ES Cell Culture Research Articles

STAT3 and SMAD1 Signaling in Medaka Embryonic Stem-Like Cells and Blastula Embryos

The activation and transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is essential for maintaining mouse embryonic stem (ES) cell cultures in an undifferentiated state. However, reports from human and monkey ES-cell culture suggest that STAT3 is dispensable for pluripotency in these systems. At the same time, BMP signaling via smad1 was shown to be able to counteract STAT3 signaling in murine ES-cell cultures, while it influences differentiation in multifaceted ways in other cellular contexts. Hence, the question arises whether the signaling situation found in mice or primates and human ES-cells represent the rule or the exception. With this study, we want to contribute an answer to this question from an evolutionary perspective. Therefore, we analyzed the expression and activation status of the Medaka (Oryzias latipes) STAT3 and SMAD1 in Medaka ES-cell-like cultures and their in vivo counterpart, the Medaka blastula embryo. While SMAD signaling is active in the culture system as well as in blastula embryos, our results indicate that STAT3 is inactive and can thus not be involved in pluripotency control of blastula cells or their derived pluripotent in vitro counterparts. These results suggest that the signaling pathways active in the mouse ES-cell culture system represent the exception, while inactivity of STAT3 is apparently the rule in vertebrate ES-cell cultures.

Development of a zebrafish spleen cell line, ZSSJ, and its growth arrest by gamma irradiation and capacity to act as feeder cells

A zebrafish spleen cell line, ZSSJ, was developed and its growth arrest by gamma radiation determined and its capacity to stimulate the proliferation of the zebrafish blastula cell line, ZEB2J, measured. ZSSJ was initiated by explant outgrowth, grew adherent with mainly an epithelial-like morphology, and stained strongly for alkaline phosphatase. ZSSJ was not only grown in L-15 with 15% fetal bovine serum at 26 degrees C to 28 degrees degrees C but also grew at room temperature. Cultures of ZSSJ have undergone approximately 40 population doublings, had few cells staining for b-galactosidase activity, which is commonly present in senescent cultures, and many cells with an aneuploid karyotype, which is frequently associated with immortalization. ZSSJ growth was arrested by 30 to 50 Gy of g-irradiation, whereas after 20 Gy, some slight growth was observed. By contrast, growth of the rainbow trout spleen stromal cell line, RTS34st, which has been used as a feeder for zebrafish ES cell cultures, was arrested completely by 20 Gy. In cocultures, nongrowth-arrested ZSSJ stimulated ZEB2J proliferation better than growth-arrested ZSSJ and better than RTS34st. ZSSJ should be useful as a feeder cell line for zebrafish ES cell cultures.

Contrasting Expression of Keratins in Mouse and Human Embryonic Stem Cells

RNA expression data reveals that human embryonic stem (hES) cells differ from mouse ES (mES) cells in the expression of RNAs for keratin intermediate filament proteins. These differences were confirmed at the cellular and protein level and may reflect a fundamental difference in the epithelial nature of embryonic stem cells derived from mouse and human blastocysts. Mouse ES cells express very low levels of the simple epithelial keratins K8, K18 and K19. By contrast hES cells express moderate levels of the RNAs for these intermediate filament proteins as do mouse stem cells derived from the mouse epiblast. Expression of K8 and K18 RNAs are correlated with increased c-Jun RNA expression in both mouse and human ES cell cultures. However, decreasing K8 and K18 expression associated with differentiation to neuronal progenitor cells is correlated with increasing expression of the Snai2 (Slug) transcriptional repression and not decreased Jun expression. Increasing K7 expression is correlated with increased CDX2 and decreased Oct4 RNA expression associated with the formation of trophoblast derivatives by hES cells. Our study supports the view that hES cells are more similar to mouse epiblast cells than mouse ES cells and is consistent with the epithelial nature of hES cells. Keratin intermediate filament expression in hES cells may modulate sensitivity to death receptor mediated apoptosis and stress.

