Elevation of Survivin levels by hematopoietic growth factors occurs in quiescent CD34+ hematopoietic stem and progenitor cells before cell cycle entry

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is overexpressed during G(2)/M phase in most cancer cells. In contrast, we previously reported that Survivin is expressed throughout the cell cycle in normal CD34(+) hematopoietic stem and progenitor cells stimulated by the combination of Thrombopoietin (Tpo), Stem Cell Factor (SCF) and Flt3 ligand (FL). In order to address whether Survivin expression is specifically up-regulated by hematopoietic growth factors before cell cycle entry, we isolated quiescent CD34(+) cells and investigated Survivin expression in response to growth factor stimulation. Survivin is up-regulated in CD34(+) cells with 2N DNA content following growth factor addition, suggesting it becomes elevated during G(0)/G(1). Survivin is barely detectable in freshly isolated umbilical cord blood (UCB) Ki-67(negative) and Cyclin D(negative) CD34(+) cells, however incubation with Tpo, SCF and FL for 20 hrs results in up-regulation without entry of cells into cell cycle. Culture of G(0) CD34(+) cells isolated based on Hoechst 33342/PyroninY staining with Tpo, SCF and FL for 48 hrs, results in significantly elevated Survivin mRNA and protein levels. Moreover, labeling of fresh G(0) CD34(+) cells with 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) before culture with growth factors for up to 72 hrs, revealed that Survivin expression was elevated in CFSE(bright) G(0) CD34(+) cells, indicating that up-regulation occurred before entry into G1. These results suggest that up-regulation of Survivin expression in CD34(+) cells is an early event in cell cycle entry that is regulated by hematopoietic growth factors and does not simply reflect cell cycle progression and cell division.     

Recruitment of primitive peripheral blood cells: synergism of interleukin 12 with interleukin 6 and stromal cell-derived FACTOR-1

In bone marrow, haematopoietic stem cells (HSC) rely on close contact with stromal cells for proliferation and differentiation. Stromal cell-derived factor (SDF-1) is a chemokine produced by bone marrow stromal cells and has been reported to be a chemoattractant for CD34(+)cells. SDF-1 was evaluated for effects on proliferation of both mature and immature human progenitor cells in vitro. Neither proliferation nor maturation of peripheral blood cells was stimulated by SDF-1 alone. Moreover, we have previously demonstrated that 5-fluorouracile (5-FU) resistant HSC require a combination of interleukin 12 (IL-12), IL-6 and SCF for the production of morphologically recognizable clonogenic elements at day 14 in semisolid medium. Our data reported a strong enhancement of the IL-6, IL-12, SCF-induced synergism (172%) by SDF-1 (296.5%). Furthermore, our data suggest that this chemokine alone had no effect on triggering quiescent cells and may preserve these cells from 5-FU cell damage or upregulate early-acting cytokine receptors. Thus, SDF-1 might play a key role in early human haematopoiesis through its potent synergistic effects in combination with early-acting cytokines. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HSC.     

Cytokine-augmented culture of haematopoietic progenitor cells in a novel three-dimensional cell growth matrix

Studies aimed at the in vitro expansion of haematopoietic progenitor cells (HPCs) have suffered from the conflict of increasing cell numbers while maintaining long-term repopulating ability. We have developed a long-term bone marrow bioreactor culture system resembling the marrow-microenvironment that cultures HPCs in an inert, three-dimensional, porous biomatrix termed Cellfoam. Previous studies have shown that the short-term culture of CD34(+)cells in Cellfoam improved the maintenance and multipotency of haematopoietic stem cells compared to cells cultured on plastic dishes. In this study, we examined the effects of low concentrations of cytokines including stem cell factor (SCF), IL-3, and Flk-2/Flt-3 ligand, on the maintenance, preservation and multipotency of CD34(+) cells cultured for 3 or 6 weeks in Cellfoam. Analysis of cell yields using flow cytometry showed that in SCF and Flk-2/Flt-3 ligand-supplemented cultures as well as cytokine-free cultures, a higher number of CD45(+)34(+) and CD45(+)34(+)38(-) cells is observed in Cellfoam cultures as compared to plastic cultures. The function of cultured cells was evaluated in colony-forming assays. The data demonstrate that Cellfoam cultures supplemented with SCF and Flk-2/Flt-3 ligand resulted in a higher output of colony activity compared to plastic cultures. Analysis of CAFC (29 days) activity also demonstrated that primitive progenitors were maintained to a greater extent in Cellfoam cultures containing either no cytokines or low concentrations of early-acting cytokines. These data suggest that culture of HPCs in three-dimensional bioreactors such as Cellfoam for extended periods may benefit from the addition of low levels of early-acting cytokines, including SCF and Flk-2/Flt-3 ligand, resulting in high yields of cells that are enriched for multipotent haematopoietic progenitors. These findings demonstrate that a three-dimensional matrix promotes the survival of primitive HPCs in culture and may modulate the in vitro effects of cytokines.     

