Transplantation Experiments In Mice Research Articles

A specific enterotype derived from gut microbiome of older individuals enables favorable responses to immune checkpoint blockade therapy

Immunotherapy has revolutionized cancer treatment, but inconsistent responses persist. Our study delves into the intriguing phenomenon of enhanced immunotherapy sensitivity in older individuals with cancers. Through a meta-analysis encompassing 25 small-to-mid-sized trials of immune checkpoint blockade (ICB), we demonstrate that older individuals exhibit heightened responsiveness to ICB therapy. To understand the underlying mechanism, we reanalyze single-cell RNA sequencing (scRNA-seq) data from multiple studies and unveil distinct upregulation of exhausted and cytotoxic Tcell markers within the tumor microenvironment (TME) of older patients. Recognizing the potential role of gut microbiota in modulating the efficacy of immunotherapy, we identify an aging-enriched enterotype linked to improved immunotherapy outcomes in older patients. Fecal microbiota transplantation experiments in mice confirm the therapeutic potential of the aging-enriched enterotype, enhancing treatment sensitivity and reshaping the TME. Our discoveries confront the prevailing paradox and provide encouraging paths for tailoring cancer immunotherapy strategies according to an individual's gut microbiome profile.

Unraveling a challenging diagnosis: Role of a TNF inhibitor

<h3>Background</h3> The generation of reporter lines for cell identity, lineage, and physiologic state has provided a powerful tool in advancing the dissection of mouse kidney morphogenesis at a molecular level. Although use of this approach is not an option for studying human development <i>in vivo</i>, its application in human induced pluripotent stem cells (iPSCs) is now feasible. <h3>Methods</h3> We used CRISPR/Cas9 gene editing to generate ten fluorescence reporter iPSC lines designed to identify nephron progenitors, podocytes, proximal and distal nephron, and ureteric epithelium. Directed differentiation to kidney organoids was performed according to published protocols. Using immunofluorescence and live confocal microscopy, flow cytometry, and cell sorting techniques, we investigated organoid patterning and reporter expression characteristics. <h3>Results</h3> Each iPSC reporter line formed well patterned kidney organoids. All reporter lines showed congruence of endogenous gene and protein expression, enabling isolation and characterization of kidney cell types of interest. We also demonstrated successful application of reporter lines for time-lapse imaging and mouse transplantation experiments. <h3>Conclusions</h3> We generated, validated, and applied a suite of fluorescence iPSC reporter lines for the study of morphogenesis within human kidney organoids. This fluorescent iPSC reporter toolbox enables the visualization and isolation of key populations in forming kidney organoids, facilitating a range of applications, including cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing approaches. These tools offer promise for enhancing our understanding of this model system and its correspondence with human kidney morphogenesis.

Experimental Research on the Antitumor Effect of Human Gastric Cancer Cells Transplanted in Nude Mice Based on Deep Learning Combined with Spleen-Invigorating Chinese Medicine.

Gastric cancer is still the fifth most common malignant tumor in the world and has the fourth highest mortality rate in the world. Gastric cancer is difficult to treat because of its unobvious onset, low resection rate, and rapid deterioration. Therefore, humans have been working hard to combat gastric cancer. At present, the most commonly used treatment method is radiotherapy. However, this method will damage the normal tissues of the irradiated area while treating malignant tumor cells. It not only has side effects of damage to the patient's skin and mucous membranes but also needs high-rate radiotherapy and has high cost for chemotherapy. In order to solve these problems, it is necessary to find new treatment methods. This article proposes the use of Chinese medicine to invigorate the spleen to inhibit human gastric cancer cells. This article combines modern machine learning technology with traditional Chinese medicine and combines traditional Chinese medicine physiotherapy with Western medicine nude mouse transplantation experiments. The treatment of tumors in Chinese medicine is based on the theory of Chinese medicine and has different characteristics. Western medicine has the advantage of permanently injuring patients. The process of the experiment is to transplant human-derived gastric cancer cells into nude mice. After grouping treatments and obtaining comparative data, deep learning techniques are used to analyze the properties of Chinese medicines for strengthening the spleen and to compare the properties of Chinese medicines for strengthening the spleen. The experimental results showed that the tumor inhibition rate of mice using fluorouracil was 18%, the tumor inhibition rate of mice using low-dose Chinese medicine was 16%, and the tumor inhibition rate of mice using high-dose Chinese medicine reached 52%. 80 days after the experiment, the survival rate of mice using high-dose Chinese medicine is 100% higher than that of mice without treatment.