Optimized mouse ES cell culture system by suspension growth in a fully defined medium

Mouse and human embryonic stem (mES and hES) cells have become one of the most intensively studied primary cell types in biomedical research. However, culturing ES cells is notoriously labor intensive. We have optimized current ES cell culture methods by growing mES cells in suspension in a defined medium. This protocol is unsurpassed in time efficiency and typically requires only 20 min of effective hands-on time per week. This protocol maintains a very high degree of pluripotent cells partly by mechanical separation of spontaneously differentiating cells. mES cells can be cultured for extended periods (>6 months) without the loss of pluripotency markers. High passage (>20) adherent mES cultures containing contaminating differentiated cells can be rescued and enriched in undifferentiated ES cells.

Hepatic Differentiation of Embryonic Stem Cells in HF/Organoid Culture

Objective The use of embryonic stem cells (ES cells) has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional mature cells from ES cells in large quantities. We applied HF/organoid culture, where cultured cells formed cylindrical multicellular aggregates (organoids) in the lumen of hollow fibers, to mouse and cynomolgus monkey ES cells for hepatic differentiation. Materials and Methods ES cells were injected into hollow fibers. The hollow fibers were centrifuged to induce organoid formation and cultured in medium including factors for hepatic differentiation. To determine the characteristics of cells in the bundle, we evaluated gene expression and liver-specific functions. Results ES cells immobilized inside hollow fibers proliferated and formed cylindrical organoids. In mouse ES cell cultures, the expression of mRNAs of hepatocyte-specific genes increased with culture time. Ammonia removal activity detected at 15 days increased with culture time. Albumin secretion activity detected at 12 days increased by 21 days. In cynomolgus monkey ES cell cultures, ES cells showed spontaneous ammonia removal functions. The maximum levels of these functions per unit volume of the hollow fibers were roughly comparable to those of primary hepatocyte-organoids. Conclusions ES cells differentiated into hepatocyte-like cells using the organoid culture technique. The results indicated that the combination of ES cells and an organoid culture technique was useful to obtain mature hepatocytes.

Embryonic Stem Cell Transplantation

Cardiovascular diseases remain the leading cause of death worldwide, and the burden is equally shared between men and women around the globe. Cardiomyocytes that die in response to disease processes or aging are replaced by scar tissue instead of new muscle cells. Although recent reports suggest an intrinsic capacity for the mammalian myocardium to regenerate via endogenous stem/progenitor cells, the magnitude of such a response appears to be minimal and has yet to be realized fully in cardiovascular patients. Despite the advances in pharmacotherapy and new biomedical technologies, the prognosis for patients diagnosed with end-stage heart failure appears to be grave. While heart transplantation is a viable option, this life-saving intervention suffers from an acute shortage of cardiac organ donors. In view of these existing issues, donor cell transplantation is emerging as a promising strategy to regenerate diseased myocardium. Studies from multiple laboratories have shown that transplantation of donor cells (e.g. fetal cardiomyocytes, skeletal myoblasts, smooth muscle cells, and adult stem cells) can improve the function of diseased hearts over a short period of time (1-4 weeks). While long-term follow-up studies are warranted, it is generally perceived that the beneficial effects of transplanted cells are mainly due to increased angiogenesis or favorable scar remodeling in the engrafted myocardium. Although skeletal myoblasts and bone marrow stem cells hold the highest potential for implementation of autologous therapies, initial results from phase I trials are not promising. In contrast, transplantation of fetal cardiomyocytes has been shown to confer protection against the induction of ventricular tachycardia in experimental myocardial injury models. Furthermore, results from multiple laboratories suggest that fetal cardiomyocytes can couple functionally with host myocytes, stimulate formation of new blood vessels, and improve myocardial function. While it is neither practical nor ethical to test the potential of fetal cardiomyocytes in clinical trials, embryonic stem (ES) cells serve as a novel source for generation of unlimited quantities of cardiomyocytes for myocardial repair. The initial success in the application of ES cells to partially repair and improve myocardial function in experimental models of heart disease has been quite promising. However, multiple hurdles need to be crossed before the potential benefits of ES cells can be translated to the clinic. In this review, we summarize the current knowledge of cardiomyocyte derivation and enrichment from ES-cell cultures and provide a brief survey of factors increasing cardiomyogenic induction in both mouse and human ES cultures. Subsequently, we summarize the current state of research using mouse and human ES cells for the treatment of heart disease in various experimental models. Furthermore, we discuss the challenges that need to be overcome prior to the successful clinical utilization of ES-derived cardiomyocytes for the treatment of end-stage heart disease. While we are optimistic that the researchers in this field will sail across the hurdles, we also suggest that a more cautious approach to the validation of ES cardiomyocytes in experimental models would certainly prevent future disappointments, as seen with skeletal myoblast studies.

