BCL9 is an essential component of canonical Wnt signaling that mediates the differentiation of myogenic progenitors during muscle regeneration.

Muscle stem cells and their progeny play a fundamental role in the regeneration of adult skeletal muscle. We have previously shown that activation of the canonical Wnt/beta-catenin signaling pathway in adult myogenic progenitors is required for their transition from rapidly dividing transient amplifying cells to more differentiated progenitors. Whereas Wnt signaling in Drosophila is dependent on the presence of the co-regulator Legless, previous studies of the mammalian ortholog of Legless, BCL9 (and its homolog, BCL9-2), have not revealed an essential role of these proteins in Wnt signaling in specific tissues during development. Using Cre-lox technology to delete BCL9 and BCL9-2 in the myogenic lineage in vivo and RNAi technology to knockdown the protein levels in vitro, we show that BCL9 is required for activation of the Wnt/beta-catenin cascade in adult mammalian myogenic progenitors. We observed that the nuclear localization of beta-catenin and downstream TCF/LEF-mediated transcription, which are normally observed in myogenic progenitors upon addition of exogenous Wnt and during muscle regeneration, were abrogated when BCL9/9-2 levels were reduced. Furthermore, reductions of BCL9/9-2 inhibited the promotion of myogenic differentiation by Wnt and the normal regenerative response of skeletal muscle. These results suggest a critical role of BCL9/9-2 in the Wnt-mediated regulation of adult, as opposed to embryonic, myogenic progenitors.

Bone marrow-derived cells are implicated as a source of lymphatic endothelial progenitors in human breast cancer.

Bone marrow-derived endothelial progenitor cells (EPCs) infiltrate into sites of neovascularization in adult tissues and mature into functional blood endothelial cells (BECs) during a process called vasculogenesis. Human marrow-derived EPCs have recently been reported to display a mixed myeloid and lymphatic endothelial cell (LEC) phenotype during inflammation-induced angiogenesis; however, their role in cancer remains poorly understood. We report the in vitro differentiation of human cord blood CD133(+)CD34(+) progenitors into podoplanin(+) cells expressing both myeloid markers (CD11b, CD14) and the canonical LEC markers vascular endothelium growth factor receptor 3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and prospero homeobox 1 (PROX-1). These podoplanin(+) cells displayed sprouting behavior comparable to that of LECs in vitro and a dual hemangiogenic and lymphangiogenic activity in vivo in an endothelial cell sprouting assay and corneal vascularization assay, respectively. Furthermore, these cells expressed vascular endothelium growth factor (VEGF) family members A, -C, and -D. Thus, bone-marrow derived EPCs stimulate hemangiogenesis and lymphangiogenesis through their ability to differentiate into LECs and to produce angiogenic factors. Importantly, plasma from patients with breast cancer induced differentiation of CD34(+) cord blood progenitors into hemangiogenic and lymphangiogenic CD11b(+) myeloid cells, whereas plasma from healthy women did not have this effect. Consistent with these findings, circulating CD11b(+) cells from breast cancer patients, but not from healthy women, displayed a similar dual angiogenic activity. Taken together, our results show that marrow-derived EPCs become hemangiogenic and lymphangiogenic upon exposure to cancer plasma. These newly identified functions of bone-marrow derived EPCs are expected to influence the diagnosis and treatment of breast cancer.

Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831)

Abstract: The study aimed to isolate, expand, differentiate and characterize progenitor cells existent in the dental pulp of agouti. The material was washed with PBS solution and dissociated mechanically with the aid of a scalpel blade on plates containing culture medium D-MEM/F-12, and incubated at 5% CO2-37⁰C. The growth curve, CFU assay, osteogenic/adipogenic differentiation and characterization were obtained from the isolation. The cells began to be released from the explant tissue around the 7th day of culture. By day 22 of culture, cells reached 80% confluence. At the UFC test, 81 colonies were counted with 12 days of cultivation. The growth curves before and after freezing showed a regular growth with intense proliferation and clonogenic potential. The cell differentiation showed formation of osteoblasts and fat in culture, starting at 15 days of culture in a specific medium. Flow cytometry (FACs) was as follows: CD34 (positive), CD14 (negative), CD45 (negative), CD73 (positive), CD79 (negative), CD90 (positive), CD105 (positive), demonstrating high specificity and commitment of isolated cells with mesenchymal stem cells strains. These results suggest the existence of a cell population of stem cells with mesenchymal features from the isolated tissue in the explants of agouti dental pulp, a potential model for study of stem cell strains obtained from the pulp tissue.

