Identification of circulating miRNAs as fracture-related biomarkers.

Fracture non-unions affect many patients worldwide, however, known risk factors alone do not predict individual risk. The identification of novel biomarkers is crucial for early diagnosis and timely patient treatment. This study focused on the identification of microRNA (miRNA) related to the process of fracture healing. Serum of fracture patients and healthy volunteers was screened by RNA sequencing to identify differentially expressed miRNA at various times after injury. The results were correlated to miRNA in the conditioned medium of human bone marrow mesenchymal stromal cells (BMSCs) during in vitro osteogenic differentiation. hsa-miR-1246, hsa-miR-335-5p, and miR-193a-5p were identified both in vitro and in fracture patients and their functional role in direct BMSC osteogenic differentiation was assessed. The results showed no influence of the downregulation of the three miRNAs during in vitro osteogenesis. However, miR-1246 may be involved in cell proliferation and recruitment of progenitor cells. Further studies should be performed to assess the role of these miRNA in other processes relevant to fracture healing.

Venous injection of a triphasic calcium-based implant in a sheep model of pulmonary embolism demonstrates minimal acute systemic effects.

PURPOSE: Implant leakage is the most common complication of vertebral augmentation. Alternative injectable materials must demonstrate intravascular safety comparable to or better than polymethyl methacrylate (PMMA). This study assessed the systemic effects of a triphasic calcium-based implant or PMMA injected directly into the femoral vein in a large animal model designed to mimic severe intravascular implant leakage. METHODS: Six skeletally mature female sheep were randomly assigned (n = 3) to either the PMMA or the triphasic implant (AGN1, composition: calcium sulfate, ß-tricalcium phosphate, brushite) treatment group. Femoral veins of each sheep were directly injected with 0.5 mL of implant material to mimic leakage volumes reported during PMMA vertebroplasty. To compare acute systemic effects of the materials, cardiovascular parameters, laboratory coagulation markers, and calcium and sulfate serum levels were monitored for 60 min after implant injection. Thrombotic and embolic events were evaluated by radiologic imaging, necropsy, and histopathology. RESULTS: Heart rate, systemic arterial blood pressure, arterial oxygenation, arterial carbon dioxide content, and coagulation markers remained within physiological range after either AGN1 or PMMA injection. No blood flow interruption in the larger pulmonary vessels was observed in either group. Lung histopathology revealed that the severity of thrombotic changes after AGN1 injection was minimal to slight, while changes after PMMA injection were minimal to massive. CONCLUSION: Acute systemic effects of intravascular AGN1 appeared to be comparable to or less than that of intravascular PMMA. Furthermore, in this preliminary study, the severity and incidence of pulmonary histological changes were lower for AGN1 compared to PMMA.

In Vitro Evaluation of a Nanoparticle-Based mRNA Delivery System for Cells in the Joint.

Biodegradable and bioresponsive polymer-based nanoparticles (NPs) can be used for oligonucleotide delivery, making them a promising candidate for mRNA-based therapeutics. In this study, we evaluated and optimized the efficiency of a cationic, hyperbranched poly(amidoamine)s-based nanoparticle system to deliver tdTomato mRNA to primary human bone marrow stromal cells (hBMSC), human synovial derived stem cells (hSDSC), bovine chondrocytes (bCH), and rat tendon derived stem/progenitor cells (rTDSPC). Transfection efficiencies varied among the cell types tested (bCH 28.4% ± 22.87, rTDSPC 18.13% ± 12.07, hBMSC 18.23% ± 14.80, hSDSC 26.63% ± 8.81) and while an increase of NPs with a constant amount of mRNA generally improved the transfection efficiency, an increase of the mRNA loading ratio (2:50, 4:50, or 6:50 w/w mRNA:NPs) had no impact. However, metabolic activity of bCHs and rTDSPCs was significantly reduced when using higher amounts of NPs, indicating a dose-dependent cytotoxic response. Finally, we demonstrate the feasibility of transfecting extracellular matrix-rich 3D cell culture constructs using the nanoparticle system, making it a promising transfection strategy for musculoskeletal tissues that exhibit a complex, dense extracellular matrix.

