Whole Bone Marrow Research Articles

Functional Niche Competition Between Normal Hematopoietic Stem and Progenitor Cells and Myeloid Leukemia Cells

Hematopoietic stem and progenitor cells (HSPCs) reside in a specialized niche that regulates their proliferative capacity and their fate. There is increasing evidence for similar roles of marrow niches on controlling the behavior of leukemic cells; however, whether normal hematopoietic stem cell (HSC) and leukemic cells reside in or functionally compete for the same marrow niche is unclear. We used the mixed lineage leukemia-AF9 (MLL-AF9) murine acute myeloid leukemia (AML) in a competitive repopulation model to investigate whether normal HSPC and leukemic cells functionally compete for the same marrow niches. Irradiated recipient mice were transplanted with fixed numbers of MLL-AF9 cells mixed with increasing doses of normal syngeneic whole bone marrow (WBM) or with purified HSPC (LSK). Survival was significantly increased and leukemic progression was delayed proportional to increasing doses of normal WBM or normal LSK cells in multiple independent experiments, with all doses of WBM or LSK cells studied above the threshold for rapid and complete hematopoietic reconstitution in the absence of leukemia. Confocal microscopy demonstrated nests of either leukemic cells or normal hematopoietic cells but not both in the marrow adjacent to endosteum. Early following transplantation, leukemic cells from animals receiving lower LSK doses were cycling more actively than in those receiving higher doses. These results suggest that normal HSPC and AML cells compete for the same functional niche. Manipulation of the niche could impact on response to antileukemic therapies, and the numbers of normal HSPC could impact on leukemia outcome, informing approaches to cell dose in the context of stem cell transplantation.

Defining Engraftment Potential within the Lineage Positive Population in Murine Marrow

The study of highly purified hematopoietic stem cells (HSCs) has dominated the field of hematopoietic stem cell biology. It is widely believed that the true stem cell population lies within the Lineage negative (Lin-) population, further sub-fractionated using positive and negative selection for surface markers such as c-Kit, Sca-1, CD150, CD41, CD48, and CD34. It is research on these highly purified subsets of HSCs that forms the foundation for almost all our knowledge of HSCs, and has led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to near homogeneity. In contrast, we have shown that a large percentage of long-term multi-lineage marrow repopulating cells in whole bone marrow (WBM) are actively cycling, that these cycling stem cells are lost during conventional HSC isolation, and that they can be found, in part, within the discarded Lineage positive (Lin+) population. Here we present data further characterizing the stem cell potential in the Lin+ fraction. We incubated WBM from B6.SJL mice with fluorescently tagged antibodies directed against TER119, B220, or T-cell markers (CD3, CD4, CD8), isolated the distinct Lin+ subsets by FACS, and then competitively engrafted each Lin+ subset into lethally irradiated C57BL/6 host mice. Donor chimerism and lineage specificity of donor cells in peripheral blood were analyzed by flow cytometry at 3 months. Although classically considered devoid of stem cell activity, we found that, when competed against equal numbers of C57BL/6 WBM, the TER119+ and B220+ B6.SJL donor cells contributed to 33% and 13% of the peripheral blood chimerism, respectively. In both cases, the engraftment was multi-lineage. When 70,000 T cell marker+ donor cells were competed with 300,000 C57BL/6 WBM, the donor cells contributed up to 1.6% of the peripheral blood multi-lineage chimerism. Given the size of the Lin+ fraction in WBM, such chimerism indicates a significant stem cell potential within this typically discarded population. Further time-points, secondary transplants and limited dilution studies are in progress to further define the prevalence and potency of this stem cell population.We have been testing mechanisms governing the loss of this stem cell population during HSC purification. First, we have previously shown that bulk Lin+ engraftment potential is due to cycling stem cells. We hypothesize that fluctuations in surface epitope expression with cell cycle transit render this population difficult to isolate with antibody-mediated strategies that rely on stable epitope expression. To begin testing this, we tracked the fluctuation of stem cell markers on Lin- cells in vitro. We isolated Lin- cells that were also negative for the stem cell markers c-Kit and Sca-1, placed them in liquid culture and, 18 hours later, re-assessed for stem cell marker expression by flow cytometry. We found that, although initially stem cell marker negative, up to 6%, 14%, and 2% of the Lin-/stem cell marker negative cells became positive for c-Kit alone, Sca-1 alone, or both c-Kit and Sca-1 expression, respectively. We are currently testing this population for a correlation between gain of c-Kit- and Sca-1 expression and stem cell function. Second, it is possible that there is a distinct subset of HSCs that are positive for both Lin+ markers and stem cell markers with stable stem cell capacity and that these distinct stem cells are thrown out in the process of lineage depletion. To begin testing this hypothesis, we have simultaneously stained WBM with antibodies directed against the Lin+ markers and conventional stem cell markers. Our preliminary data indicate that each Lin+ fraction tested to date has a subpopulation that is also positive for c-Kit and Sca-1. For example, 21% of CD3+ cells, 6.2% of CD4+ cells, 2.26% of CD8+ cells, 0.5% of B220+, and 0.45% of TER119+ cells express both c-Kit and Sca-1. We suspect these two populations have distinct functional phenotypes and experiments characterizing the molecular phenotype and engraftment capacity of these subpopulations are ongoing. In sum, our data indicate that stem cell purification skews isolation towards a small population of quiescent stem cells, underrepresenting a potentially large pool of actively cycling HSCs that are found within the Lin+ fraction. These data underscore the need to re-evaluate the total hematopoietic stem cell potential in marrow on a population level. DisclosuresNo relevant conflicts of interest to declare.

The murine long-term multi-lineage renewal marrow stem cell is a cycling cell

Prevailing wisdom holds that hematopoietic stem cells (HSCs) are predominantly quiescent. Although HSC cycle status has long been the subject of scrutiny, virtually all marrow stem cell research has been based on studies of highly purified HSCs. Here we explored the cell cycle status of marrow stem cells in un-separated whole bone marrow (WBM). We show that a large number of long-term multi-lineage engraftable stem cells within WBM are in S/G2/M phase. Using bromodeoxyuridine, we show rapid transit through the cell cycle of a previously defined relatively dormant purified stem cell, the long-term HSC (LT-HSC; Lineage(-)/c-kit(+)/Sca-1(+)/Flk-2(-)). Actively cycling marrow stem cells have continually changing phenotype with cell cycle transit, likely rendering them difficult to purify to homogeneity. Indeed, as WBM contains actively cycling stem cells, and highly purified stem cells engraft predominantly while quiescent, it follows that the population of cycling marrow stem cells within WBM are lost during purification. Our studies indicate that both the discarded lineage-positive and lineage-negative marrow cells in a stem cell separation contain cycling stem cells. We propose that future work should encompass this larger population of cycling stem cells that is poorly represented in current studies solely focused on purified stem cell populations.