The development of macrophages from human CD34+ haematopoietic stem cells in serum-free cultures is optimized by IL-3 and SCF

The derivation of human macrophages from peripheral blood monocytes remains a convenient method for the study of macrophage biology. However, for macrophage differentiation, a significant proportion of development has occurred prior to the monocyte stage; monocyte subsets also have varying potential for differentiation. Differentiation of macrophages from a less mature precursor, such as CD34⁺ haematopoietic stem cells, can further inform with regard to the development of macrophage-lineage cells. CD34⁺ cells were cultured in serum-free medium containing Flt3L, SCF, IL-3, IL-6 and M-CSF. Using differing combinations of growth factors, the effect on cell proliferation and differentiation to adherent macrophage-like cells was determined. The proliferative response of CD34⁺ cells to M-CSF was determined during the initial phase of cell culture. Thirteen combinations of SCF, IL-3, IL-6 and M-CSF were then compared to determine the optimum combination for proliferation. Adherence was used to isolate mature macrophages, and the macrophage-like phenotype was confirmed by analyses of surface markers, histo-morphology and phagocytosis. This study refines the means by which large numbers of macrophages are obtained under serum-free conditions from haematopoietic precursors.     

Chimeric cytokine receptor can transduce expansion signals in interleukin 6 receptor alpha (IL-6Ralpha)-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors

We generated transgenic mice expressing chimeric receptors, which comprise extracellular domains of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor and transmembrane and cytoplasmic domains of the mouse leukemia inhibitory factor receptor. In suspension cultures of lineage-negative (Lin(-)), 5-fluorouracil-resistant bone marrow cells of the transgenic mice, a combination of hGM-CSF and stem cell factor (SCF) induced exponential expansions of mixed colony-forming unit. The combination of hGM-CSF and SCF was effective on enriched, Lin(-)Sca-1(+)c-kit(+) progenitors and increased either mixed colony-forming unit or cobblestone area-forming cells. In case of stimulation with hGM-CSF and SCF, interleukin-6 (IL-6) and SCF, or IL-11 and SCF, the most efficient expansion was achieved with hGM-CSF and SCF. When Lin(-)Sca-1(+)c-kit(+)CD34(-) further enriched progenitors were clone sorted and individually incubated in the presence of SCF, hGM-CSF stimulated a larger number of cells than did IL-6, IL-6 and soluble IL-6 receptor (IL-6R), or IL-11. These data suggest the presence of IL-6Ralpha-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors. Such primitive progenitors are equipped with signal transduction molecules and can expand when these chimeric receptors are genetically introduced into the cells and stimulated with hGM-CSF in the presence of SCF.     

Elevation of Survivin Levels by Hematopoietic Growth Factors Occurs in Quiescent CD34+ Hematopoietic Stem and Progenitor Cells Before Cell Cycle Entry

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is over-expressed during G2/M phase in most cancer cells. In contrast, we previously reported that Survivin is expressed throughout the cell cycle in normal CD34+ hematopoietic stem and progenitor cells stimulated by the combination of Thrombopoietin (Tpo), Stem Cell Factor (SCF) and Flt3 ligand (FL). In order to address whether Survivin expression is specifically up-regulated by hematopoietic growth factors before cell cycle entry, we isolated quiescent CD34+ cells and investigated Survivin expression in response to growth factor stimulation. Survivin is up-regulated in CD34+ cells with 2N DNA content following growth factor addition, suggesting it becomes elevated during G0/G1. Survivin is barely detectable in freshly isolated umbilical cord blood (UCB) Ki-67negative and Cyclin Dnegative CD34+ cells, however incubation with Tpo, SCF and FL for 20 hrs results in up-regulation without entry of cells into cell cycle. Culture of G0 CD34+ cells isolated based on Hoechst 33342/PyroninY staining with Tpo, SCF and FL for 48 hrs, results in significantly elevated Survivin mRNA and protein levels. Moreover, labeling of fresh G0 CD34+ cells with 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) before culture with growth factors for up to 72 hrs, revealed that Survivin expression was elevated in CFSEbright G0 CD34+ cells, indicating that up-regulation occurred before entry into G1. These results suggest that up-regulation of Survivin expression in CD34+ cells is an early event in cell cycle entry that is regulated by hematopoietic growth factors and does not simply reflect cell cycle progression and cell division.