In This Issue

In This Issue

Primary human hepatocyte gene editing: Prometheus’ chains are loosening

Primary human hepatocytes (PHHs) isolated from livers retain proliferative properties when xeno-transplanted into chimeric mouse models1 or other species. Serial transplantation experiments in mice showed that PHH populations multiply by at least 100-fold per round,2 illustrating their greater than million-fold expansion potential. This has sparked widespread research into therapeutic hepatocyte transplantation for patients with liver conditions. Two major roadblocks that have limited clinical progress with hepatocyte transplantation are the lack of renewable high-quality human hepatocyte sources and the inability to efficiently correct inborn errors in PHHs.

Crosstalk between invadopodia and the extracellular matrix

The scaffold protein Tks5α is required for invadopodia-mediated cancer invasion both in vitro and in vivo. We have previously also revealed a role for Tks5 in tumor cell growth using three-dimensional (3D) culture model systems and mouse transplantation experiments. Here we use both 3D and high-density fibrillar collagen (HDFC) culture to demonstrate that native collagen-I, but not a form lacking the telopeptides, stimulated Tks5-dependent growth, which was dependent on the DDR collagen receptors. We used microenvironmental microarray (MEMA) technology to determine that laminin, fibronectin and tropoelastin also stimulated invadopodia formation. A Tks5α-specific monoclonal antibody revealed its expression both on microtubules and at invadopodia. High- and super-resolution microscopy of cells in and on collagen was then used to place Tks5α at the base of invadopodia, separated from much of the actin and cortactin, but coincident with both matrix metalloprotease and cathepsin proteolytic activity. Inhibition of the Src family kinases, cathepsins or metalloproteases all reduced invadopodia length but each had distinct effects on Tks5α localization. These studies highlight the crosstalk between invadopodia and extracellular matrix components, and reveal the invadopodium to be a spatially complex structure.

T Cell Acute Lymphoblastic Leukemia as a Consequence of Thymus Autonomy.

Thymus autonomy is the capacity of the thymus to maintain T lymphocyte development and export independently of bone marrow contribution. Prolonging thymus autonomy was shown to be permissive to the development of T cell acute lymphoblastic leukemia (T-ALL), similar to the human disease. In this study, performing thymus transplantation experiments in mice, we report that thymus autonomy can occur in several experimental conditions, and all are permissive to T-ALL. We show that wild type thymi maintain their function of T lymphocyte production upon transplantation into recipients with several genotypes (and corresponding phenotypic differences), i.e., Rag2 - / - γc - / -, γc - / -, Rag2 - / - IL-7rα - / -, and IL-7rα - / - We found that the cellularity of the thymus grafts is influenced exclusively by the genotype of the host, i.e., IL-7rα-/- versus γc -/- Nonetheless, the difference in cellularity detected in thymus autonomy bore no impact on onset, incidence, immunophenotype, or pathologic condition of T-ALL. In all tested conditions, T-ALL reached an incidence of 80%, demonstrating that thymus autonomy bears a high risk of leukemia. We also analyzed the microbiota composition of the recipients and their genetic background, but none of the differences found influenced the development of T-ALL. Taken together, our data support that IL-7 drives cellular turnover non-cell autonomously, which is required for prevention of T-ALL. We found no influence for T-ALL in the specific combination of the genotypic mutations tested (including the developmental block caused by Rag deficiency), in microbiota composition, or minor differences in the genetic background of the strains.

TRAIL-Expressing Monocyte/Macrophages Are Critical for Reducing Inflammation and Atherosclerosis

SummaryCirculating tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) levels are reduced in patients with cardiovascular disease, and TRAIL gene deletion in mice exacerbates atherosclerosis and inflammation. How TRAIL protects against atherosclerosis and why levels are reduced in disease is unknown. Here, multiple strategies were used to identify the protective source of TRAIL and its mechanism(s) of action. Samples from patients with coronary artery disease and bone-marrow transplantation experiments in mice lacking TRAIL revealed monocytes/macrophages as the main protective source. Accordingly, deletion of TRAIL caused a more inflammatory macrophage with reduced migration, displaying impaired reverse cholesterol efflux and efferocytosis. Furthermore, interleukin (IL)-18, commonly increased in plasma of patients with cardiovascular disease, negatively regulated TRAIL transcription and gene expression, revealing an IL-18-TRAIL axis. These findings demonstrate that TRAIL is protective of atherosclerosis by modulating monocyte/macrophage phenotype and function. Manipulating TRAIL levels in these cells highlights a different therapeutic avenue in the treatment of cardiovascular disease.