Hematopoietic stem cells are a critical sub-population of whole bone marrow in the treatment of myocardial infarction

Recent studies suggest that whole bone marrow (WBM) derived stem cells may facilitate recovery following myocardial infarction. However, the sub-population of WBM responsible for recovery remains uncertain. By adjusting the abundance of CD34+LinNeg cells in human bone marrow we examined the relative significance of hematopoietic stem cells (HSC) in the recovery of cardiac function in a murine model of induced myocardial infarction. Enrichment of HSC by ~100-fold in WBM transplanted into mice significantly increased recovery of heart function and reduced scar size compared to transplantation of WBM depleted in HSC by ~10-fold (P P P P

Regenerative Injection Therapy with Whole Bone Marrow Aspirate for Degenerative Joint Disease: A Case Series

Regenerative therapeutic strategies for joint diseases usually employ either enriched concentrates of bone marrow-derived stem cells, chondrogenic preparations such as platelet-rich plasma, or irritant solutions such as hyperosmotic dextrose. In this case series, we describe our experience with a simple, cost-effective regenerative treatment using direct injection of unfractionated whole bone marrow (WBM) into osteoarthritic joints in combination with hyperosmotic dextrose. Seven patients with hip, knee or ankle osteoarthritis (OA) received two to seven treatments over a period of two to twelve months. Patient-reported assessments were collected in interviews and by questionnaire. All patients reported improvements with respect to pain, as well as gains in functionality and quality of life. Three patients, including two whose progress under other therapy had plateaued or reversed, achieved complete or near-complete symptomatic relief, and two additional patients achieved resumption of vigorous exercise. These preliminary findings suggest that OA treatment with WBM injection merits further investigation.

Myelodysplastic/Myeloproliferative Neoplasm in Crebbp+/− Mice Is a Transplantable Disease in Which Multiple Hematopoietic Cell Populations Are Involved, As Well As the Microenvironment

Abstract 1705Myelodysplastic/myeloproliferative neoplasms (MDS/MPNs) are myeloid malignancies that display features of both MDS and MPN, but cannot be properly assigned to either MDS or MPN. It is currently not known whether it originates from the hematopoietic stem cell (HSC) compartment (like MDS), from a more committed myeloid progenitor population, or a combination thereof. Fifteen to 40% of MDS/MPN patients develop acute myeloid leukemia (AML); whether the transformation occurs in a particular cell population is also unknown. We previously demonstrated that mice heterozygous for the CREB binding protein gene (Crebbp) develop MDS/MPN at 9–12 months of age and ∼40% of them progress to develop a hematologic malignancy. Thus, Crebbp+/− mice are an excellent model to address the before mentioned questions, which is important for the development of better strategies to treat MDS/MPN.For this purpose, we harvested and combined bone marrow from 1.5-year old Crebbp+/− mice (10 donors per experiment, thereby ensuring that the marrow of ∼4 donors harbored malignant hematopoietic cells) and transplanted it into lethally irradiated, wild-type recipients. Groups of mice either received unfractionated whole bone marrow (WBM) or populations purified by fluorescence-activated cell sorting. Naive Crebbp+/− mice had demonstrated functional and/or quantitative abnormalities in long-term and short-term HSCs, common myeloid progenitors (CMPs) and granulocyte/macrophage progenitors (GMPs) and we therefore focused on these populations. All transplant recipients also received unfractionated wild-type “helper cells” to increase survival. Mice were closely monitored and those suspected of having developed a hematopoietic disease were sacrificed and their hematopoietic system analyzed. Four independent experiments were performed and data were combined for analysis. Among the 18 recipients who received Crebbp+/− WBM, 8 recipients (44%) developed an early-onset AML with myelofibrosis, 2–7 months after the transplant, which was not preceded by MDS. The other 10 recipients (56%) developed MDS/MPN, 12–18 months after the transplant. These mice displayed ineffective hematopoiesis, evidenced by a normocellular bone marrow, significant leukopenia, and trilineage dysplasia. One of these 10 Crebbp+/− WBM recipients that developed MDS/MPN subsequently progressed to AML. In contrast, none of the 15 recipients of Crebbp+/− HSCs (defined as Lin−Sca-1+c-Kit++ (LSK) cells, including long-term and short-term progenitors, as well as lymphoid-restricted progenitors) developed early-onset AML. Instead, 1 developed MDS/MPN while the remainder developed MDS by 11–17 months after the transplant, with one of them progressing to a disease resembling human mature T-cell leukemia. Transplantation of Crebbp+/− CMPs and GMPs also failed to cause early-onset AML and, as expected, gave rise to extremely low long-term reconstitution. Thus, these mice were mostly reconstituted by the co-transplanted wild-type “helper cells”. However, unexpectedly, 9 out of 24 (38%) showed <10% dysplastic cells in 1 or more lineages, while 4 (17%) developed overt MDS, i.e. >10% dysplastic cells and 2 (8%) developed MPD or AML with myelofibrosis. Control mice, i.e., recipients of wild-type BM cells remained healthy for the duration of the experiments.The results of these transplantation experiments show that in this mouse model, MDS/MPN is transplantable. However, it requires transplantation of WBM, since the transplantation of LSK cells resulted in MDS, suggesting that the microenvironment may play a crucial role in the etiology of MDS/MPN. This notion is in concordance with our previous study, demonstrating that Crebbp+/− mice transplanted with wild-type cells developed MPD that originated from the transplanted wild-type cells. This notion is further supported by the outcome of the CMP and GMP transplantation experiments, suggesting that abnormal myeloid progenitors are also important factors for MDS/MPN disease development. Moreover, malignant transformation seems to occur in a non-LSK cell that is more differentiated than CMPs and GMPs. Alternatively, malignant transformation requires all hematopoietic and non-hematopoietic cells to be present, again suggesting that MDS/MPN is a complex disease where both the hematopoietic compartment and its bone marrow microenvironment are affected. Disclosures:No relevant conflicts of interest to declare.