Mesodermal Patterning Activity of the Transcription Factor SCL.

The transcription factor SCL is critically required for the establishment of the hematopoietic lineage as well as for proper endothelial development, however it is not required for maintenance of HSCs or endothelial cells in the adult. Conflicting data exists regarding the developmental function of SCL, namely whether it acts as a master regulator, actively patterning mesoderm toward hematopoietic/endothelial development at the expense of other lineages, or is merely necessary to maintain the earliest committed hematopoietic and endothelial precursors. To answer this question in a mammalian model, we have engineered murine ES cells with a conditional doxycycline-inducible SCL transgene, and evaluated the effects of pulses of SCL expression at defined time points during in vitro development. During embryoid body differentiation, a pulse of SCL expression during mesodermal patterning results in enhanced hematopoiesis at later time points, as assayed by CFC activity and FACS analysis, as well significantly increasing the frequency of bipotent hematopoietic/endothelial hemangioblast CFCs. Concomitantly, SCL represses cardiac development, assayed by beating cardiomyocyte frequency, and molecular markers of the cardiac lineage. SCL also represses paraxial mesodermal developmental potential, severely reducing both the earliest PDGFαR+/Flk-1neg paraxial progenitors, as well as repressing levels of later markers including Pax3. Through chimeric embryoid bodies in which SCL cells are mixed with GFP-labelled ES cells, we show formally that this effect is cell-autonomous. Finally, we tested whether expression of SCL would enable hematopoiesis during ES cell monolayer differentiation. Hematopoiesis requires inter-germ layer inductive signaling which occurs in 3 dimensions but not in monolayer culture, thus ES cells do not spontaneously differentiate into blood in this 2 dimensional culture. We show that when mesoderm is produced during monolayer culture of wild-type ES cells, or control cells not expressing SCL, it is biased towards paraxial or cardiac differentiation. However when given a pulse of SCL expression, these pathways are repressed, PDGFαRneg/Flk-1+ presumptive lateral plate progenitors are produced, and abundant hematopoietic differentiation occurs. This data in the mammalian system thus strongly supports the former hypothesis, of an active role for SCL in mesodermal patterning.

Defined, Feeder‐Independent Medium for Human Embryonic Stem Cell Culture

The developmental potential of human ES cells makes them an important tool in developmental, pharmacological, and clinical research. For human ES cell technology to be fully exploited, however, culture efficiency must be improved, large-scale culture enabled, and safety ensured. Traditional human ES cell culture systems have relied on serum products and mouse feeder layers, which limit the scale, present biological variability, and expose the cells to potential contaminants. Defined, feeder-independent culture systems improve the safety and efficiency of ES cell technology, enabling translational research. The protocols herein are designed with the standard research laboratory in mind. They contain recipes for the formulation of mTeSR (a defined medium for human ES cell culture) and detailed protocols for the culture, transfer, and passage of cells grown in these feeder-independent conditions. They provide a basis for routine feeder-independent culture, and a starting point for additional optimization of culture conditions.