A Microcavity Array-Based 3D Model System of the Hematopoietic Stem Cell Niche.

Despite huge advances in recent years, the interaction between hematopoietic stem and progenitor cells (HSPCs) and their niches in the bone marrow is still far from being fully understood. One reason is that hematopoiesis is a multi-step maturation process leading to HSPC heterogeneity. Subpopulations of HSPCs can be identified by clonogenic assays or in serial transplantation experiments in mice following sublethal irradiation, but it is very complex to reproduce or even maintain stem cell plasticity in vitro. Advanced model systems have been developed that allow to precisely control and analyze key components of the physiologic microenvironment for not only fundamental research purposes but, as a long-term goal, also for clinical applications. In this chapter, we describe our approach of building an artificial hematopoietic stem cell niche in the form of polymer film-based microcavities with a diameter of 300μm and a depth of up to 300μm and arranged in a 634-cavity array. The polymer films are provided with 3μm pores and thus allow perfusion of the culture medium. The microcavity arrays can be inserted into a microbioreactor where a closed circulation loop can be tightly controlled with regard to medium flow and gas supply. The microcavity arrays were used for a three-dimensional (3D) co-culture of MSCs and HSPCs in a defined ratio over a time period of up to 21days. With this setup, it could be demonstrated that the HSPCs maintained their stem cell characteristics more efficiently as compared to conventional monolayer co-culture controls.

Molecular Mechanism of Mutant CALR-Mediated Transformation.

Elf and colleagues used an elegant series of functional and biochemical assays to investigate the molecular mechanism of mutant calreticulin (CALR)-driven cellular transformation in myeloproliferative neoplasms (MPN). Mutant CALR is sufficient to induce MPN in mouse transplantation experiments, and transformation is dependent upon physical interaction mediated by the positive electrostatic charge of the mutant CALR C-terminal domain and the thrombopoietin receptor MPL.

Sugars and cell adhesion: The role of ST6GalNAc1 in prostate cancer progression

O-linked glycans become altered in cancer cells, leading to changes in cell adhesive properties and contributing to metastasis. But the mechanisms driving these changes and how these carbohydrate groups are involved in tumour spread remain poorly understood. We recently identified the sialyltransferase gene ST6GalNAc1 as a novel androgen-regulated gene in prostate cancer. Expression of ST6GalNAc1 in prostate cancer cells induced expression of the cancer-associated sTn antigen, reduced cell adhesion, and dramatically inhibited the formation of stable tumour masses in vivo after orthotopic transplantation experiments in mice. Although ST6GalNAc1 is significantly upregulated in primary prostate carcinoma tissue, there is a striking downregulation of this gene in metastatic prostate tissue, suggesting an important yet transient role for ST6GalNAc1 in prostate cancer progression. Here we discuss mechanistically how changes in sialylation could alter prostate tumour cell behaviour and contribute to cancer cell dissemination.

Repopulation dynamics of single haematopoietic stem cells in mouse transplantation experiments: Importance of stem cell composition in competitor cells

The transplantation of blood tissues from bone marrow into a lethally irradiated animal is an experimental procedure that is used to study how the blood system is reconstituted by haematopoietic stem cells (HSC). In a competitive repopulation experiment, a lethally irradiated mouse was transplanted with a single HSC as a test cell together with a number of bone marrow cells as competitor cells, and the fraction of the test cell progeny (percentage of chimerism) was traced over time. In this paper, we studied the stem cell kinetics in this experimental procedure. The balance between symmetric self-renewal and differentiation divisions in HSC determined the number of cells which HSC produce and the length of time for which HSC live after transplantation. The percentage of chimerism depended on the type of test cell (long-, intermediate-, or short-term HSC), as well as the type and number of HSC included in competitor cells. We next examined two alternative HSC differentiation models, one-step and multi-step differentiation models. Although these models differed in blood cell production, the percentage of chimerism appeared very similar. We also estimated the numbers of different types of HSC in competitor cells. Based on these results, we concluded that the experimental results inevitably include stochasticity with regard to the number and the type of HSC in competitor cells, and that, in order to detect different types of HSC, an appropriate number of competitor cells needs to be used in transplantation experiments.