Cycling Marrow Stem Cells Are Lost with Purification.

Abstract 2308Hematopoietic stem cell biologists have amassed a tremendous depth of knowledge about the biology of the marrow stem cell over the past few decades, facilitating invaluable basic scientific and translational advances in the field. Most of the studies to date have focused on highly purified populations of marrow cells, with emphasis placed on the need to isolate increasingly restricted subsets of marrow cells within the larger population of resident bone marrow cells in order to get an accurate picture of the true stem cell phenotype. Such studies have led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to homogeneity. However, work from our laboratory, focusing on the stem cell potential in un-separated whole bone marrow (WBM), supports an alternate view of marrow stem cell biology in which a large population of marrow stem cells are actively cycling, continually changing phenotype with cell cycle transit, and therefore, cannot be purified to homogeneity. Our studies separating WBM into cell cycle-specific fractions using Hoechst 33342/Pyronin Y or exposing WBM to tritiated thymidine suicide followed by competitive engraftment into lethally irradiated mice revealed that over 50% of the long-term multi-lineage engraftment potential in un-separated marrow was due to cells in S/G2/M. This is in stark contrast to studies showing that highly purified stem cell populations such as LT-HSC (Lineage–c-kit+sca-1+flk2−) engraft predominantly when in G0. Additionally, by performing standard isolation of a highly purified population of stem cells, SLAM cells (Lineage–c-kit+sca-1+flk2−CD150+CD41−CD48−), and testing the engraftment potential of different cellular fractions created and routinely discarded during this purification process, we found that 90% of the potential engraftment capacity in WBM was lost during conventional SLAM cell purification. Incubation of the Lineage-positive and Lineage-negative fractions with tritiated thymidine, a DNA analogue which selectively kills cells traversing S-phase, led to dramatic reductions in long-term multi-lineage engraftment potential found within both cellular fractions (over 95% and 85% reduction, respectively). This indicates that the discarded population of stem cells during antibody-based stem cell purification is composed largely of cycling cells. In sum, these data strongly support that 1) whole bone marrow contains actively cycling stem cells capable of long-term multi-lineage engraftment, 2) these actively cycling marrow stem cells are lost during the standard stem cell purification strategies, and 3) the protean phenotype of actively cycling cells as they transit through cell cycle will render cycling marrow stem cells difficult to purify to homogeneity. Given the loss of a large pool of actively cycling HSC during standard stem cell isolation techniques, these data underscore the need to re-evaluate the total hematopoietic stem cell pool on a population level in addition to a clonal level in order to provide a more comprehensive study of HSC biology. Disclosures:No relevant conflicts of interest to declare.

Leukemic and Hematopoietic Stem Cells Compete for the Same Functional Marrow Niche in a MLL-AF9 Murine Leukemia Model

AbstractAbstract 4897Much is known regarding the location, cellular composition, signaling pathways, and functional role of the normal hematopoietic stem cell (HSC) niche in the bone marrow microenvironment. Microenvironmental cells including osteoblasts, other specialized mesenchymal cells, and vascular endothelial cells exert control over HSC self-renewal, differentiation, and engraftment. Niche occupancy appears to be competitive and limiting in terms of controlling the number of HSCs per organism. Leukemia stem cells (LSCs), through their inherent properties of quiescence and resistance to chemotherapeutic agents, are thought to be one of the principal mechanisms underlying disease relapse in patients. Much less is known regarding the interaction of LSCs and the marrow microenvironment. It is not clear whether LSCs localize to the same niches as HSCs, compete with HSCs for niche occupancy, or share dependence on niche signals, and whether those signals affect tumor responses to chemotherapy. Using a human pre-B ALL xenograft mouse model, Colmone et al (Science 2008) recently showed that leukemic cells may alter the normal microenvironment, resulting in initial homing of transplanted normal HSPCs in distinct atypical niches. Shiozawa et al (JCI 2011) showed that metastatic prostate cancer cells, a tumor type known to target bone, impeded HSC engraftment in a murine model, suggesting competition for the same niche.To investigate the relationship between HSC and LSC niche localization and functional occupancy, we used murine progenitor cells transduced with an MLL-AF9 vector expressing GFP in a murine syngeneic competitive transplantation model. MLL-AF9 cells are highly enriched for LSCs, particularly the c-kit+ compartment (Somervaille Cancer Cell 2006). We found that between approximately 21% and 24% of cells were c-kit+ by FACS in 2 separate experiments. In our model, mice transplanted with unsorted MLL-AF9 cells (1×107) died of AML with a latency of 11–14 days.We cotransplanted a fixed number of MLL-AF9-GFP cells (1×106) with increasing numbers of normal mouse whole bone marrow (WBM) cells, derived from dsRed transgenic mice to facilitate distinction from the GFP+ MLL-AF9 cells, into mice irradiated with 1000 rads: 1×105 [group 1], 1×106 [group 2], 1×107 [group 3], 5×107 [group 4]. Control groups received 1×105 and 1×106 normal WBM cells only. Survival was monitored daily. The control group receiving 1×105 cells only all died with median time to death of 16.5 days from lack of count recovery, those receiving 1×106 cells are still alive 35 days after transplant, indicating that 1×106 cells is adequate to rescue from irradiation. Mice were bled weekly until death and samples were analyzed by flow cytometry. Complete blood counts, blood smears, and splenic sections were obtained from these mice. As expected, there were no circulating blasts detected 7 days post transplant and all mice were healthy. However, 14 days after transplant the percentages of GFP+ leukemic cells detected in the blood were inversely proportional to the number of normal dsRed WBM cells transplanted (group 1 vs. group 2 vs. group 3 vs. group 4 mean percentage of GFP+ cells, 83.97 v 66.53 v 18.73 v 9.275 p< 0.0001). At day 15, mice from group 1, but not from groups 2 to 4, became moribund and were sacrificed. Spleens in this group were heavier than in those mice transplanted with 1×105 normal WBM cells alone and 2 out of 3 showed leucocytosis compared to leucopenia in all mice in the group transplanted with normal cells alone. When mice in the other groups had blood samples taken for analysis while moribund, GFP+ cells were greater than 80% suggesting that mice in group 1 died from complications relating to leukemic infiltration. Confocal microscopy confirmed the colocalization of normal HSPCs and MLL-AF9-GFP LSCs in the niche. Most interestingly, survival was proportional to the numbers of normal WBM cells transplanted, with a continuous delay in leukemic death proportional to the number of normal WBM cells cotransplanted with the same dose of MLL-AF9 cells (Figure 1). Hence, this murine model of leukemia suggests that normal and leukemic cells compete for the same functional niche, that manipulation of the niche could impact on response to anti-leukemic therapies, and that cell dose in the context of stem cell transplantation for leukemia may have an impact on outcome via niche competition. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Bone Marrow-Derived Progenitor Cells Augment Venous Remodeling in a Mouse Dorsal Skinfold Chamber Model