ES cell culture medium

ES cell culture medium

Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis

BackgroundAngiogenesis assays are important tools for the identification of regulatory molecules and the potential development of therapeutic strategies to modulate neovascularization. Although numerous in vitro angiogenesis models have been developed in the past, they exhibit limitations since they do not recapitulate the entire angiogenic process or correspond to multi-step procedures that are not easy to use. Convenient, reliable, easily quantifiable and physiologically relevant assays are still needed for pharmacological screenings of angiogenesis.ResultsHere, we have optimized an angiogenesis model based on ES cell differentiation for screening experiments. We have established conditions leading to angiogenic sprouting of embryoid bodies during ES cell differentiation in type I three-dimensional collagen gels. Immunostaining experiments carried out during these cultures showed the formation of numerous buds comprising CD31 positive cells, after 11 days of culture of ES cells. Moreover, this one-step model has been validated in response to activators and inhibitors of angiogenesis. Sprouting was specifically stimulated in the presence of VEGF and FGF2. Alternatively, endothelial sprouting induced by angiogenic activators was inhibited by angiogenesis inhibitors such as angiostatin, TGFβ and PF4. Sprouting angiogenesis can be easily quantified by image analysis after immunostaining of endothelial cells with CD31 pan-endothelial marker.ConclusionTaken together, these data clearly validate that this one-step ES differentiation model constitutes a simple and versatile angiogenesis system that should facilitate, in future investigations, the screening of both activators and inhibitors of angiogenesis.

Bone Morphogenetic Protein-Mediated Modulation of Lineage Diversification During Neural Differentiation of Embryonic Stem Cells

Embryonic stem cells (ES cells) can give rise to a broad spectrum of neural cell types. The biomedical application of ES cells will require detailed knowledge on the role of individual factors modulating fate specification during in vitro differentiation. Bone morphogenetic proteins (BMPs) are known to exert a multitude of diverse differentiation effects during embryonic development. Here, we show that exposure to BMP2 at distinct stages of neural ES cell differentiation can be used to promote specific cell lineages. During early ES cell differentiation, BMP2-mediated inhibition of neuroectodermal differentiation is associated with an increase in mesoderm and smooth muscle differentiation. In fibroblast growth factor 2-expanded ES cell-derived neural precursors, BMP2 supports the generation of neural crest phenotypes, and, within the neuronal lineage, promotes distinct subtypes of peripheral neurons, including cholinergic and autonomic phenotypes. BMP2 also exerts a density-dependent promotion of astrocyte differentiation at the expense of oligodendrocyte formation. Experiments involving inhibition of the serine threonine kinase FRAP support the notion that these effects are mediated via the JAK/STAT pathway. The preservation of diverse developmental BMP2 effects in differentiating ES cell cultures provides interesting prospects for the enrichment of distinct neural phenotypes in vitro.

Induction of melanocyte precursors from neural crest cells surrounding the neural tube-like structures developed in vitro using mouse ES cell culture

Neural crest cells differentiate into various cell types including melanocytes. In this study, we wanted to induce neural crest cells locating outside of neural tube by culturing mouse ES cells with retinoic acid (RA) and bone morphologic protein 4 (BMP4) in vitro, which mimicked the appearance of neural crest cells in the fetus. Histological examination of the cell aggregates composed of ES cells treated with RA and BMP4 disclosed emergence of neural tube-like structures surrounded by slug expressing neural crest cells. We detected melanocyte associated mRNAs such as pax3, sox10, Mitf and tyrosinase by RT-PCR of the aggregates. The neural crest cells expressed KIT and tyrosinase, indicating their differentiation to the melanocyte lineage. To accelerate the melanocyte induction, the cells were further treated by stem cell factor and endothelin 3 and then were transplanted to mouse femoral quadriceps muscles. They adhered to the recipient muscle tissue and retained the characteristics of melanocyte precursors, including expression of slug, KIT, endothelin receptor B and tyrosinase. Collectively, the neural crest cells derived from ES cells have a potency to differentiate into melanocyte precursors and they may be applicable for use in certain disease conditions such as vitiligo vulgaris in future.