Differential Regulation of circRNA, miRNA, and piRNA during Early Osteogenic and Chondrogenic Differentiation of Human Mesenchymal Stromal Cells.

The goal of the present study is to identify the differential expression of circular RNA (circRNA), miRNA, and piwi-interacting RNA (piRNA) after lineage commitment towards osteo- and chondrogenesis of human bone marrow mesenchymal stromal cells (hMSCs). The cells were maintained for 7 days in either osteogenic or chondrogenic medium. RNA sequencing was performed to assess the expression of miRNA and piRNA, while RNA hybridization arrays were used to identify which circRNA were differentially expressed. qPCR validation of a selection of targets for both osteogenic and chondrogenic differentiation was carried out. The differential expression of several circRNA, miRNA, and piRNA was identified and validated. The expression of total and circular isoforms of FKBP5 was upregulated both in osteo- and chondrogenesis and it was influenced by the presence of dexamethasone. ZEB1, FADS2, and SMYD3 were also identified as regulated in differentiation and/or by dexamethasone. In conclusion, we have identified a set of different non-coding RNAs that are differentially regulated in early osteogenic and chondrogenic differentiation, paving the way for further investigation to understand how dexamethasone controls the expression of those genes and what their function is in MSC differentiation.

Mesenchymal Stem Cell Homing Into Intervertebral Discs Enhances the Tie2-positive Progenitor Cell Population, Prevents Cell Death, and Induces a Proliferative Response.

STUDY DESIGN: Experimental study with human mesenchymal stem cells (MSCs) and intervertebral disc (IVD) tissue samples. OBJECTIVE: This study aimed to characterize the effect of MSC homing on the Tie2-positive IVD progenitor cell population, IVD cell survival, and proliferation. SUMMARY OF BACKGROUND DATA: Homing of human MSCs has been described as potential alternative to MSC injection, aiming to enhance the regenerative capacity of the IVD. IVD cells expressing Tie2 (also known as CD202b or Angiopoietin-1 receptor TEK tyrosine kinase) represent a progenitor cell population with discogenic differentiation potential. However, the fraction of Tie2-positive progenitor cells decreases with aging and degree of IVD degeneration, resulting in a potential loss of the IVD's regenerative capacity. METHODS: Human MSCs, isolated from vertebral bone marrow aspirates, were labeled and seeded onto the endplate of bovine IVDs and human IVD tissue. Following MSC migration for 5 days, IVD cells were isolated by tissue digestion. The fractions of Tie2-positive, dead, apoptotic, and proliferative IVD cells were evaluated by flow cytometry and compared to untreated IVDs. For human IVDs, 3 groups were investigated: nondegenerated (organ donors), IVDs of patients suffering from spinal trauma, and degenerative IVD tissue samples. RESULTS: MSC homing enhanced the fraction of Tie2-positive IVD cells in bovine and human IVD samples. Furthermore, a proliferative response and lower fraction of dead cells were observed after MSC homing in both bovine and human IVD tissues. CONCLUSION: Our findings indicate that MSC homing enhances the survival and regenerative capability of IVD cells, which may be mediated by intercellular communication. MSC homing could represent a potential treatment strategy to prevent the onset of the degenerative cascade in IVDs at risk such as IVDs adjacent to a fused segment or IVDs after herniation. LEVEL OF EVIDENCE: N/A.

Phenotypic Characterization of Bone Marrow Mononuclear Cells and Derived Stromal Cell Populations from Human Iliac Crest, Vertebral Body and Femoral Head.

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14-91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45-CD34-CD73+, CD45-CD34-CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head.

Shear and Dynamic Compression Modulates the Inflammatory Phenotype of Human Monocytes in vitro.