The delivery of bone marrow-derived cells (BMDCs) has been widely used to stimulate angiogenesis and arteriogenesis. We identified a progenitor-enriched subpopulation of BMDCs that is able to augment venular remodeling, a generally unexplored area in microvascular research. Two populations of BMDCs, whole bone marrow (WBM) and Lin−/Sca-1+ progenitor cells, were encapsulated in sodium alginate and delivered to a mouse dorsal skinfold chamber model. Upon observation that encapsulated Sca-1+ progenitor cells enhance venular remodeling, the cells and tissue were analyzed on structural and molecular levels. Venule walls were thickened and contained more nuclei after Sca-1+ progenitor cell delivery. In addition, progenitors expressed mRNA transcript levels of chemokine (C-X-C motif) ligand 2 (CXCL2) and interferon gamma (IFNγ) that are over 5-fold higher compared to WBM. Tissues that received progenitors expressed significantly higher protein levels of vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), and platelet derived growth factor-BB (PDGF-BB) compared to tissues that received an alginate control construct. Nine days following cell delivery, tissue from progenitor recipients contained 39% more CD45+ leukocytes, suggesting that these cells may enhance venular remodeling through the modulation of the local immune environment. Results show that different BMDC populations elicit different microvascular responses. In this model, Sca-1+ progenitor cell-derived CXCL2 and IFNγ may mediate venule enlargement via modulation of the local inflammatory environment.

A Fully Automated Method for Isolating Highly Enriched Population of Multiple Myeloma Plasma Cell From Whole Bone Marrow for Enhanced Sensitivity in Downstream Assays

Abstract 5064Introduction, Multiple myeloma (MM) is characterized by a huge clinical heterogeneity despite the homogenous morphologic appearance of malignant plasma cells (PCs). The advent of interphase fluorescence in situ hybridization (FISH) or MicroArrays (MA) allows an increased rate of aberration detection and identification of some recurrent cryptic changes, which have been increasingly implemented as additional diagnostic and prognostic factors. To heighten sensitivity of Single Nucleotide Polymorphism (SNP) arrays, or FISH it is necessary to have a purified population of cells as starting material. Screening must be performed systematically on the purified CD138+ PCs.After testing different systems for cell purification, we encountered some challenges. We didn't obtain enough PCs for FISH and SNP array studies. This was due to excess M-protein accumulating in the blood stream, increasing hyper viscosity and also due to the morphology and size variations of PCs at various stages of differentiation. Additionally, downstream DNA extraction can be a challenge since EDTA found in most buffers is an inhibitor for chemical PCR reaction for some MA chips.Given the challenges, CERBA laboratory and Miltenyi Biotec GmbH have developed a fully automated process (FAP) for purification for CD138+ PCs. In a study of 100 BM patient samples, we compared the specificity, efficiency, performance, purity, ease of use, technologists’ time and the quality of DNA after CD138+ PCs purification. Two methods were compared. In the first method, cells were directly purified from bone marrow samples by FAP using Automated Magnetic Cell Sorter (AMCS). In the second method, mononuclear cells from fresh whole bone marrow (WBM) were enriched by Ficoll, followed by cell selection procedure with anti-CD138+ MicroBeads using the AutoMACS®. Before separation and following the separation, the percentage of PCs was determined by Flow cytometry (FC) on WBM by multiparameter FC (MFC) for CD138/CD38 expression. Additionally, DNA quality on separated cells was assessed by Nanodrop. A fraction of the CD138+ PCs were used after hypotonic shock and Carnoy fixation, applied to glass slides for FISH application and another fraction for DNA extraction for MA (SNP.6 Affymetrix®) FISH was performed with the recommended unbalanced alterations & reciprocal rearrangements: del(13) (q14)(D13S25), del(17)(p13)(TP53),+3(D3Z), +9(D9Z1), +15(D15Z14), t(4;14)(p16;q32)/IGH-FGFR3.Results, the specificity and purity were the same for both process but the efficiency and performance were considerably better for FAP than mononuclear cells enriched by Ficoll (MCEFicoll) process. With FAP, in 95% of the MM cases we obtained enough PCs for performance of the recommended panel of FISH and for 50% of them we could extract DNA for SNP array. For the MCEFicoll, we observed inferior performance, with very few plasma cells after isolation. Having enough PSc for only 65% of the cases and we could only extract DNA for 28% of them. The quality of DNA was the same for both process and the technologists' time was longer by 30' /patient for MCEFicoll process than for FAP.Currently in CERBA lab, we realize more than 20 plasma cells isolation per week for patients with MM and from October 2007 to July 2011 we have separated more than 5.000 specimens using CD138 Whole Blood MicroBeads (CD 138 WBMB) from Miltenyi Biotec, in combination with the AMCS. This has allowed isolation directly from WBM without any sample preparation required, such as density gradient centrifugation (ficoll) or erythrocyte lysis.The detection rate of chromosomal abnormalities and the number of abnormalities per case in MM and PCs dyscrasia significantly improves when there are enough CD138+PCs for analysis.Conclusion, in this report we describe the benefits of fully automated isolations of CD138+ cells from WBM. We have developed an SOP for an automated reliable and standardized method which allows the processing of multiple samples in a single day, while maintaining sample integrity and increasing sensitivity of FISH analysis and WG arrays for a diagnosis lab. Disclosures:No relevant conflicts of interest to declare.