Prospective Isolation of Mesenchymal Stem Cells from Human Bone Marrow and Placenta Using a Frizzled-9-Specific Monoclonal Antibody.

We have recently shown that Frizzled-9 (FZD9) is expressed on the cell surface of multipotential mesenchymal stromal cells derived from human bone marrow (BM) and placenta (PL). To test whether FZD9 is also expressed on primary mesenchymal stem cells (MSC) with CFU-F (colony forming unit fibroblast) capacity, we used the FZD9-specific monoclonal antibody W3C4E11 to fractionate BM and PL cells into FZD9+ and FZD9- populations. Defined numbers of sorted cells were then cultured in a serum free bFGF-containing medium, originally designed fur the culture of human ES cells. 14 days after culture, the resulting fibroblastoid cells were stained with Giemsa staining solution and colonies consisting of > 50 cells were enumerated. In the FZD9+ fraction, a 70 fold increase of the CFU-F frequency in PL and a 80–90 fold increase in BM was observed. Cultured FZD9+ cells also expressed the stem cell markers Oct-4, nanog-3, and nestin, as well as SSEA-4, TRA1-2-49/6E, CD105, CD13, CD164, CD318, and W8B2B10 antigen. After culture under appropriate conditions, FZD9+ cells could be differentiated into cells of the ectodermal (neuronal like cells), endodermal (pancreatic like cells), and mesodermal (adipocytes and osteoblasts) lineages, indicating the multi-lineage differentiation capacity of these cells. In conclusion, FZD9 is a novel marker for the prospective isolation of MSC from human BM and PL.

Pharmacological Control of Sendai Viral Transgene Expression in Primate Embryonic Stem Cells.

<Background> Sendai virus (SeV) is an enveloped virus with a negative-stranded RNA genome and is a member of the family Paramyxoviridae. SeV-based vectors replicate exclusively in the cytoplasm of transduced cells, and do not go through a DNA phase; therefore, there is no concern about the unwanted integration of foreign sequences into the host chromosomal DNA, which indeed caused a life-threatening disease in a few limited ceases.Human ES cells are expected to have clinical applications as well as to serve as a basic research tool. Foreign genes are often transferred into ES cells for genetic marking or regulating differentiation. We have previously shown that SeV vector can introduce a foreign gene efficiently and stably into cynomolgus monkey ES cells while preserving the pluripotency. The transgene expression, however, should be suppressed in cases of unexpected toxicity or when only a temporal transgene expression is necessary. Since SeV is an RNA virus, we have examined whether anti-RNA virus agents are able to remove the SeV vector from transduced ES cells.<Methods> The F-defective SeV vector carrying the GFP gene was constructed. Cynomolgus ES cells were transduced with the vector at 10 MOI per cell (transduction efficiency 60%). Fluorescent ES-cell colonies were plucked 1 month post-transduction and propagated in the regular FBS-supplemented medium (GFP-positive cells > 90%). The cells were treated with anti-viral agents which were reported to suppress the replication of SeV; ribavirin (12–20 μM), papaverine (1–100 μM), and/or prostaglandin A1 (PGA1; 0.3–30 μM). The regular culture medium was DMEM/F12 with 15% FBS. The serum-free culture medium was DMEM/F12 with 20% Knockout serum replacement (KSR, Invitrogen).<Results and Discussion> Although the GFP expression in ES cells was suppressed by treatment with either ribavirin or papaverine, the cells showed differentiated phenotypes. Treatment with PGA1 led to the apoptosis of ES cells at lower concentrations than those required to suppress the GFP expression. Treatment with a mixture of these three agents successfully suppressed the GFP expression in the SSEA-4-negative, differentiated population. On the other hand, cultivation of the transduced ES cells in the KSR-supplemented medium, a widely used serum-free recipe for ES cell cultures, resulted in the complete suppression of GFP expression in ES cells in the undifferentiated state. The SeV genome and GFP gene were undetectable even by RNA-PCR. KSR was originally developed to maintain ES cells in an undifferentiated state, and this is the first report about its unexpected antiviral activities. The anti-viral effect of KSR was cancelled by the addition of 15% FBS. KSR, however, had no effect on the SeV-mediated expression of GFP in LLC-MK2, a standard control cell line for SeV transduction. Unfortunately, information on the composition of KSR is not available, and we cannot explain the mechanism by which KSR eradicates the SeV genome.<Conclusions> Although none of the antiviral agents alone; ribavirin, papaverine, or PGA1, is effective at suppressing SeV vector-mediated transgene expression in ES cells, a mixture of the three agents may be useful for suppressing the transgene expression after the differentiation of the transduced ES cells. On the other hand, KSR should be useful in terms of eradicating the SeV genome from transduced ES cells as well as maintaining the pluripotency.

