Lars Martin Jakt

Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells

Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2Rα (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+Sox17+ definitive endoderm and Gsc−Sox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.

Intrinsic transition of embryonic stem-cell differentiation into neural progenitors

The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521.

In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility

Endothelial cells (ECs) are thought to be derived mainly from the vascular endothelial growth factor receptor 2 (VEGFR‐2)+ lateral mesoderm during early embryogenesis. In this study, we specified several pathways for EC differentiation using a murine embryonic stem (ES) cell differentiation culture system that is a model for cellular processes during early embryogenesis. Based on the results of in vitro fate analysis, we show that, in the main pathway, committed ECs are differentiated through the VEGFR‐2+ platelet‐derived growth factor receptor α (PDGFR‐α)− single‐positive (VSP) population that is derived from the VEGFR‐2+PDGFR‐α+ double‐positive (DP) population. This major differentiation course was also confirmed using DNA microarray analysis. In addition to this main pathway, however, ECs also can be generated from the VEGFR‐2−PDGFR‐α+ single‐positive (PSP) population, which represents the paraxial mesodermal lineage and is also derived from the DP population. Our results strongly suggest that, even after differentiation from the common progenitor DP population into the VSP and PSP populations, these two populations continue spontaneous switching of their surface phenotype, which results in switching of their eventual fates. The rate of this interlineage conversion between VSP and PSP is unexpectedly high. Because of this potential to undergo fate switch, we conclude that ECs can be generated via multiple pathways in in vitro ES cell differentiation.

Embryonic stem-cell culture as a tool for developmental cell biology

The cell biology of the early processes of mammalian embryogenesis, such as germ-layer formation, has been technically challenging to study owing to the size and accessibility of mammalian embryos. Embryonic stem cells, which can generate the three germ layers in vitro, are useful for studying embryogenesis at the cellular level. So, how can the study of embryonic stem cells and their differentiation provide a deeper understanding of the cell biology of early development?

Effective generation of iPS cells from CD34+ cord blood cells by inhibition of p53

OBJECTIVEnCord blood banks provide fully human leukocyte antigen-typed cells, from which a set of standard induced pluripotent stem (iPS) cells for use in allogenic transplantation can be derived. Hence, the ability to generate iPS cells from cord blood cells has the potential to provide a suitable source for clinical transplantation. The aim of this work is to determine the reprogramming methods, culture conditions, and cell fractions that can be used to generate iPS cells from cord blood cells effectively.nnnMATERIALS AND METHODSnCD34(+), mononucleated, and derived adherent cells from cord blood were cultured in hematopoietic medium (X-vivo10 containing 50 ng/mL interleukin-6, 50 ng/mL soluble interleukin-6 receptor, 50 ng/mL stem cell factor, 10 ng/mL thrombopoietin, and 20 ng/mL Flit3/4 ligand) 3 days prior to viral infection. Cells were then infected with retroviral constructs driving the expression of OCT3/4, SOX2, Krüppel-like factor 4, c-MYC, and enhanced green fluorescent protein together with or without the p53 knockdown lentiviral construct Shp53 pLKO.1-puro. Infected cells were then cultured for an additional 4 days in hematopoietic culture medium before being transferred onto mouse embryonic fibroblast (MEF) or SNL76/7 feeder cells in human embryonic stem cell medium (Dulbeccos modified Eagle medium/F-12 containing 20% knockout serum replacement, 200 mM L-glutamine, 1% non-essential amino acids (NEAA), 0.1 mM 2-mercaptoethanol, and 4 ng/mL basic fibroblast growth factor). Subsequently, the number of embryonic stem cell-like colonies that emerged in the following 4 weeks was scored. Expression of a number of pluripotency makers were examined by immunochemistry and reverse transcriptase polymerase chain reaction. Finally, the differentiation potential of selected colonies was determined by teratoma formation in severe combined immunodeficient mice and in vitro culture.nnnRESULTSnRepression of p53 expression by the addition of a lentiviral p53 short-hairpin RNA expression vector increased the frequency of formation of iPS-like colonies from 1 (on average) to around 100 per 2 x 10(4) cells when infected cells were grown on SNL feeder cells.nnnCONCLUSIONSniPS cells can be generated easily from CD34(+) cord blood cells through the addition of p53 inhibition to standard reprogramming conditions.

Necdin restricts proliferation of hematopoietic stem cells during hematopoietic regeneration.