Pre-treatment of allogeneic bone marrow recipients with the CXCR4 antagonist AMD3100 transiently enhances hematopoietic chimerism without promoting donor-specific skin allograft tolerance

Hematopoietic chimerism established by allogeneic bone marrow transplantation is known to promote donor-specific organ allograft tolerance; however, clinical application is limited by the need for toxic host conditioning and “megadoses” of donor bone marrow cells. A potential solution to this problem has been suggested by the observation that recipient bone marrow mobilization by the CXCR4 antagonist AMD3100 promotes chimerism in congenic bone marrow transplantation experiments in mice. Here we report that a single subcutaneous dose of 10mg/kg AMD3100 in recipient C57BL/6 mice was able to enhance hematopoietic chimerism when complete MHC-mismatched BALB/c donor bone marrow cells were transplanted 1h after drug dosing. However, levels of chimerism measured 30days post-transplantation were not sustained when mice were reexamined on day 90 post-transplantation. Moreover, transient chimerism induced by this protocol did not support robust donor-specific skin allograft tolerance. Using the same transient immunosuppression protocol, we confirmed that “megadoses” of donor bone marrow cells could induce durable chimerism associated with donor-specific skin allograft tolerance without AMD3100 pre-treatment. We conclude that in this protocol AMD3100 pretreatment may empty bone marrow niches that become reoccupied by allogeneic donor hematopoietic progenitor cells but not by true long-lived donor hematopoietic stem cells, resulting in short-lived chimerism and failure to support durable donor-specific allograft tolerance.

Cardiology: Race for healthy hearts.

Transplantation experiments in mice reveal that the increased risk of congenital heart disease in the pups of older mothers is not conferred by ageing eggs, but by the mothers' age, and can be mitigated by exercise. See Letter p.230 Patrick Jay and colleagues investigate the known increased risk of congenital heart disease with increased maternal age. By transplanting ovaries between young and old mice they establish that the maternal age-associated risk of congenital heart disease in pups harbouring a mutation of the cardiac transcription factor gene Nkx2-5 is independent of the age of the ovaries and depends on the age of the mother. They further show that maternal exercise can mitigate this age-associated risk, although the mechanisms at work are not yet known.

Integrated Analysis Of CRLF2 Signaling In Acute Lymphoblastic Leukemia Identifies Polo-Like Kinase 1 As a Therapeutic Target