HOXA11 mutation in amegakaryocytic thrombocytopenia with radio-ulnar synostosis syndrome inhibits megakaryocytic differentiation in vitro

Homeobox genes encode for regulatory proteins central to hematopoietic differentiation and proliferation. Previously, we identified an inherited syndrome of congenital amegakaryocytic thrombocytopenia and radio-ulnar synostosis that is associated with a point mutation in the third helix of HOXA11 homeodomain (HOXA11-ΔH3). Here, we demonstrate that this mutation results in a significantly truncated protein with impaired DNA-binding efficiency. Electrophoretic mobility shift assays (EMSA) confirm that wild-type HOXA11 (HOXA11-WT) interacts in vitro with the DNA-binding consensus sequence for HOXA11, and that this interaction is most efficient when the TALE transcription factor, Meis1b, is also present. However, the binding between HOXA11-ΔH3 and DNA is abrogated even in the presence of Meis1b, suggesting the point mutant causes a disruption in the DNA-binding capacity. We investigated whether the point mutation also affected the physical protein–protein interaction between HoxA11 and Meis1b. Using GST pulldown assays, we find Meis1b interactions with both HOXA11-WT and HOXA11-ΔH3 in the presence of DNA. DNAse treatment decreased these interactions, suggesting that the interaction is a protein–protein association, and DNA may serve to stabilize this interaction. Stable expression of FLAG-HOXA11-WT or -ΔH3 in K562 cells significantly impacts megakaryocytic differentiation. Staurosporine (STSP) induced K562 cells to differentiate into a megakaryocytic phenotype. Treatment leads to an increase in surface expression of the megakaryocytic/platelet-specific antigen, CD61, and causes morphological changes consistent with megakaryocytic differentiation. CD61 surface expression on STSP treated HOXA11-WT and -ΔH3 expressing cells was significantly reduced as compared to untransfected K562 cells. Interestingly, we found only a slight difference in CD61 expression between wild-type and mutant HOXA11 K562. These data suggest that HoxA11 inhibition of differentiation may involve nonhomeodomain sequences. Finally, our laboratory has detected a small amount of HoxA11 mRNA in cells isolated from unfractionated human cord blood and murine ES cell culture cocultured on OP9 for 6 days in the absence of leukemia inhibitory factor (LIF). This finding suggests HoxA11 may be endogenously expressed in very early hematopoietic precursor cells. Taken together, these data begin to give us insight into the molecular mechanisms by which HoxA11 may be involved in regulating megakaryocytic differentiation.