Hematopoietic stem cell (HSC) proliferation is tightly regulated by a poorly understood complex of positive and negative cell-cycle regulatory mechanisms. Necdin (Ndn) is an evolutionally conserved multifunctional protein that has been implicated in cell-cycle regulation of neuronal cells. Here, we provide evidence that necdin plays an important role in restricting excessive HSC proliferation during hematopoietic regeneration. We identify Ndn as being preferentially expressed in the HSC population on the basis of gene expression profiling and demonstrate that mice deficient in Ndn show accelerated recovery of the hematopoietic system after myelosuppressive injury, whereas no overt abnormality is seen in steady-state hematopoiesis. In parallel, after myelosuppression, Ndn-deficient mice exhibit an enhanced number of proliferating HSCs. Based on these findings, we propose that necdin functions in a negative feedback loop that prevents excessive proliferation of HSCs during hematopoietic regeneration. These data suggest that the inhibition of necdin after clinical myelosuppressive treatment (eg, chemotherapy, HSC transplantation) may provide therapeutic benefits by accelerating hematologic recovery.

ALCAM (CD166) Is a Surface Marker for Early Murine Cardiomyocytes

ALCAM (activated leukocyte cell adhesion molecule, CD166) belongs to the immunoglobulin superfamily and is involved in axon guidance, hematopoiesis, immune response and tumor metastasis. During embryogenesis, mRNA encoding ALCAM was expressed in the cardiac crescent and the neural groove at embryonic day (E) 7.75 and predominately in the tubular heart at E8.5. A newly generated monoclonal antibody against the ALCAM molecule (ALC-48) exclusively stained cardiomyocytes at E8.25–10.5. However, ALCAM expression was lost by cardiomyocytes by E12.5 and its expression shifts to a variety of organs during later stages. ALCAM was found to be a prominent surface marker for cardiomyocytes in early embryonic hearts. The transient expression of ALCAM during early developmental stages marks specific developmental stages in cardiomyocyte differentiation.

Transcriptomic landscape of the primitive streak.

In birds and mammals, all mesoderm cells are generated from the primitive streak. Nascent mesoderm cells contain unique dorsoventral (D/V) identities according to their relative ingression position along the streak. Molecular mechanisms controlling this initial phase of mesoderm diversification are not well understood. Using the chick model, we generated high-quality transcriptomic datasets of different streak regions and analyzed their molecular heterogeneity. Fifteen percent of expressed genes exhibit differential expression levels, as represented by two major groups (dorsal to ventral and ventral to dorsal). A complete set of transcription factors and many novel genes with strong and region-specific expression were uncovered. Core components of BMP, Wnt and FGF pathways showed little regional difference, whereas their positive and negative regulators exhibited both dorsal-to-ventral and ventral-to-dorsal gradients, suggesting that robust D/V positional information is generated by fine-tuned regulation of key signaling pathways at multiple levels. Overall, our study provides a comprehensive molecular resource for understanding mesoderm diversification in vivo and targeted mesoderm lineage differentiation in vitro.

A competitive protein interaction network buffers Oct4-mediated differentiation to promote pluripotency in embryonic stem cells

Pluripotency in embryonic stem cells is maintained through the activity of a small set of transcription factors centred around Oct4 and Nanog, which control the expression of ‘self‐renewal’ and ‘differentiation’ genes. Here, we combine single‐cell quantitative immunofluorescence microscopy and gene expression analysis, together with theoretical modelling, to investigate how the activity of those factors is regulated. We uncover a key role for post‐translational regulation in the maintenance of pluripotency, which complements the well‐established transcriptional regulatory layer. Specifically, we find that the activity of a network of protein complexes involving Nanog, Oct4, Tcf3, and β‐catenin suffices to account for the behavior of ES cells under different conditions. Our results suggest that the function of the network is to buffer the transcriptional activity of Oct4, which appears to be the main determinant to exit pluripotency. The protein network explains the mechanisms underlying the gain and loss of function in different mutants, and brings us closer to a full understanding of the molecular basis of pluripotency.

The novel protein kinase Vlk is essential for stromal function of mesenchymal cells

From a list of protein kinases (PKs) that are newly induced upon differentiation of mouse embryonic stem cells to mesendoderm, we identified a previously uncharacterized kinase, Vlk (vertebrate lonesome kinase), that is well conserved in vertebrates but has no homologs outside of the vertebrate lineage. Its kinase domain cannot be classified into any of the previously defined kinase groups or families. Although Vlk is first expressed in E-cadherin-positive anterior visceral endoderm and mesendoderm, its expression is later confined to E-cadherin-negative mesenchyme. Vlk is enriched in the Golgi apparatus and blocks VSVG transport from the Golgi to the plasma membrane. Targeted disruption of Vlk leads to a defect in lung development and to delayed ossification of endochondral bone. Vlk-/- mice display neonatal lethality due to respiratory failure, with a suckling defect arising from a cleft palate. Our results demonstrate that Vlk is a novel vertebrate-specific PK that is involved in the regulation of the rate of protein export from the Golgi, thereby playing an important role in the formation of functional stroma by mesenchymal cells.