Key Words:

Survivin, Cord blood, CD34+ cells, Cell cycle     

Cell cycle distribution of primitive haematopoietic cells stimulated in vitro and in vivo

A novel approach is used to study the proliferating behaviour of primitive haematopoietic cell populations in response to different stimuli. A mathematical model based on the average proportion of apoptotic, dividing and quiescent cells in primitive haematopoietic cell populations is developed to describe the mitotic history of 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester-labelled cells. The cell cycle distributions in different cytokine-supplemented cultures of primitive human and mouse bone marrow cells are determined and compared with those found in vivo. The results indicate that a combination of flt-3 ligand, Steel factor and interleukin-11 or hyper-interleukin-6 provide a level of mitogenic stimulation similar to that existing in vivo after a myeloablative radiation dose. The comparison of the cell cycle distribution obtained for different cultures of human bone marrow CD34(+)(45RA/71)(-) cells demonstrates that the addition of flt-3 ligand in these cultures decreases apoptosis significantly but does not reduce quiescence. In addition, in vivo and in vitro, it was found that more than 3 days of stimulation are required to recruit a maximum number of quiescent cells into active cell cycle. These kinetics of cell cycle activation are found to be similar to those identified for the haematopoietic stem cells compartment in the same cultures. This mathematical analysis provides a useful tool for the development of haematopoietic stem cell culture processes for clinical applications.     

IL-9 enhances the growth of human mast cell progenitors under stimulation with stem cell factor

We examined the effects of IL-9 on human mast cell development from CD34(+) cord blood (CB) and peripheral blood cells in serum-deprived cultures. IL-9 apparently enhanced cell production under stimulation with stem cell factor (SCF) from CD34(+) CB cells. A great majority of the cultured cells grown with SCF + IL-9 became positive for tryptase at 4 wk. In methylcellulose cultures of CD34(+) CB cells, IL-9 increased both the number and size of mast cell colonies grown with SCF. Furthermore, SCF + IL-9 caused an exclusive expansion of mast cell colony-forming cells in a 2-wk liquid culture of CD34(+) CB cells, at a level markedly greater than for SCF alone. Clonal cell cultures and RT-PCR analysis showed that the targets of SCF + IL-9 were the CD34(+)CD38(+) CB cells rather than the CD34(+)CD38(-) CB cells. IL-9 neither augmented the SCF-dependent generation of progeny nor supported the survival of 6-wk-cultured mast cells. Moreover, there was no difference in the appearance of tryptase(+) cells and histamine content in the cultured cells between SCF and SCF + IL-9. The addition of IL-9 increased numbers of mast cell colonies grown with SCF from CD34(+) peripheral blood cells in children with or without asthma. It is of interest that mast cell progenitors of asthmatic patients responded to SCF + IL-9 to a greater extent than those of normal controls. Taken together, IL-9 appears to act as a potent enhancer for the SCF-dependent growth of mast cell progenitors in humans, particularly asthmatic patients.     

Up-regulation of miR-210 by vascular endothelial growth factor in ex vivo expanded CD34+ cells enhances cell-mediated angiogenesis