Monocytes and their derived macrophages are found at the site of remodeling tissue, such as fracture hematoma, that is exposed to mechanical forces and have been previously implicated in the reparative response. However, the mechanoresponsive of monocytes and macrophages to skeletal tissue-associated mechanical forces and their subsequent contribution to skeletal repair remains unclear. The aim of this study was to investigate the potential of skeletal tissue-associated loading conditions to modulate human monocyte activation and phenotype. Primary human monocytes or the human monocyte reporter cell line, THP1-Blue, were encapsulated in agarose and exposed to a combination of shear and compressive loading for 1 h a day for 3 consecutive days. Exposure of monocytes to mechanical loading conditions increased their pro-inflammatory gene and protein expression. Exposure of undifferentiated monocytes to mechanical loading conditions significantly upregulated gene expression levels of interleukin(IL)-6 and IL-8 compared to free swelling controls. Additionally, multiaxial loading of unstimulated monocytes resulted in increased protein secretion of TNF-α (17.1 ± 8.9 vs. 8 ± 7.4 pg/ml) and MIP-1α (636.8 ± 471.1 vs. 124.1 ± 40.1 pg/ml), as well as IL-13 (42.1 ± 19.8 vs. 21.7 ± 13.6) compared monocytes cultured under free-swelling conditions. This modulatory effect was observed irrespective of previous activation with the M1/pro-inflammatory differentiation stimuli lipopolysaccharide and interferon-γ or the M2/anti-inflammatory differentiation factor interleukin-4. Furthermore, mechanical shear and compression were found to differentially regulate nitric oxide synthase 2 (NOS2) and IL-12B gene expression as well as inflammatory protein production by THP1-Blue monocytes. The findings of this study indicate that human monocytes are responsive to mechanical stimuli, with a modulatory effect of shear and compressive loading observed toward pro-inflammatory mediator production. This may play a role in healing pathways that are mechanically regulated. An in depth understanding of the impact of skeletal tissue-associated mechanical loading on monocyte behavior may identify novel targets to maximize inflammation-mediated repair mechanisms.

Five Days Granulocyte Colony-Stimulating Factor Treatment Increases Bone Formation and Reduces Gap Size of a Rat Segmental Bone Defect: A Pilot Study.

Bone is an organ with high natural regenerative capacity and most fractures heal spontaneously when appropriate fracture fixation is provided. However, additional treatment is required for patients with large segmental defects exceeding the endogenous healing potential and for patients suffering from fracture non-unions. These cases are often associated with insufficient vascularization. Transplantation of CD34+ endothelial progenitor cells (EPCs) has been successfully applied to promote neovascularization of bone defects, however including extensive ex vivo manipulation of cells. Here, we hypothesized, that treatment with granulocyte colony-stimulating factor (G-CSF) may improve bone healing by mobilization of CD34+ progenitor cells into the circulation, which in turn may facilitate vascularization at the defect site. In this pilot study, we aimed to characterize the different cell populations mobilized by G-CSF and investigate the influence of cell mobilization on the healing of a critical size femoral defect in rats. Cell mobilization was investigated by flow cytometry at different time points after five consecutive daily G-CSF injections. In a pilot study, bone healing of a 4.5-mm critical femoral defect in F344 rats was compared between a saline-treated control group and a G-CSF treatment group. In vivo microcomputed tomography and histology were applied to compare bone formation in both treatment groups. Our data revealed that leukocyte counts show a peak increase at the first day after the last G-CSF injection. In addition, we found that CD34+ progenitor cells, including EPCs, were significantly enriched at day 1, and further increased at day 5 and day 11. Upregulation of monocytes, granulocytes and macrophages peaked at day 1. G-CSF treatment significantly increased bone volume and bone density in the defect, which was confirmed by histology. Our data show that different cell populations are mobilized by G-CSF treatment in cell specific patterns. Although in this pilot study no bridging of the critical defect was observed, significantly improved bone formation by G-CSF treatment was clearly shown.

Isolation of high-quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization.