Endoderm patterning in embryos and in human ES cell cultures

Endoderm patterning in embryos and in human ES cell cultures

Homologous Recombination in Zebrafish ES Cells

Targeted insertion of a plasmid by homologous recombination was demonstrated in zebrafish ES cell cultures. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene. One selection strategy involved the manual isolation of targeted cell colonies that were identified by the loss of fluorescent protein gene expression. A second strategy used the diphtheria toxin A-chain gene in a positive-negative selection approach. Homologous recombination was confirmed by PCR, sequence and Southern blot analysis and colonies isolated using both selection methods were expanded and maintained for multiple passages. The results demonstrate that zebrafish ES cells have potential for use in a cell-mediated gene targeting approach.

Generation of cerebellar neuron precursors from embryonic stem cells

Here, we report in vitro generation of Math1 + cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1 +. The induced Math1 + cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1 + cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7 +/Calbindin-D28K + neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system.

Tie2Cre B-Rafflox/flox Mice Reveal a Role for B-Raf in Tpo-Mediated Thrombopoiesis and Survival of Mature Megakaryocytes in Adult Hematopoiesis.

Thrombopoietin (Tpo) is the primary cytokine regulator of megakaryocytopoiesis. Tpo engagement of its receptor, Mpl, activates the classic MAP kinase (Raf/MEK/ERK) pathway, but the biological role of MAP kinase signaling in megakaryocytopoiesis remains poorly defined. Raf family kinases control signal flow through the classic MAP kinase pathway from activated cytokine receptors. We therefore undertook a genetic approach to understand the role of MAP kinase signaling and to identify which Raf family member is important for megakaryocytopoiesis. Using data from in vitro B-raf-/- ES cell cultures, fetal liver hematopoietic cells from mid-gestation B-raf-/- embryos, and B-raf-/- chimeric mice, we recently reported that B-Raf acts in a cell autonomous manner to quantitatively affect megakaryocytopoiesis. However, limitations of the chimeric mouse model, and mid-gestation lethality of B-raf-/- embryos precluded a detailed mechanistic understanding of B-Raf activity or the evaluation of B-Raf in adult megakaryocytopoiesis. We therefore generated tissue-restricted B-Raf deficient mice by crossing B-rafflox/flox mice with mice expressing Cre recombinase controlled by the Tie2 promoter/enhancer. The Tie2Cre+/B-rafflox/flox mice were born with normal Mendelian genetics and without gross abnormalities. Circulating leukocytes demonstrated complete recombination of the floxed B-Raf allele, and western blots showed undetectable B-Raf expression in platelet, spleen, and thymus lysates, consistent with complete hematopoietic Cre-mediated recombination. Steady state platelet counts were not altered in the B-Raf deficient animals at baseline: Tie2Cre+/B-rafflox/flox mice = 788 +/−57 x 103/mm3; Tie2Cre-/B-rafflox/flox mice 800 +/−40 x 103/mm3 (p=0.76, n=4). However, Tie2Cre+/B-rafflox/flox mice had a markedly impaired platelet count rise following Tpo injection, with peak counts of 3,375 +/−752 x 103/mm3 compared with 5,320 +/−606 x 103/mm3 for Tie2Cre-/B-rafflox/flox mice (p=0.0147, n=4) at 6 days post injection. In vitro expansion of CD41+ cells from Tie2Cre+/B-rafflox/flox bone marrow was only a third that of control mice, suggesting that the impaired in vivo platelet rise following Tpo reflects, at least in part, a decreased expansion of megakaryocyte lineage cells. Day 4 Tie2Cre+/B-rafflox/flox bone marrow cultures also yielded decreased low (2N-8N) and high (>32N) ploidy CD41+ cells compared with marrow from Tie2Cre-/B-rafflox/flox mice, while intermediate (16N-32N) ploidy CD41+ megakaryocytes were relatively preserved. TUNEL analysis revealed increased apoptotic death of the high ploidy (>32N) cells, a second possible mechanism contributing to the impaired platelet rise following Tpo injection. Together, our data demonstrate that the B-Raf/MAP kinase pathway is required for normal adult Tpo-induced thrombopoiesis through its effect on megakaryocyte lineage expansion and apoptotic cell death of mature megakaryocytes. Additional analysis is now underway to more fully define the role of B-Raf in megakaryocytopoiesis, including detailed biochemical studies to determine how the absence of B-Raf impacts intracellular signaling during this complex developmental process.