Ex vivo culture has been proposed as a means to augment and repair autologous cells in patients with chronic diseases, but the mechanisms governing improvement in cell function are not well understood. Although microRNAs (miRs) are increasingly appreciated as key regulators of cellular function, a role for these factors in CD34+ cell-mediated angiogenesis has not been elucidated. Vascular endothelial growth factor (VEGF) was previously shown to induce expression of certain miRs associated with angiogenesis in endothelial cells and promote survival and number of vascular colony forming units of haematopoietic stem cells (HSCs). We sought to evaluate the role of VEGF in expansion and angiogenic function of CD34+ cells and to identify specific miRs associated with angiogenic properties of expanded cells. Umbilical cord blood CD34+ cells were effectively expanded (18- to 22-fold) in culture medium containing stem cell factor (SCF), Flt-3 ligand (Flt-3), thrombopoietin (TPO) and interleukin-6 (IL-6) with (postEX/+VEGF) and without VEGF (postEX/noVEGF). Tube formation in matrigel assay and tissue perfusion/capillary density in mice ischaemic hindlimb were significantly improved by postEX/+VEGF cells compared with fresh CD34+ and postEX/noVEGF cells. MiR-210 expression was significantly up-regulated in postEX/+VEGF cells. MiR-210 inhibitor abrogated and 210 mimic recapitulated the pro-angiogenic effects by treatment of postEX/+VEGF and postEX/noVEGF cells respectively. Collectively, these observations highlight a critical role for VEGF in enhancing the angiogenic property of expanded cells, and identify miR-210 as a potential therapeutic target to enhance CD34+ stem cell function for the treatment of ischaemic vascular disease.     

Expression of the cell adhesion molecule E-cadherin by the human bone marrow stromal cells and its probable role in CD34(+) stem cell adhesion.

Bone marrow stroma is the physical basis of the haematopoietic microenvironment and regulates several key features of stem cell proliferation and differentiation. It plays a crucial role in maintaining haematopoietic homeostasis. Earlier studies have shown that this is achieved through interactions with the extracellular matrix and specific molecules called the cell adhesion molecules (CAMs). In this paper, we show that E-cadherin, a cell adhesion molecule which plays a crucial role in cell-cell aggregation during development, is also present in the bone marrow stroma. The expression of the CAM can also be demonstrated on a subset of CD34(+)stem cells. Stromal expression of E-cadherin is decreased when treated with lymphokine mixture, phytohaemagglutinin-treated-leukocyte-conditioned medium (PHA-LCM). This is the reverse of ICAM-I expression, which increases with PHA-LCM treatment. E-cadherin shows homotypic and homophilic interaction and its presence on a subset of CD34(+)cells leads to speculation on whether this CAM has a role in adherence of primitive stem cells to the marrow stroma.     

Modulation of haematopoietic progenitor development by FLT-3 ligand.

The Flt-3 receptor is expressed in primitive haematopoietic cells and its ligand exerts proliferative effects on these cells in vitro in synergy with other cytokines. To increase our knowledge of the functional properties of the human Flt-3 ligand (FL) as relating to in vitro expansion of haematopoietic stem cells, the effects on murine haematopoiesis of FL alone or in combination with other growth factors were studied. Analysis of Flk-2/Flt-3 mRNA expression indicated that Flk-2/Flt-3 was preferentially expressed in primitive haematopoietic cell populations. To examine the expression of the Flk-2/Flt-3 receptor on megakaryocyte progenitors (CFU-Meg), Flk-2/Flt-3 positive and negative CD34(+)populations were separated from human bone marrow and cultured in a plasma clot culture system. CFU-Meg colonies were found in the Flk-2/Flt-3 negative fraction. Myeloid (CFU-GM) derived colonies appeared in the presence of FL alone. Neither FL+IL-3 nor FL+IL-3+IL-6 had any effect on the generation of megakaryocyte colonies (CFU-MK), due to the lack of FL receptor expression on megakaryocyte progenitors. Bone marrow cells remaining after 5-fluorouracil (5-FU) treatment of mice represent a very primitive population of progenitors enriched for reconstituting stem cells. This cell population expressed FL receptors, as revealed by RT-PCR analysis. Addition of FL alone did not enhance the replication of such cells in liquid cultures as compared to controls. However, a significantly greater generation of myeloid progenitors (CFU-GM) in clonogenic assays was observed in the presence of FL+IL-3, FL+GM-CSF or FL+CSF-1. In addition, the effects of FL on in vitro expansion of murine haematopoietic stem cells were studied using lineage-negative (lin(-)) Sca-1 positive (Sca-1(+)) c-kit positive (c-kit(+)) marrow cells from 5-FU treated mice. FL enhanced the survival of primitive murine lin(-)Sca-1(+)c-kit(+)cells. FL and IL-6 were able to significantly expand murine progenitor stem cells in vitro and promote their survival. These studies strongly suggest that FL significantly and selectively enhanced the generation of myeloid progenitors in vitro and increased myeloid progenitor responsiveness to later acting growth factors. In addition, FL synergized with IL-6 to support in vitro expansion of haematopoietic progenitors and promoted the survival of lin(-)Sca-1(+)c-kit(+)cells.     