Pak A Proteins Are Essential for Hematopoietic Stem/Progenitor Cell (HSC/P) Engraftment Through Regulation of Signaling Pathways Controlling HSC/P Proliferation, Survival, Migration, and the Actin Cytoskeleton

Abstract 917Rho GTPases, including Rac, integrate multiple extracellular signals and play important regulatory roles in HSC/P functions such as engraftment, retention, migration, adhesion, proliferation, and survival (Gu et al. Science, 2003). Our studies now focus on identifying potential Rac downstream effector proteins important for normal HSC/P function(s). The p21-activated kinases (Pak) are serine/threonine kinases that interact with and are major downstream targets of Rac and Cdc42. There are six human Paks (Pak1–6), which are grouped based on homology into Group A (Pak 1–3) and Group B (4–6) Paks. Paks regulate cytoskeletal organization including stress fiber dissolution, lamellipodia formation and focal adhesion disassembly and mediate activation of MAPK pathways. To identify the possible role(s) of Pak proteins in engraftment, freshly isolated LSK cells from WT (CD45.1+/CD45.2+) BM were transduced with retrovirus containing the Pak Inhibitory Domain (PID), which inhibits Group A Pak protein function or empty vector control (Mieg3); both constructs co-express GFP. 1.0×105 GFP+ LSK+ cells were then isolated and co-transplanted with 5.0×105 BoyJ (CD45.1+) whole bone marrow (WBM) into lethally irradiated C57Bl/6J (CD45.2+) recipients. Percent chimerism was measured at 3- to 24- weeks post BMT. PID transduced LSK+ cells were incapable of contributing to recipient hematopoietic reconstitution (Table 1). To explore the underlying mechanism of this engraftment failure we performed in vivo homing assays and found a 4- and 16- fold decrease, respectively, in BM homing of PID transduced LSK+ vs controls at 12 and 48 hours (p<0.05, for both time points). Altered cell migration of LSK+ cells was confirmed by live imaging microscopy which showed a 4-fold decrease in overall cell displacement in SDF-1-stimulated directed migration in the PID-expressing LSK+ compared to controls and was associated with a two-fold increase in random cell migration of PID-transduced LSK+ cells in transwell migration assays. PID-expressing LSK+ cells also demonstrated abnormal lamellipodia associated with significant increases in both cell surface area and cell perimeter. Because cytoskeletal changes may be linked to alterations in cell growth, we next examined the effect of Pak inhibition on cell survival and proliferation. PID-expressing LSK+ cells had decreased proliferation (17.7% vs 36.8% of cells in S-phase, p<0.05) and increased apoptosis (48.1% vs 16.7% AnnexinV+ cells, p<0.05) when compared to controls, respectively. These phenotypic changes were associated with decreased pERK and pAKT in PID-expressing LSK+. To confirm the importance of Pak activation of these proteins in HSC/P, we performed experiments to rescue the observed engraftment defect by co-transducing PID or Mieg3 with a constitutively active-ERK (ca-MEK1) or ca-AKT. We found ca-MEK1, but not ca-AKT, was able to increase proliferation in vitro (% proliferating cells for PID + empty vector = 6.1% and PID+ ca-MEK1 = 9.5%; p<0.05) and partially but only transiently rescue Pak-deficient HSC/P engraftment (% donor cells for LSK+ transduced with: PID + empty vector =1.5%, PID + ca-MEK1 =15.8%, and PID + ca-AKT =0.5% at 3-weeks post-BMT; p<0.05 for empty vector vs ca-MEK1). Finally, to determine which PakA pathway is critical in HSC engraftment we studied Pak genetic knock-out cells. We found that Pak2Δ/Δ -but not Pak1−/− -cells resulted in a profound HSC/P engraftment defect (% Pak2Δ/Δ vs Pak2flox/flox and Pak1−/− vs Pak1wt/wt: 1.0% vs 26.5% and 35.8% vs 37.4%; p<0.05 and p=ns, respectively at 3-weeks). Taken together, these data suggest that Pak A proteins regulate multiple HSC/P functions and link Rac GTPases to actin cytoskeletal rearrangements, directed cell migration, and proliferation/survival of HSC/P during engraftment.TABLE 1:Percentage of GFP+ cells in peripheral blood of recipient mice at indicated time points post BMT3-weeks6-weeks10-weeks14-weeks24-weeksWT-Mieg331.6% (±6.69)27.1% (±8.6)34.6% (±15.0)43.2% (±16.3)22.2% (±10.7)WT-PID0.22 (±0.19)0.07% (0.06)0%0%0%**Data represent mean ± s.d., n=10 recipients per group, p<0.05 for all time points, two independent experiments. Disclosures:No relevant conflicts of interest to declare.

Characterization of Clonotypic B Cell Progenitors in Bone Marrow of Multiple Myeloma Patients,

Abstract 4002The lack of specific molecules to define malignant B progenitor cells in multiple myeloma (MM) has hampered the evaluation of minimal residual disease (MRD). We have identified a bone marrow (BM) CD138- subset that co-express CD19+ with identical κ or λ light chain (LC) restriction as the abnormal plasma cell (PC), as previously shown by others. The majority of LC restricted (LCR) B progenitors are CD19+/CD34- and <0.5% of whole BM (WBM) cells exhibit an immature phenotype: CD19+/CD34+ with aberrant CD27 expression. Immature B cell precursors are undetectable in peripheral blood (PB). LCR CD138-/CD19+ cells represent only 0.72± 0.5% of WBM in newly diagnosed patients (n=23) and do not increase (0.47± 0.51%) in patients with relapsed disease (n=21). The κ/λ LC ratio is 1.46±0.6 regardless of disease stage suggesting that conventional LC ratios for PCs (> 4 or <0.5) may not apply in B progenitors. LCR B progenitors (CD19+/34+ or CD19+/34-) are CD117+, Notch+ and Survivin+ as MM patient’s hematopoietic stem cells (HSC). ALDH enzymatic activity is 3.1% (0.1-–7.26%) in LCR B cells. Flow sorted CD138+ did not grow in a colony formation assay (methylcellulose with PHA-LCM), whereas CD19+/CD34- or CD19+/CD34+ grew colonies with efficiency of 1:25,000 or 1:10000 respectively. Cells harvested from colonies have a lympho-plasmacytoid appearance and LCR B progenitors differentiated into CD138+ PC (80±5%) compared to HSC (10±5%). Colony efficiency was optimized (3 fold) using conditioned medium (CM) from HS5-stroma. Isolated CD138-/CD19+ cells were relatively bortezomib and melphalan resistant compared to CD138+ PC. We hypothesize that CD138-/CD19+/CD34+ cells contains earlier progenitor B cells that differentiate into the malignant PC. Surrogate assays for stem cell activity and xenotransplant models should determine cancer stem cell activity of immature B cell precursors. Research studies of MM putative progenitor cells will allow developing novel treatments to eradicate potential MM MRD reservoir. Disclosures:Baz:Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding.