CD41+ and Vascular Endothelial (VE)-Cadherin+ Cells: Two Developmental Origins of Hematopoietic Cell Lineages That Show Different Hematogenic Potentials.

It has been proposed that the definitive hematopoietic cell lineages are derived from hemogenic endothelial cells. Recently, CD41 was identified as an earliest cell surface marker of hematopoietic progenitor cells during mouse embryogenesis. We examined relationship between VE-cadherin+ hemogenic endothelial cells and CD41+ progenitors as developmental origins of hematopoietic cells by using in vitro differentiation system of ES cells as well as mouse embryos.FACS analyses on ES cells differentiating on OP9 stromal cells identified two cell populations, CD41+CD45− and CD41lowVE-cadherin+CD45−. The CD41+ cell population was derived from Flk1+ cells that represent lateral plate mesoderm but not from PDGFRa+ cells that represent paraxial mesoderm. CD41 expression on CD41+CD45− cells was weak at Day4 of ES cell culture. CD41+CD45− cells rapidly increased in number and CD41 expression became higher after Day5. CD45+ cells became detectable as a subpopulation of CD41+ cells two days after the appearance of the CD41+CD45− cell population. A significant proportion of the purified CD41lowVE-cadherin+CD45− cells differentiated to cells with CD41+CD45− phenotype in short-term culture, while CD41+CD45− cells did not differentiate into VE-cadherin+ cells. Unsurprisingly only CD41lowVE-cadherin+CD45− population had potential to produce endothelial cell colonies on OP9 cell layer. Liquid cultures and methylcellulose colony assay with a proper combination of cytokines showed that primitive erythroid colony forming cells were highly enriched in the CD41+CD45− cell population. CD41+CD45− cells also differentiated to Ter119+ definitive erythrocytes and Gr-1+ and Mac-1+ myeloid cells. In contrast, CD41lowVE-cadherin+CD45− cells produced only few hematopoietic cells in the same condition. However, CD41lowVE-cadherin+CD45− cells were capable of differentiating into multi-lineage hematopoietic cells including B lymphocytes when cultured with OP9 stromal cells. CD41+CD45− cells did not show any B lymphogenic potentials even when cultured with OP9 cells.We examined hemogenic potentials of phenotypically equivalent cells purified from mouse embryos. FACS analyses on cells dissociated from yolk sac and lower trunk of embryos proper revealed two distinct populations, CD41+CD45− cells and CD41−/lowVE-cadherin+CD45− cells. Both populations were already detectable in 8.5 dpc embryos. CD41−/lowVE-cadherin+CD45− cells but not CD41+CD45− cells produced endothelial cell colonies in vitro. The CD41−/lowVE-cadherin+CD45− cell population isolated from yolk sac was able to differentiate into multi-lineage hematopoietic cells when cultured with OP9 cells. However, the same population that was isolated from embryos proper had very poor potential to generate erythroid and myeloid cells although it still initiated robust production of B lymphocytes. Nevertheless, hemogenic activities of this population declined to undetectable level on 11.5 dpc. In contrast, CD41+CD45− cells isolated from yolk sac and embryos proper gave rise to multilineage hematopoietic cells and those potentials were stronger than that of yolk sac-derived CD41−/lowVE-cadherin+CD45− cells. Our results suggest that two distinct precursors, hemogenic endothelial cells and CD41+ progenitor cells, may contribute to the initiation of definitive hematopoiesis in mouse ontogeny, although activity of hemogenic endothelial cells in embryo proper might be unexpectedly limited.