The isolation of high-quality RNA from the intervertebral disc and especially from the nucleus pulposus is challenging due to the low cellularity and high proteoglycan content of this tissue. In this study, we report a simple modification of the standard guanidinium thiocyanate-phenol-chloroform extraction method, which involves enzymatic predigestion of the tissue prior to standard RNA isolation. Yield, purity and integrity of RNA isolated from bovine nucleus pulposus, inner annulus fibrosus and outer annulus fibrosus were compared among complete matrix digestion, predigestion and pulverization, pulverization alone, and pulverization followed by on-column purification. With predigestion, the average yield of RNA obtained from bovine nucleus pulposus was 8.82 ± 2.05 ng/mg of wet tissue with 260/280 and 260/230 optical density ratios of 1.91 ± 0.15 and 1.84 ± 0.30, respectively. RIN analysis indicated that RNA quality was best preserved with the predigestion method (RNA integrity number > 7), and the extracted RNA was suitable for real-time polymerase chain reaction. This method is of importance for gene expression studies on intervertebral disc development, degeneration and repair, and we anticipate that it may be further applied to other tissues rich in proteoglycans.

Monitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probes.

Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be observed in a non-destructive manner. In addition, the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed analysis of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective analysis of differentiation is also possible, which will lead to a greater understanding of MSC differentiation.

An In Vitro Investigation of Platelet-Rich Plasma-Gel as a Cell and Growth Factor Delivery Vehicle for Tissue Engineering.

Platelet-rich plasma (PRP) has been used for different applications in human and veterinary medicine. Many studies have shown promising therapeutic effects of PRP; however, there are still many controversies regarding its composition, properties, and clinical efficacy. The aim of this study was to evaluate the influence of different platelet concentrations on the rheological properties and growth factor (GF) release profile of PRP-gels. In addition, the viability of incorporated bone marrow-derived human mesenchymal stem cells (MSCs) was investigated. PRP (containing 1000 × 10(3), 2000 × 10(3), and 10,000 × 10(3) platelets/µL) was prepared from human platelet concentrates. Platelet activation and gelification were achieved by addition of human thrombin. Viscoelastic properties of PRP-gels were evaluated by rheological studies. The release of GFs and inflammatory proteins was measured using a membrane-based protein array and enzyme-linked immunosorbent assay. MSC viability and proliferation in PRP-gels were assessed over 7 days by cell viability staining. Cell proliferation was examined using DNA quantification. Regardless of the platelet content, all tested PRP-gels showed effective cross-linking. A positive correlation between protein release and the platelet concentration was observed at all time points. Among the detected proteins, the chemokine CCL5 was the most abundant. The greatest release appeared within the first 4 h after gelification. MSCs could be successfully cultured in PRP-gels over 7 days, with the highest cell viability and DNA content found in PRP-gels with 1000 × 10(3) platelets/µL. The results of this study suggest that PRP-gels represent a suitable carrier for both cell and GF delivery for tissue engineering. Notably, a platelet concentration of 1000 × 10(3) platelets/µL appeared to provide the most favorable environment for MSCs. Thus, the platelet concentration is an important consideration for the clinical application of PRP-gels.

Three-dimensional culture and characterization of mononuclear cells from human bone marrow.

BACKGROUND AIMS: The diverse phenotypic changes and clinical and economic disadvantages associated with the monolayer expansion of bone marrow-derived mesenchymal stromal cells (MSCs) have focused attention on the development of one-step intraoperative cells therapies and homing strategies. The mononuclear cell fraction of bone marrow, inclusive of discrete stem cell populations, is not well characterized, and we currently lack suitable cell culture systems in which to culture and investigate the behavior of these cells. METHODS: Human bone marrow-derived mononuclear cells were cultured within fibrin for 2 weeks with or without fibroblast growth factor-2 supplementation. DNA content and cell viability of enzymatically retrieved cells were determined at days 7 and 14. Cell surface marker profiling and cell cycle analysis were performed by means of multi-color flow cytometry and a 5-ethynyl-2'-deoxyuridine incorporation assay, respectively. RESULTS: Total mononuclear cell fractions, isolated from whole human bone marrow, was successfully cultured in fibrin gels for up to 14 days under static conditions. Discrete niche cell populations including MSCs, pericytes and hematopoietic stem cells were maintained in relative quiescence for 7 days in proportions similar to that in freshly isolated cells. Colony-forming unit efficiency of enzymatically retrieved MSCs was significantly higher at day 14 compared to day 0; and in accordance with previously published works, it was fibroblast growth factor-2-dependant. CONCLUSIONS: Fibrin gels provide a simple, novel system in which to culture and study the complete fraction of bone marrow-derived mononuclear cells and may support the development of improved bone marrow cell-based therapies.