Unique populations of lung mast cells are required for antigen‐mediated bronchoconstriction

Studies in both human and mouse indicate that mediators released by mast cells can lead to bronchoconstriction, and thus these are important effector cells in lifethreatening anaphylaxis. Much of our understanding of the various functions of mast cells emanates from the study of mice lacking these cells, particularly mice carrying mutations in the tyrosine kinase gene Kit. Definitive evidence for the role of mast cells in the altered immune response requires the demonstration that this response can be normalized by reconstitution of the mice with cultured bone marrow-derived mast cells (BMMCs). While many mast cell niches can be restored with BMMCs, this has not been demonstrated for mast cells present in the airways of the lung, cells poised to mediate bronchoconstriction during allergic responses. To determine if mast cell-deficient Kit(Wsh/Wsh) reconstituted lines are an appropriate model for the study of the role of these cells in bronchoconstriction associated with allergic responses. Kit(Wsh/Wsh) mice were reconstituted with either whole bone marrow (WBM) or BMMCs and responses to IgE-mediated mast cell activation were determined; including systemic hypothermia, mediator release, and bronchoconstriction in anaesthetized, mechanically ventilated animals. Engraftment of Kit(Wsh/Wsh) mice with WBM and BMMCs results in reconstitution of the central airways with mast cells. While the treatment of the two groups of animals resulted in systemic changes when challenged with IgE/Ag in a model of passive anaphylaxis, bronchoconstriction was observed only in kit(Wsh/Wsh) animals, which had received a bone marrow transplant. While BMMCs can populate the lung, they cannot restore IgE/Ag-mediated bronchoconstriction to mast cell-deficient animals. This suggests that the mast cell population, which mediates this function, may be unique, and to fill this niche in the lung cells must undergo a specific developmental programme, one that is no longer available to cultured mast cells.

Unique populations of lung mast cells are required for antigen-mediated bronchoconstriction (86.11)

Abstract Several studies implicate mast cells as important effector cells of anaphylaxis. Much of our understanding of the functions of mast cells emanates from the study of mice lacking theses cells, particularly mice carrying mutations in c-Kit. Definitive evidence for the role of mast cells in these mice requires the normalization of altered immune responses by reconstitution with cultured mast cells (BMMC). While many niches can be restored with BMMC, this has not been demonstrated for mast cells present in the airways of the lung, cells poised to mediate bronchoconstriction during allergic responses. To determine if mast cell reconstitution is an appropriate model for the study of bronchoconstriction associated with allergic responses, KitWsh/Wsh mice were engrafted with either whole bone marrow (WBM) or BMMC and responses to IgE-mediated anaphylaxis determined. Engraftment of KitWsh/Wsh mice with WBM or BMMC results in reconstitution of the central airways with mast cells. While the treatment of both groups results similar systemic changes in a model of passive anaphylaxis, bronchoconstriction was observed only in animals which received WBM. While BMMC can populate the lung, they cannot restore IgE/Ag-mediated bronchoconstriction to mast cell-deficient animals. This suggests the mast cell population mediating this function may be unique, and to fill this niche in the lung cells must undergo a specific developmental program that is no longer available to cultured mast cells.

Genome-Wide Chip-Seq Analysis of Histone Methylation Reveals Modulators of NF-κB Signaling and the Histone Demethylase JMJD3 as Implicated in Disease Progression in Myelodysplastic Syndrome (MDS).

Abstract 291Although cytogenetic abnormalities are common in MDS, search for genetic alterations has been less informative with few prevalent abnormalities thus far known. To identify genes aberrantly activated in MDS, we developed a novel approach based on chromatin immuno-precipitation combined with massive parallel sequencing (CHIP-Seq) using the Solexa 1G sequencing technology. To our knowledge this is the first example of the use of this technology in primary human samples. For CHIP analysis we used an antibody against H3K4me3 (histone-H3-lysine 4-trimethylation). H3K4me3 is a chromatin mark of gene activation that localizes to active gene promoter regions. CHIP-Seq was performed in CD34+, CD34 neg cells and whole bone marrow (WBM) from 6 patients with MDS and 4 normal controls. In total 30 samples were sequenced. Patients samples were obtained at the time of initial referral at MDACC and were sorted immediately using standard separation procedures. When compared to normal controls for each cellular compartment, we identified 36, 156 and 32 potential active gene promoters associated with H3K4me3 in CD34+, CD34 neg cells and WBM respectively. Of importance, gene promoter regions identified did not overlap among the different cellular compartments analyzed (differences were observed comparing normal vs MDS but also among different MDS compartments), indicating that chromatin structure and gene expression profiles are aberrant and distinct in non-CD34+ cells that may also contribute to the pathobiology of MDS. Here we focus on H3K4me3-associated gene promoters in CD34+ cells. To confirm the results obtained with the CHIP-seq approach, we studied the expression levels of the top 9 CHIP-Seq identified genes in an independent cohort of in CD34+ cells obtained from 54 MDS at the time of initial diagnosis. Patient characteristics have been previously reported (Leukemia, in press): 11 (20%) low risk, 20 (37%) int-1, 15 (27%) int-2 and 8 (14%) high risk by IPSS. We confirmed gene expression up-regulation of 7 (C5AR1, FPR1, FPR2, AQ9, FYB, FCAR, IL8RA) of 9 genes detected by CHIP-Seq. Using Ingenuity Pathway Analysis of the 36 genes identified in CD34+ cells revealed NF-κB as central activated knot in CD34+ cells. This was confirmed by phospho-p65 immuno-staining in primary cells. Furthermore up-regulation of all 10 NF-κB activation associated genes was confirmed in MDS CD34+ cells by Q-RT-PCR. Transfection of OCI-AML3 cells with a siRNAs cocktail targeting 4 of the CD34+ NF-κB activation genes dramatically repressed NF-κB activation as well as expression and promoter NF-κB association of JMJD3 gene, a known NF-κB transcriptional target. JMJD3 encodes a Jmjc-domain K27me3 demethylase, which positively regulates H3K4me3. We further characterized expression levels of 17 known histone demethylases known in human in 35 patients with MDS and identified JMJD3 as the only histone demethylase overexpressed in MDS CD34+ cells. siRNA targeting JMJD3 reduced expression and promoter H3K4me3 levels of several CHIP-Seq detected MDS- CD34+-NF-κB activation genes. Finally expression profile of JMJD3 and the panel CD34+-NF-κB activation genes in the 54 patients with MDS indicated that expression levels were consistently overexpressed in patients with higher-risk (high and int-2) disease compared to patients with lower (low and int-1) risk disease. In view of the known antiapoptotic and proliferative role of the NF-κB pathway, this data indicates that expression of upstream and downstream modulators of NF-κB signaling, regulated at the chromatin level by JMJD3, have a role in MDS progression and could serve as therapeutic targets. Through this novel in vivo CHIP-Seq analysis, we demonstrated that a positive regulatory loop exists in MDS CD34+ cells. This loop contains JMJD3 promoted gene activation through positive regulation of H3K4me3, which leads to NF-κB signaling activation, and then further promotion of JMJD3 expression and activation of the whole signaling cascade. Our study also demonstrates that in vivo CHIP-Seq can be used to discover disease specific targets. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Transplant Induces Pulmonary Vascular Remodeling in Mice.