Expression, regulation and function of AC133, a putative cell surface marker of primitive human haematopoietic cells

To explore the physiological significance of AC133 expression on human haematopoietic cells, we phenotyped normal and malignant human haematopoietic cells for AC133 expression, evaluated the utility of AC133 for isolating human stem/progenitor cells in comparison to other known early haematopoietic cell markers, investigated the role of AC133 in regulating hematopoiesis, and evaluated the possibility that MYB might regulate AC133. We found that while human CD34+ progenitor cells expressed AC133, expression was rapidly downregulated during differentiation. In apparent contrast, AC133 mRNA was detectable in cells isolated from CFU-Mix, BFU-E, CFU-GM and CFU-Meg colonies. Human cord blood CD34+ cells expressed AC133 at higher levels than their normal bone marrow counterparts. In apparent contrast to normal primitive haematopoietic cells, the AC133 protein was undetectable on cells from 24 different human haematopoietic cells lines, even though the majority of these cells expressed AC133 mRNA. Since CD34, AC133 and the c-kit (KIT) receptor are all co-expressed on human stem/progenitor cells, we compared the ability of monoclonal antibodies directed against each of these proteins to isolate early progenitor cells. Using these antibodies and magnetized particles in a standard immunoaffinity isolation protocol, we found that anti-CD34 and anti-KIT MoAbs could isolate > 80-90% of the clonogeneic cell population present in a given marrow sample. Anti-AC133 MoAbs recovered approximately 75-80% of CFU-GM and CFU-Meg, but only about 30% of CFU-Mix and BFU-E. Perturbation of AC133 expression with antisense oligodeoxynucleotides (AS ODN) resulted in transient downregulation of AC133 protein on human CD34+ cells but no apparent effect on cell survival or cloning efficiency ex vivo. Finally, downregulation of MYB expression with AS ODN had no effect on the AC133 expression at either the mRNA or protein level. Based on these results, we conclude that AC133 offers no distinct advantage over CD34 or c-kit as a target for immunoaffinity based isolation of primitive hematopoietic cells, that AC133 expression is not required for normal hematopoietic progenitor cell development in vitro, and finally that AC133 expression may not be MYB-dependent.     

Stem cell factor and interleukin-3 induce stepwise generation of erythroid precursor cells from a basic fibroblast growth factor-dependent hematopoietic stem cell line, A-6

A multipotent immature myeloid cell population was produced from a basic fibroblast growth factor (bFGF)-dependent hematopoietic stem cell line, A-6, when cultured with stem cell factor (SCF) replacing bFGF. Those cells were positive for stem cell markers, c-kit and CD34, and a myeloid cell marker, F4/80. Some cell fractions were also positive for Mac-1, a macrophage marker or Gr-1, a granulocytic maker, but negative for an erythroid marker TER119. They also showed the expression of mRNA for the myeloid-specific PU.1 but did not that for the erythroid-specific GATA-1. Among various cytokines, interleukin-3 (IL-3) induced erythroid precursor cells that expressed the erythroid-specific GATA-1 and beta-major globin. The quantitative analysis showed that erythroid precursor cells were newly produced from the immature myeloid cells by cultivation with IL-3. SCF and IL-3 induced stepwise generation of erythroid precursor cells from an A-6 hematopoietic stem cell line.     

Positive and negative hematopoietic cytokines produced by bone marrow endothelial cells