IntroductionAcute lymphoblastic leukemia is the most common cancer in children, and the five-year survival rate among patients older than 20 years old is still poor. Recently, Cytokine Receptor-Like Factor 2 (CRLF2) was identified as a prognostic factor in 15% of precursor B cell-acute lymphoblastic leukemia (B-ALL) that lack typical gene rearrangements (Yoda et al., Proc Natl Acad Sci U S A. 2010). In addition, several independent studies also demonstrated that CRLF2 acts as an oncoprotein (Mullighan et al., Nat Genet. 2009; Russell et al., Blood. 2009; Chapiro et al., Leukemia. 2010). The Children's Oncology Group also found that high expression of CRLF2 confers an adverse prognosis in high-risk B-ALL patients (Chen et al., Blood. 2012). The mechanism of CRLF2-associated leukemogenesis, however, is still not clear. In CRLF2-rearranged B-ALL cases, CRLF2 is overexpressed because of genetic rearrangements. Interestingly, one-half of cases co-harbor an activating mutation in exon 16 of JAK2, most commonly involving R683 or I682. In a murine progenitor B cell line, BaF3, it has been shown that the combination of CRLF2 overexpression and JAK2 R683G confers IL-3-independent growth. This illustrates that CRLF2 overexpression could synergize with JAK2 R683G to trigger leukemic proliferation. In our previous phosphoproteomics analysis of BaF3 cells expressing CRLF and JAK2 R683G, the combination upregulated phosphorylation of Protein phosphatase 1, regulatory subunit 12A (Ppp1r12a) (van Bodegom et al., Blood. 2012), which is a negative regulator of Polo-like kinase 1 (Plk1) (Yamashiro et al., Dev Cell. 2008). In this study, we aimed to focus on novel approaches to target CRLF2 signaling in B-ALL. MethodsTo identify effective kinase inhibitors against leukemic CRLF2 signaling, we subjected BaF3 cells with CRLF2 overexpression and JAK2 R683G (BJC cells) to a kinase inhibitor screening system that consisted of 73 kinase inhibitors and other small molecules. An in vitro cell proliferation assay was coupled to observe growth inhibition at 37°C for 3 days (Tyner et al., Cancer Res. 2012). To validate ex vivo efficacy of the Plk1 inhibitor (BI-2536), which scored in the screen, we injected BJC cells with/without pre-treatment for 24 hours at 100 nM into the NOD-scid IL2Rγnull (NSG) mice and compared spleen/liver sizes 15 days after injection. To compare in vitro efficacy of Plk1 inhibitor in CRLF2-rearranged B-ALL xenografts, we used human hTERT-transformed bone marrow stromal cells as a feeder layer and green fluorescent dye (488 nm) viability staining for flow cytometry analysis 48 hours after treatment. To investigate the downstream effect of dysregulated Plk1 in BJC cells, morphology of centrosomes was examined using anti-g-tubulin immunofluorescence staining. ResultsThe kinase inhibitor screening showed that the Plk1 inhibitor BI-2536 is particularly potent against BJC cells with an IC50 of 11 nM. To validate this screening result and test the efficacy of BI-2536 ex vivo, NSG mouse transplantation experiments showed that BJC cells that were pretreated with BI-2536 had significantly smaller spleen (0.47% vs. 1.45% of total body weight, p<0.01) and liver (5.53% vs. 7.62% of total body weight, p<0.01) sizes compared with BJC cells without treatment. The efficacy of BI-2536 against human CRLF2-rearranged B-ALL xenografts was evaluated in vitro. In comparison with vehicle control, the viability of CRLF2-rearranged B-ALL cells treated with BI-2536 was lower (48.8% of vehicle) than a wild-type CRLF2 B-ALL with BI-2536 (105.3% of vehicle). Because Plk1 is a major regulator of centrosome, we carried out immunofluorescence staining of γ-tubulin, a specific marker for centrosomes. While morphology of centrosomes showed normal patterns in BaF3 cells, in cells treated with BI-2536, replicated centrosomes failed to part from each other, surrounded by M phase chromosomes. ConclusionsBy integrating results from prior phosphoproteomics analysis, kinase inhibitor screening, ex vivo therapy in mouse transplantation experiments and in vitro viability assay using human xenografts, Plk1 inhibitor BI-2536 was shown to have potent activity against CRLF2-rearranged B-ALL cells. Our result suggests future therapy for this subset of B-ALL could be directed against Plk1 and its associated signaling pathways. Disclosures:Tyner:Incyte Corporation: Research Funding.

Cigarette smoke induces epithelial to mesenchymal transition and increases the metastatic ability of breast cancer cells

BackgroundRecent epidemiological studies demonstrate that both active and involuntary exposure to tobacco smoke increase the risk of breast cancer. Little is known, however, about the molecular mechanisms by which continuous, long term exposure to tobacco smoke contributes to breast carcinogenesis because most previous studies have focused on short term treatment models. In this work we have set out to investigate the progressive transforming effects of tobacco smoke on non-tumorigenic mammary epithelial cells and breast cancer cells using in vitro and in vivo models of chronic cigarette smoke exposure.ResultsWe show that both non-tumorigenic (MCF 10A, MCF-12A) and tumorigenic (MCF7) breast epithelial cells exposed to cigarette smoke acquire mesenchymal properties such as fibroblastoid morphology, increased anchorage-independent growth, and increased motility and invasiveness. Moreover, transplantation experiments in mice demonstrate that treatment with cigarette smoke extract renders MCF 10A cells more capable to survive and colonize the mammary ducts and MCF7 cells more prone to metastasize from a subcutaneous injection site, independent of cigarette smoke effects on the host and stromal environment. The extent of transformation and the resulting phenotype thus appear to be associated with the differentiation state of the cells at the time of exposure. Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44hi/CD24low population in MCF 10A cells and of CD44+ and CD49f + MCF7 cells, indicating that cigarette smoke causes the emergence of cell populations bearing markers of self-renewing stem-like cells. The phenotypical alterations induced by cigarette smoke are accompanied by numerous changes in gene expression that are associated with epithelial to mesenchymal transition and tumorigenesis.ConclusionsOur results indicate that exposure to cigarette smoke leads to a more aggressive and transformed phenotype in human mammary epithelial cells and that the differentiation state of the cell at the time of exposure may be an important determinant in the phenotype of the final transformed state.

Abstract 2860: A critical role of bone marrow-derived cells for hedgehog-mediated carcinogenesis.