Abstract 4480 ObjectivePulmonary complications are common in bone marrow transplant (BMT) recipients and often of an infectious etiology or attributed to intensive conditioning regimens. Cases of pulmonary arterial hypertension have been described in the setting of autologous BMT but are believed to be the result of acquired infections or previously-administered chemotherapy. Whether transplanted marrow cells themselves are toxic to the recipient's lungs is unknown. MethodsTo address this, non-irradiated female BALB/C mice were transplanted with 5-6×107 whole bone marrow (WBM) cells from male BALB/C mice daily for four days on week zero, then again on week eight (4.5×108 cells total/mouse), or an equal volume of diluent (control). On week 24, transplanted mice received 1000 cGy of chest-only radiation or no radiation. On week 32, histochemical and immunohistochemical analyses were performed on recipient's lungs. ResultsBone marrow chimerism was not significantly different in the irradiated and non-irradiated cohorts at the time of sacrifice (average 45.11+6.25% Y chromosome+, all mice). Recipient lungs contained few non-hematopoietic donor marrow-derived cells. These cells were exclusively epithelial (Y+/cytokeratin+), primarily type II pneumocytes (Y+/prosurfactant C+), while no endothelial (Y+/von Willebrand+) or vascular smooth muscle (Y+/alpha-actin+) cells were identified. Irradiated and non-irradiated cohorts had similar quantities of these cells (0.80+0.22% vs. 0.51+0.08% Y+/cytokeratin+; 0.37+0.08% vs. 0.32+0.12% Y+/prosurfactant C+, p>0.2). Pulmonary vessel wall thickness-to-blood vessel diameter ratios (PVWT/D) were significantly increased in the non-irradiated cohort compared with controls and these ratios were further increased in the irradiated cohort (141+5.75%, 161+5.34% of control, p<0.05 comparing all groups). ConclusionsThese data indicated that marrow cell infusion alone results in pulmonary vascular remodeling and this effect is augmented by radiation injury. These changes are independent of transplanted marrow cell conversion to pulmonary vascular endothelial, smooth muscle cells. These studies suggest that transplanted cells may be lung toxic entities in the setting clinical BMT. Disclosures:No relevant conflicts of interest to declare.

Marrow Cell Infusion Attenuates Vascular Remodeling in a Murine Model of Monocrotaline-Induced Pulmonary Hypertension

There have been reports of marrow cells converting into pulmonary epithelial cells after marrow transplantation in irradiated mice. We evaluated the impact of whole bone marrow (WBM) infusion in mice, with or without total body irradiation (TBI), treated with saline or monocrotaline (MCT), which induces pulmonary hypertension (PH). C57BL/6 mice were injected with MCT or saline weekly for 4 weeks. Cohorts were then infused with saline vehicle (vehicle) or WBM from C57BL/-Tg(UBC-GFP)30Scha/J mice, with or without previous TBI (WBM or WBM/TBI). Four weeks later, right ventricular peak pressures (RVPP), right ventricular free wall-to-body weight ratios (RV/BW), and pulmonary vessel wall thickness-to-blood vessel diameter ratios (PVWT/D) were determined. WBM infusion and WBM following TBI induced increases in RVPP and RV/BW in saline-treated mice, while only TBI-exposed mice showed additional increases in PVWT/D. MCT increased RVPP, RV/BW, and PVWT/D in mice given vehicle or WBM alone, but not in mice given WBM/TBI. RVPP and RV/BW were not significantly lower in MCT mice given WBM/TBI than in MCT mice treated with vehicle, but MCT-treated mice given WBM or TBI/WBM had significantly lower PVWT/D compared to MCT-treated mice given saline vehicle. No donor WBM-derived pulmonary vascular cells were detected, suggesting that the observed effects of WBM infusion may be due to paracrine effects separate from cell conversions. The observation of PH after marrow infusion suggests an additional mechanism for lung toxicity seen in marrow transplantation. In conclusion, WBM alone appears to increase RVPP and RV/BW in normal mice but the combination of WBM and TBI attenuates MCT-induced PH.

Angiogenesis induced by autologous whole bone marrow stem cells transplantation.

It has been presumed that unknown cells and growth factors in bone marrow might promote angiogenesis, so angiogenesis effect could be enhanced by autologous whole bone marrow (WBM) stem cell transplantation. We compared capillary ratio induced by autologous WBM and bone marrow-mononuclear cells (BM-MNCs) to evaluate the anigiogenic effect of auotologous WBM. In addition, the combined effect of WBM transplantation and granulocyte colony-stimulating factor (G-CSF) injection was examined in an ischemic canine model. After creating ischemic limb model, autologous WBM and isolated BM-MNCs were transplanted into the ischemic muscle. In other experiments, autologous WBM with recombinant human G-CSF (rhG-CSF) and autologous WBM without rhG-CSF were transplanted into the ischemic muscle. In this study, normal saline was injected into the contralateral sites in each ischemic model as a control group. After 8 weeks of transplantation, angiography and muscle harvest were performed, and then the anigiographic findings and capillary density, as assessed by immunohistochemical staining, were investigated and analyzed. In comparison with the control group, BM-MNCs and WBM transplantation groups showed higher ratios of the capillary density (1.5±0.01 times, p<0.001 and 1.6±0.15 times, p=0.005, respectively). Between the BM-MNCs and WBM transplantation groups, the capillary ratio was 1.2 folds higher in the WBM group than that in the BM-MNCs group, but there was no significantly different (p=0.116). The angiogensis ratios of both the WBM without G-CSF group and the WBM with G-CSF groups were higher (1.6±0.15 times, p=0.004 and 1.8 ±0.01 times, p=0.005, respectively) than that of the control groups. In comparison with the WBM without G-CSF group, the WBM with G-CSF transplantation group revealed a 1.1 folds higher angiogenesis ratio, but there was no statistically significant difference (p=0.095). Autologous WBM transplantation is a simpler method and it is not inferior for inducing therapeutic angiogenesis as compared with isolated BM-MNCs transplantation. In addition to autologous WBM transplantation, intravenous G-CSF injection enhances the angiogenic effect of autologous WBM in an ischemic limb.