Recently, cytokines and interleukins such as SCF, GM-CSF, G-CSF, TGF-beta, IL-6, IL-7, IL-8, IL-11 have been reported to be elaborated by endothelial cells. For further study, serum free bone marrow endothelial cell conditioned medium (BMEC-CM) was collected and ultrafiltrated by using a centriprep 10. The concentrated retentate (R-BMEC-CM) contained some substances whose molecular weight was more than 10 000 daltons. The filtrate (F-BMEC-CM) contained some substances whose molecular weight was less than 10 000 daltons. The effects of R-BMEC-CM and F-BMEC-CM on the growth of haematopoietic progenitors and the expression of cytokine and interleukin mRNAs of BMEC were investigated. The results showed that R-BMEC-CM stimulated the growth of CFU-GM, HPP-CFC, BFU-E, CFU-E, and CFU-Meg; while F-BMEC-CM inhibited the growth of these progenitors. Using the method of hybridizing to the Atlas cDNA Array, we were able to detect the presence of mRNAs of cytokines and interleukins in bone marrow endothelial cells. Our finding of the existence of mRNAs of SCF, GM-CSF, IL-6, TGF-beta, IL-1, and IL-11 in these cells was in agreement with the data reported previously. Furthermore, we detected mRNAs of MIP-2, Thymosion-beta4, PDGF, MSP-1, IFN-gamma, IL-13 and inhibin, which are related to haematopoiesis. Among these cytokines and interleukins, SCF, GM-CSF, IL-6, IL-1, and IL-11 are haematopoietic stimulators which may be responsible for the stimulative effects on the growth of haematopoietic progenitors. One of our new findings, the thymosin-beta4, is a small molecular haematopoietic inhibitor. It may be responsible for the inhibitory effect of F-BMEC-CM on haematopoietic progenitors. The presence of mRNAs of BMP, MSP-1, MIP-2, PDGF and IL-13 suggests that bone marrow endothelial cells might elaborate these substances. Their influence on haematopoietic progenitors needs further study.     

Simultaneous knockdown of p18INK4C, p27Kip1 and MAD1 via RNA interference results in the expansion of long-term culture-initiating cells of murine bone marrow cells in vitro

A combination of extrinsic hematopoietic growth regulators, such as stem cell factor (SCF), interleukin (IL)-3 and IL-6, can induce division of quiescent hematopoietic stem cells (HSCs), but it usually impairs HSCs' self-renewal ability. However, intrinsic negative cell cycle regulators, such as p18^INK4c (p18) p27^Kip1 (p27) and MAD1, can regulate the self-renewal of HSCs. It is unknown whether the removal of some extrinsic regulators and the knockdown of intrinsic negative cell cycle regulators via RNA interference (RNAi) induce ex vivo expansion of the HSCs. To address this question, a lentiviral vector-based RNAi tool was developed to produce two copies of small RNA that target multiple genes to knockdown the intrinsic negative cell cycle regulators pl8, p27 and MAD1. Colony-forming cells, long-term culture-initiating cells (LTC-IC) and engraftment assays were used to evaluate the effects of extrinsic and intrinsic regulators. Results showed that the medium with only SCF, but without IL-3 and IL-6, could maintain the sca-1⁺c-kit⁺ bone marrow cells with high LTC-IC frequency and low cell division. However, when the sca-1⁺c-kit⁺ bone marrow cells were cultured in a medium with only SCF and simultaneously knocked down the expression of pl8, p27 and MAD1 via the lentiviral vector-based RNAi, the cells exhibited both high LTC-IC frequency and high cell division, though engraftment failed. Thus, the simultaneous knockdown of pl8, p27 and MAD1 with a medium of only SCF can induce LTC-IC expansion despite the loss of engraftment ability.     

Bone marrow stromal cell interaction reduces Syndecan-1 expression and induces kinomic changes in myeloma cells

CD138 (Syndecan 1) is a heparan sulfate proteoglycan that concentrates heparan sulfate-binding growth factors on the surface of normal and malignant plasma cells (multiple myeloma, MMC). Recent studies have shown the presence of a CD138-negative fraction of MMC within myelomatous bone marrow (BM). We employed kinome array technology to characterize this fraction at a molecular level, using a myeloma cell line model. Compared to CD138-positive cells, CD138-negative MMC showed (i) a reduced activity of kinases involved in cell cycle progression, in agreement with a decreased labeling index and (ii) reduced Rho signaling to F-actin. Interestingly, CD138 mRNA and protein expression was reduced upon interaction of MM cells with stromal cell lines and primary mesenchymal cultures, which was accompanied by the acquisition of an increased Bcl6/Blimp1 ratio. Co-culture induced an increased activity of kinases involved in adhesion and a decreased S-phase transition in both CD138-positive and -negative fractions. In addition, CD138-negative MMC demonstrated an increased STAT3 and ERK1/2 activation compared to CD138+ MMC, in agreement with a lower sensitivity to compound exposure. The presence of a less mature, more resistant CD138-negative myeloma cell fraction within bone marrow microniches might contribute to high incidence of relapse of Myeloma patients.