CD34/CD133 enriched bone marrow progenitor cells promote neovascularization of tissue engineered constructs in vivo.

Vascularization is critical for 3D tissue engineered constructs. In large size implants the ingrowth of vessels often fails. The purpose of this study was to identify an easily accessible, clinically relevant cell source able to promote neovascularization in engineered implants in vivo and to establish an autologous culture method for these cells. MSCs (mesenchymal stem cells) and an endothelial progenitor containing cell (EPCC) population were obtained from human bone marrow aspirates. The expression of endothelial-markers, uptake of acetylated low density lipoprotein (acLDL) and tube-like structure formation capability of EPCCs were analyzed after expansion in endothelial growth medium or medium supplemented with autologous platelet lysate (PL). EPCCs were co-seeded with MSCs on hydroxyapatite-containing polyurethane scaffolds and then implanted subcutaneously in nude mice. Human EPCCs displayed typical characteristics of endothelial cells including uptake of acLDL and formation of tube-like structures on Matrigel™. In vivo, EPCCs cultured with PL triggered neovascularization. MSC/EPCC interactions promoted the maturation of newly formed luminal structures, which were detected deep within the scaffold and partly perfused, demonstrating a connection with the host vascular system. We demonstrate that this population of cells, isolated in a clinically relevant manner and cultured with autologous growth factors readily promoted neovascularization in tissue engineered constructs in vivo enabling a potential translation into the clinic.

Modulation of the murine CD8 gene complex following the targeted integration of human CD2-locus control region sequences.

The human CD2 (hCD2) locus control region (LCR) inserted in the mouse CD8 gene complex activates expression of the CD8 genes in T cell subsets in which the CD8 locus is normally silenced (e.g., CD4(+) single-positive T cells). In this article, we show that, in conditional mCD8/hCD2-LCR (CD8/LCR) knock-in mice, the continuous presence of the hCD2-LCR is required for this effect. Deletion of the inserted hCD2-LCR in a developmental stage and cell lineage-specific manner revealed that the temporary presence of the LCR during early development does not permanently alter the expression pattern of the CD8 genes. As a result, cells that have been affected by the insertion of the LCR can convert to their destined phenotype once the LCR is removed. DNaseI hypersensitive sites 1 and 2 of the hCD2-LCR influence the expression of the CD8 genes in a similar manner as does the full LCR, whereas insertion of hypersensitive site 3 alone of the LCR does not result in a changed expression pattern. This analysis revealed a dynamic interaction between the hCD2-LCR and the endogenous regulatory elements of the CD8 genes.

Pre-TCR signaling and CD8 gene bivalent chromatin resolution during thymocyte development.

The CD8 gene is silent in CD4(-)CD8(-) double-negative thymocytes, expressed in CD4(+)CD8(+) double-positive cells, and silenced in cells committing to the CD4(+) single-positive (SP) lineage, remaining active in the CD8(+) SP lineage. In this study, we show that the chromatin of the CD8 locus is remodeled in C57BL/6 and B6/J Rag1(-/-) MOM double-negative thymocytes as indicated by DNaseI hypersensitivity and widespread bivalent chromatin marks. Pre-TCR signaling coincides with chromatin bivalency resolution into monovalent activating modifications in double-positive and CD8 SP cells. Shortly after commitment to CD4 SP cell lineage, monovalent repressive characteristics and chromatin inaccessibility are established. Differential binding of Ikaros, NuRD, and heterochromatin protein 1α on the locus during these processes may participate in the complex regulation of CD8.