Abstract Amounting evidence indicate that the tissue microenvironment in a developing tumor is created by the tumor to orchestrates molecular and cellular interactions. As a major pathway involved in tumorigenesis, Hedgehog (Hh) signaling is known to regulate tumor microenvironment, but the underlying molecular mechanisms in hedgehog-driven tumors remain unclear. Here we show that smoothened (SMO)-mediated tumor development is accompanied by TGFβ activation in a variety of cell types of the tumor microenvironment, such as keratinocytes, fibroblasts and myeloid-derived suppressor cells. To determine the role of TGFβ signaling for SMO-mediated tumor development, we generated cell type specific TGFβR2flp/flp mice in which SmoM2 expression is accompanied by TGFβR2 knocking out. We found that while K14cre-mediated TGFβRII knockout promoted tumor development, Mx-1cre-mediated TGFβRII knockout suppressed tumor development, indicating a tumor promoting effect of stromal TGFβ signaling. Using EGFP expressing mice for bone marrow transplantation, we determined that most stromal cells with TGFβ signaling activation are derived from bone marrow. To further determine the role of stromal TGFβ signaling for hedgehog signaling-mediated tumor development, we performed bone marrow transplantation experiments in mice that can be induced to develop basal cell carcinomas (BCC). We found that bone marrow cells with TGFβR2 knockout, but not the wild type cells, inhibited tumor development and reduced the size of developed tumors. Reduction of tumor development was associated with decreased expression of multiple inflammation stimulatory cytokines as well as the number of myeloid-derived suppressor cells (MDSCs). The tumor promoting effect of TGFβ signaling is not limited to BCC because tumor growth of B16-F10 cells was also affected by TGFβRII elimination in bone marrow-derived cells. Our data indicate that TGFβ signaling in the tumor microenvironment, not in the keratinocytes, is critical for maintaining the tumor microenvironment to foster tumor development and growth. Citation Format: Jingwu Xie, Eric Qipeng Fan, Dongsheng Gu, Hailan Liu, Xiaoli Zhang, Mark Kaplan, Merv Yoder. A critical role of bone marrow-derived cells for hedgehog-mediated carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2860. doi:10.1158/1538-7445.AM2013-2860

MicroRNA miR-125b causes leukemia

MicroRNA miR-125b has been implicated in several kinds of leukemia. The chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b of up to 90-fold normal. Moreover, miR-125b is also up-regulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative neoplasm, suggesting an important role for miR-125b in early hematopoiesis. Furthermore, coexpression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared with control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus, we show that overexpression of miR-125b is sufficient both to shorten the latency of BCR-ABL-induced leukemia and to independently induce leukemia in a mouse model.

MicroRNA Mir-125b Causes Leukemia

AbstractAbstract 3158MicroRNA miR-125b has been shown to be involved in different kind of leukemia. Indeed, the chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b up to 90 fold. Moreover, miR-125b is also upregulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All of the mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half of them died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative disorder, suggesting an important role of miR-125b in myeloid and lymphoid lineages. Co-expression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared to control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus we showed that the overexpression of miR-125b is sufficient to induce leukemia in vivo and decrease the latency of BCR-ABL -induced leukemia. Disclosures:No relevant conflicts of interest to declare.

Identification of human placenta‐derived mesenchymal stem cells involved in re‐endothelialization

Human placenta is an attractive source of mesenchymal stem cells (MSC) for regenerative medicine. The cell surface markers expressed on MSC have been proposed as useful tools for the isolation of MSC from other cell populations. However, the correlation between the expression of MSC markers and the ability to support tissue regeneration in vivo has not been well examined. Here, we established several MSC lines from human placenta and examined the expression of their cell surface markers and their ability to differentiate toward mesenchymal cell lineages. We found that the expression of CD349/frizzled-9, a receptor for Wnt ligands, was positive in placenta-derived MSC. So, we isolated CD349-negative and -positive fractions from an MSC line and examined how successfully cell engraftment repaired fractured bone and recovered blood flow in ischemic regions using mouse models. CD349-negative and -positive cells displayed a similar expression pattern of cell surface markers and facilitated the repair of fractured bone in transplantation experiments in mice. Interestingly, CD349-negative, but not CD349-positive cells, showed significant effects on recovering blood flow following vascular occlusion. We found that induction of PDGFβ and bFGF mRNAs by hypoxia was greater in CD349-negative cells than in CD349-positive cells while the expression of VEGF was not significantly different in CD349-negative and CD349-positive cells. These findings suggest the possibility that CD349 could be utilized as a specialized marker for MSC isolation for re-endothelialization.