Tissue-Specific Gene Expression of Marrow Cells Co-Cultured with Various Murine Organs

We have previously demonstrated that murine bone marrow cells co-cultured with lung opposite a cell-impermeable membrane express high levels of lung cell-specific genes (Aliotta et al, Stem Cells , 2007; 25(9):2245–56). Greater changes in gene expression were noted in marrow cells co-cultured with radiation-injured lung. A factor smaller than the pore size of the cell-impermeable membrane (0.4μm) is responsible for these changes as cell-free conditioned media (CM) had a similar effect on co-cultured marrow cells. Lung-derived microvesicles were found to enter marrow cells in culture and may be among the factors responsible for these phenotypic changes. We wished to determine if these observations could be generalized to co-cultures using other murine organs and whether these changes in gene expression are tissue-specific. Whole bone marrow (WBM) cells were isolated from C57BL/6 mice and co-cultured with lung, liver, heart and WBM cells from C57BL/6 mice exposed to 1200 centigrey (cGy) of total body irradiation (TBI) or no radiation. Control WBM cells were co-cultured with no tissue (control). Co-cultured WBM cells were analyzed 7 or 14 days later by Real Time RT-PCR and fold difference in target gene expression was determined (relative to control cells). In addition, cell-free CM made from the same organs was co-cultured for 7 or 14 days with WBM cells which were then analyzed by RT-PCR. Alternatively, CM was analyzed for the presence of microvesicles by electron microscopy (EM) of ultracentrifuged (UCF) pelleted material. WBM co-cultured only with lung had increased gene expression of surfactant proteins A (Sp-A) and C (Sp-C, 89 and 334-fold increase vs. control, respectively) whereas WBM co-cultured only with brain had increased gene expression of Glial Fibrillary Acidic Protein (GFAP, 4.6-fold increase vs. control). Slight increases in Albumin expression were seen in all co-culture groups but expression was markedly elevated in WBM co-cultured with liver (162,657-fold increase vs. control). Expression of heart-specific markers, including Troponin I and T2, was seen in WBM co-cultured with heart but these levels were not significantly different from those of other co-culture groups. Radiation injury augmented expression of certain genes in co-cultured WBM, including Sp-A (1019 vs. 89-fold increase) in lung co-cultures and GFAP (24 vs. 4.6-fold increase) in brain co-cultures. WBM co-cultured with CM from all organs demonstrated similar changes in gene expression. In addition, pelleted material from UCF CM contained RNA that was specific to the tissue from which the CM was made. EM of UCF CM demonstrated numerous membranebound particles 50–200nm in size that were typical of microvesicles in appearance. These data suggest that changes seen in gene expression of co-cultured WBM are largely tissue-specific, depending on the tissue they are co-cultured with. Microvesicles released by various tissues in co-culture may be among the mediators responsible for the changes seen in WBM gene expression.

Actinomycin D Blocks Transcription of Surfactant B mRNA in Marrow Cells Co-Cultured with Lung

We have previously described that RNA-filled microvesicles are shed from lung cells and are capable of entering whole bone marrow (WBM) cells in culture (Aliotta et al, Stem Cells , 2007; 25(9):2245–56). WBM cells cultured with these lung-derived microvesicles appear to express lung cell-specific genes after 7 days in culture. However, it is not known if the lung cell-specific mRNA detected in these cells represents de novo transcription or previously transcribed mRNA carried into the cell via microvesicles. To address this issue, we co-cultured WBM cells extracted from C57BL/6 mice and co-cultured them opposite lung from C57BL/6 mice exposed to 500 centigrey (cGy) of total body irradiation (TBI) or no radiation. The two cell types were separated by a cell-impermeable membrane (pore size, 0.4μm). After 7 days of co-culture, WBM cells were removed, extensively washed and cultured alone for an additional 36 hours, with or without the transcriptional blocking agent Actinomycin-D (Act-D, 10 ug/ml). Control WBM cells were co-cultured without lung and not exposed to Act-D (control). WBM cells were then analyzed for the presence of a variety of hematopoietic and lung-specific markers by Real Time RT-PCR and fold difference in target gene expression was determined (relative to control cells). WBM cells co-cultured with or without lung expressed high levels of hematopoietic markers including Sca-1, c-kit and CD45. This expression was nearly or completely eliminated after exposure to Act-D in culture. Expression of the surfactant protein B (Sp-B) gene was also significantly decreased in WBM co-cultured with lung (irradiated or non-irradiated) after exposure to Act-D (mean ± SE, from 16±4.9 to 1.1±0.4-fold increase vs. control, p=0.012). Interestingly, exposure to Act-D resulted in an increase in the expression of other lung-specific genes, including surfactant protein C (Sp-C) and clara cell specific protein (CCSP), in WBM co-cultured with lung (from 165±103 to 3,700±1,625 and 2±0.9 to 93±38-fold increases vs. control, p<0.015). In separate experiments, WBM cells co-cultured with cell-free lung conditioned media (CM) that was pre-treated with RNase had nearly 70% lower Sp-B expression compared with WBM cells co-cultured with untreated cell-free CM. These data suggest that lung cells release a transcriptional factor that induces marrow cell expression of the Sp-B gene in culture. This transcriptional factor is RNase-sensitive and could potentially represent microRNA transferred to marrow cells via lung-derived microvesicles. The increase in expression of other lung-specific genes after Act-D exposure suggests alternative transcriptional control mechanisms for lung-specific genes other than Sp-B.