Fate mapping of IL-17-producing T cells in inflammatory responses.

Here we describe a reporter mouse strain designed to map the fate of cells that have activated interleukin 17A (IL-17A). We found that IL-17-producing helper T cells (T(H)17 cells) had distinct plasticity in different inflammatory settings. Chronic inflammatory conditions in experimental autoimmune encephalomyelitis (EAE) caused a switch to alternative cytokines in T(H)17 cells, whereas acute cutaneous infection with Candida albicans did not result in the deviation of T(H)17 cells to the production of alternative cytokines, although IL-17A production was shut off in the course of the infection. During the development of EAE, interferon-γ (IFN-γ) and other proinflammatory cytokines in the spinal cord were produced almost exclusively by cells that had produced IL-17 before their conversion by IL-23 ('ex-T(H)17 cells'). Thus, this model allows the actual functional fate of effector T cells to be related to T(H)17 developmental origin regardless of IL-17 expression.

Replication allows inactivation of a knocked-in locus control region in inappropriate cell lineages.

To study the influence of a locus control region (LCR) on the expression of a highly characterized, developmentally regulated locus, we have targeted the hCD2-LCR as a single copy into the endogenous mouse CD8 gene complex. Two knock-in mouse lines that differ in the integration site of the hCD2-LCR within the mCD8 gene complex were generated, and the influence on expression of the CD8 coreceptor was assessed. In these mice the normal developmental silencing of the CD8 genes in the CD4 lineage is deregulated, and the mice develop CD4(+) cells that also express the CD8 genes. This is accompanied by the physical maintenance of the CD8 genes within an extended loop away from their subchromosomal territory. Further analysis of these mice revealed unexpected fluid chromatin dynamics, whereby the LCR can be initially dominant over the endogenous CD8 gene-repressive regulatory processes present in CD4(+) cells but is continuously contested by them, resulting in the eventual inactivation of the inserted LCR, probably as a result of multiple rounds of replication.

HP1-beta is required for development of the cerebral neocortex and neuromuscular junctions.

HP1 proteins are thought to be modulators of chromatin organization in all mammals, yet their exact physiological function remains unknown. In a first attempt to elucidate the function of these proteins in vivo, we disrupted the murine Cbx1 gene, which encodes the HP1-beta isotype, and show that the Cbx1(-/-) -null mutation leads to perinatal lethality. The newborn mice succumbed to acute respiratory failure, whose likely cause is the defective development of neuromuscular junctions within the endplate of the diaphragm. We also observe aberrant cerebral cortex development in Cbx1(-/-) mutant brains, which have reduced proliferation of neuronal precursors, widespread cell death, and edema. In vitro cultures of neurospheres from Cbx1(-/-) mutant brains reveal a dramatic genomic instability. Our results demonstrate that HP1 proteins are not functionally redundant and that they are likely to regulate lineage-specific changes in heterochromatin organization.

Mll has a critical role in fetal and adult hematopoietic stem cell self-renewal.

The Mixed Lineage Leukemia (Mll) gene is a homolog of Drosophila Trithorax commonly rearranged in infant leukemia. Comprehensive analysis of the role of Mll in hematopoiesis in fetal and adult knockout mice has been prevented by the lethality of Mll(-/-) mice. We have established a conditional deletion model that allows us to study adult hematopoiesis in the absence of Mll. In this study, Mll(-/-) embryos survive to E16.5 and have reduced numbers of HSCs. The quiescent fraction of these HSCs is greatly reduced, and they are unable to compete with wild-type cells in transplantation assays. Mice with Mll expression conditionally deleted in the hematopoietic system have grossly normal hematopoiesis in bone marrow, thymus, and spleen. However, transplanted Mll-deficient bone marrow cells are highly compromised in their ability to competitively reconstitute irradiated recipients. These results suggest a critical role for Mll in regulating stem cell self-renewal.