor (MGDF, a pegylated, truncated molecule related to the endogenous Mpl ligand). The megakaryocytic development of HPC cultured in contact with a stromal layer (contact cultures) was compared with cultures in which HPC were grown separated from a stromal layer by a microporous membrane (non-contact cultures). A significantly lower number of megakaryocytes (CD41- and CD61-positive cells) was obtained from contact cultures compared to non-contact cultures. Furthermore, the expression of CD42b was higher in non-contact cultures, as compared to contact cultures, indicating an increase in megakaryocyte maturation in non-contact cultures. In contrast, no difference in clonogenic capacity was observed (CFU-GM, BFU-E, CFU-Mk). The possibility that direct contact between HPC and stroma induces the production of a soluble inhibitory cytokine as an explanation for the diminished megakaryocytic development in contact cultures, was excluded by performing transwell experiments in which HPC were cultured in direct contact and in a transwell simultaneously. The percentage of megakaryocytes raised from HPC present in the transwell did not decrease, irrespective of the presence of HPC simultaneous below the transwell in direct contact with stroma. It is concluded that both megakaryocytic development out of HPC and megakaryocytic differentiation is reduced in contact cultures, as compared to non-contact cultures. This is due to modulation by adhesive interactions with stroma, stroma-derived ECM or cytokines bound to the membrane of stromal cells, rather than resulting from the production of diffusible cytokines by stromal cells.

Ex Vivo Culture of CD34+/Lin−/DR− Cells in Stroma-Derived Soluble Factors, Interleukin-3, and Macrophage Inflammatory Protein-1α Maintains Not Only Myeloid But Also Lymphoid Progenitors in a Novel Switch Culture Assay

We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1α (MIP-1α). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin−/DR−cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin−/DR− cells assayed in SNC cultures supplemented with IL-3 and MIP-1α. When CD34+/Lin−/DR− progeny from the SNC culture were plated sequentially into “NK cell progenitor switch” conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by “NK cell differentiation” conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3− NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1α directly to “NK cell differentiation” conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33− cells present after SNC cultures with IL-3 and MIP-1α, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33− population similar to fresh sorted CD34+/Lin−/DR− cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1α, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.

Ex Vivo Culture of CD34+/Lin−/DR− Cells in Stroma-Derived Soluble Factors, Interleukin-3, and Macrophage Inflammatory Protein-1α Maintains Not Only Myeloid But Also Lymphoid Progenitors in a Novel Switch Culture Assay

AbstractWe have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1α (MIP-1α). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin−/DR−cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin−/DR− cells assayed in SNC cultures supplemented with IL-3 and MIP-1α. When CD34+/Lin−/DR− progeny from the SNC culture were plated sequentially into “NK cell progenitor switch” conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by “NK cell differentiation” conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3− NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1α directly to “NK cell differentiation” conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33− cells present after SNC cultures with IL-3 and MIP-1α, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33− population similar to fresh sorted CD34+/Lin−/DR− cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1α, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.

Primitive Long-Term Culture Initiating Cells (LTC-ICs) in Granulocyte Colony-Stimulating Factor Mobilized Peripheral Blood Progenitor Cells Have Similar Potential for Ex Vivo Expansion as Primitive LTC-ICs in Steady State Bone Marrow

AbstractWe have recently shown that more than 90% of long-term culture initiating cells (LTC-IC) mobilized in the peripheral blood (PB) of normal individuals express HLA-DR and CD38 antigens and can sustain hematopoiesis for only 5 weeks. However, 10% of LTC-IC in mobilized PB are CD34+HLA-DR− and CD34+CD38− and can sustain hematopoiesis for at least 8 weeks. We now examine the ex vivo expansion potential of CD34+HLA-DR+ cells (rich in mature LTC-IC) and CD34+HLA-DR− cells (rich in primitive LTC-IC) in granulocyte colony-stimulating factor (G-CSF ) mobilized PB progenitor cells (PBPC). Cells were cultured in contact with M2-10B4 cells (contact) or in transwells above M2-10B4 (noncontact) without and with interleukin-3 (IL-3) and macrophage inflammatory protein (MIP-1α) for 2 and 5 weeks. Progeny were evaluated for the presence of colony-forming cells (CFC) and LTC-IC. When CD34+HLA-DR+ PB cells were cultured in contact cultures without cytokines, a threefold expansion of CFC was seen at 2 weeks, but an 80% decrease in CFC was seen at week 5. Further, the recovery of LTC-IC at week 2 was only 17% and 1% at week 5. This confirms our previous observation that although CD34+HLA-DR+ mobilized PB cells can initiate long-term cultures, they are relatively mature and cannot sustain long-term hematopoiesis. In contrast, when CD34+HLA-DR− mobilized PB cells were cultured in contact cultures without cytokines, CFC expansion persisted until week 5 and 49% and 11% of LTC-IC were recovered at week 2 and 5, respectively. As we have shown for steady state bone marrow (BM) progenitors, recovery of LTC-IC was threefold higher when CD34+HLA-DR− PBPC were cultured in noncontact rather than contact cultures, and improved further when IL-3 and MIP-1α were added to noncontact cultures (96 ± 2% maintained at week 5). We conclude that although G-CSF mobilizes a large population of “mature” CD34+HLA-DR+ LTC-IC with a limited proliferative capacity, primitive CD34+HLA-DR− LTC-IC present in mobilized PB have similar characteristics as LTC-IC from steady state BM: (1) they can be maintained in noncontact cultures containing IL-3 and MIP-1α for at least 5 weeks; (2) they are subject to the same proliferation inhibitory influences of contact with stroma. Since the absolute number of primitive LTC-IC (week 8 LTC-IC) per mL of G-CSF mobilized PB is similar to that per mL of steady state BM, these studies further confirm that G-CSF mobilized PBPC may have similar long-term repopulating abilities as steady state BM.