Use of RAPD-PCR to identify true hybrid plants from crosses between closely related progenitors

RAPD-PCR molecular markers were used to identify common bean and soybean hybrid plants derived from crosses between closely related progenitors, with no apparent phenotypic differences. Primers OP-F12 and OP-0O3 were used to identify true hybrids derived from crosses between common bean cultivars Rudá (A 285) and AN 910408, and soybean cultivars Cristalina and Bossier, respectively. Each primer generated one polymorphic DNA band which was present in the male progenitor and absent in the female progenitor. As RAPD bands are normally inherited as dominant characters, the presence of these bands in the F1 plants confirmed their status.

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

Identification of renin progenitors in the mouse bone marrow that give rise to B-cell leukaemia

cell of origin and triggering events for leukaemia are mostly unknown. Here we show that the bone marrow contains a progenitor that expresses renin throughout development and possesses a B-lymphocyte pedigree. This cell requires RBP-J to differentiate. Deletion of RBP-J in these renin-expressing progenitors enriches the precursor B-cell gene programme and constrains lymphocyte differentiation, facilitated by H3K4me3 activating marks in genes that control the pre-B stage. Mutant cells undergo neoplastic transformation, and mice develop a highly penetrant B-cell leukaemia with multi-organ infiltration and early death. These reninexpressing cells appear uniquely vulnerable as other conditional models of RBP-J deletion do not result in leukaemia. The discovery of these unique renin progenitors in the bone marrow and the model of leukaemia described herein may enhance our understanding of normal and neoplastic haematopoiesis.

Els drets dels infants vistos pels progenitors i mestres de nens i nenes d'entre deu i dotze anys. 'Los derechos de la infancia vistos por los progenitores y maestros de niños y niñas de entre diez y doce años'.

Basado en el resumen de los autores en catalán

Detailed molecular characterization of cord blood-derived endothelial progenitors

Objective. Given their involvement in pathological and physiological angiogenesis, there has been growing interest in understanding and manipulating endothellial progenitor cells (EPC) for therapeutic purposes. However, detailed molecular analysis of EPC before and during endothelial differentiation is lacking and is the subject of the present study. Materials and Methods. We report a detailed microarray gene-expression profile of freshly isolated (day 0) human cord blood (CB)-derived EPC (CD133(+)KDR(+) or CD34(+)KDR(+)), and at different time points during in vitro differentiation (early: day 13; late: day 27). Results. Data obtained reflect an EPC transcriptome enriched in genes related to stem/progenitor cells properties (chromatin remodeling, self-renewal, signaling, cytoskeleton organization and biogenesis, recruitment, and adhesion). Using a complementary DNA microarray enriched in intronic transcribed sequences, we observed, as well, that naturally transcribed intronic noncoding RNAs were specifically expressed at the EPC stage. Conclusion. Taken together, we have defined the global gene-expression profile of CB-derived EPC during the process of endothelial differentiation, which can be used to identify genes involved in different vascular pathologies. (C) 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Directed Differentiation of Neural Progenitors into Neurons Is Accompanied by Altered Expression of P2X Purinergic Receptors

Neural differentiation has been extensively studied in vitro in a model termed neurospheres, which consists of aggregates of neural progenitor cells. Previous studies suggest that they have a great potential for the treatment of neurological disorders. One of the major challenges for scientists is to control cell fate and develop ideal culture conditions for neurosphere expansion in vitro, without altering their features. Similar to human neural progenitors, rat neurospheres cultured in the absence of epidermal and fibroblast growth factors for a short period increased the levels of beta-3 tubulin and decreased the expression of glial fibrillary acidic protein and nestin, compared to neurospheres cultured in the presence of these factors. In this work, we show that rat neurospheres cultured in suspension under mitogen-free condition presented significant higher expression of P2X2 and P2X6 receptor subunits, when compared to cells cultured in the presence of growth factors, suggesting a direct relationship between P2X2/6 receptor expression and induction of neuronal differentiation in mitogen-free cultured rat neurospheres.

Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

Reduction of erythroid progenitors in protein-energy malnutrition

Protein-energy malnutrition is a syndrome in which anaemia together with multivitamin and mineral deficiency may be present. The pathophysiological mechanisms involved have not, however, yet been completely elucidated. The aim of the present study was to evaluate the pathophysiological processes that occur in this anaemia in animals that were submitted to protein-energy malnutrition, in particular with respect to Fe concentration and the proliferative activity of haemopoietic cells. For this, histological, histochemical, cell culture and immunophenotyping techniques were used. Two-month-old male Swiss mice were submitted to protein-energy malnutrition with a low-protein diet (20g/kg) compared with control diet (400 g/kg). When the experimental group had attained a 20% loss of their original body weight, the animals from both groups received, intravenously, 20IU erythropoietin every other day for 14 d. Malnourished animals showed a decrease in red blood cells, Hb concentration and reticulocytopenia, as well as severe bone marrow and splenic atrophy. The results for serum Fe, total Fe-binding capacity, transferrin and erythropoietin in malnourished animals were no different from those of the control animals. Fe reserves in the spleen, liver and bone marrow were found to be greater in the malnourished animals. The mixed colony-forming unit assays revealed a smaller production of granulocyte-macrophage colony-forming units, erythroid burst-forming units, erythroid colony-forming units and CD45, CD117, CD119 and CD71 expression in the bone marrow and spleen cells of malnourished animals. These findings suggest that, in this protein-energy malnutrition model, anaemia is not caused by Fe deficiency or erythropoietin deficiency, but is a result of ineffective erythropoiesis.

Progesterone upregulates GATA-1 on erythroid progenitors cells in liquid culture

Steroids hormones modify the hematological features of homozygous sickle cell disease, including the levels of fetal hemoglobin. We used semi-quantitative RT-PCR analysis of GATA-1, GATA-2, NF-E2, and gamma-globin mRNA levels in a two-phase liquid culture system of human adult erythroid cells in order to assay the effect of progesterone upon gene expression. The levels of expression of GATA-1 and gamma-globin mRNA were significantly increased in cells treated with progesterone compared to untreated cells (1.7- to 2.0-fold). Progesterone treatment did not produce any stimulatory effect upon GATA-2 and NF-E2 mRNA expression. Differences in the synthesis of HbF protein could not be detected by flow cytometry, although we observed a small difference in mean intensity fluorescence between cells treated and cells untreated with progesterone on days 7 and 9. Using anti-transferrin receptor and anti-glycophorin A antibodies, we verified that addition of progesterone did not cause any change in erythroid proliferation and differentiation. In conclusion, it is possible that the increased expression of gamma-globin mRNA after progesterone treatment observed in this study may be related to the increased GATA-1 mRNA expression. Interactions of the steroid receptors with the basal transcriptional machinery and with transcription factors might mediate their transcriptional effects. (C) 2002 Elsevier B.V. (USA).

Vascular wall stem cells. Selection and conditioning of progenitors useful for cell therapy. A pathological case study

The arterial wall contains MSCs with mesengenic and angiogenic abilities. These multipotent precursors have been isolated from variously-sized human adult segments, belying the notion that vessel wall is a relatively quiescent tissue. Recently, our group identified in normal human arteries a vasculogenic niche and subsequently isolated and characterized resident MSCs (VW-MSCs) with angiogenic ability and multilineage potential. To prove that VW-MSCs are involved in normal and pathological vascular remodeling, we used a long-term organ culture system; this method was of critical importance to follow spontaneous 3-D vascular remodeling without any influence of blood cells. Next we tried to identify and localize in situ the VW-MSCs and to understand their role in the vascular remodeling in failed arterial homografts. Subsequently, we isolated this cell population and tested in vitro their multilineage differentiation potential through immunohistochemical, immunofluorescence, RT-PCR and ultrastructural analysis. From 25-30cm2 of each vascular wall homograft sample, we isolated a cell population with MSCs properties; these cells expressed MSC lineage molecules (CD90, CD44, CD105, CD29, CD73), stemness (Notch-1, Oct-4, Sca-1, Stro-1) and pericyte markers (NG2) whilst were negative for hematopoietic and endothelial markers (CD34, CD133, CD45, KDR, CD146, CD31 and vWF). MSCs derived from failed homografts (H-MSCs) exhibited adipogenic, osteogenic and chondrogenic potential but scarce propensity to angiogenic and leiomyogenic differentiation. The present study demonstrates that failed homografts contain MSCs with morphological, phenotypic and functional MSCs properties; H-MSCs are long-lived in culture, highly proliferating and endowed with prompt ability to differentiate into adipocytes, osteocytes and chondrocytes; compared with VW-MSCs from normal arteries, H-MSCs show a failure in angiogenic and leiomyogenic differentiation. A switch in MSCs plasticity could be the basis of pathological remodeling and contribute to aneurysmal failure of arterial homografts. The study of VW-MSCs in a pathological setting indicate that additional mechanisms are involved in vascular diseases; their knowledge will be useful for opening new therapeutic options in cardiovascular diseases.