No abstract

A method for stimulating the differentiation of human pluripotent stem cells (hPSCs) into kidney lineages remains to be developed. Most cells in kidney are derived from an embryonic germ layer known as intermediate mesoderm (IM). Here we show the establishment of an efficient system of homologous recombination in hPSCs by means of bacterial artificial chromosome (BAC)-based vectors and single nucleotide polymorphism (SNP) array-based detection. This system allowed us to generate human induced pluripotent stem cell (hiPSC) lines containing green fluorescence protein (GFP) knocked into OSR1, a specific marker for IM. We have also established a robust induction protocol for IM, which produces up to 90% OSR1+ cells. These human IM cells can differentiate into multiple cell types of IM-derived organs in vitro and in vivo, thereby supplying an unprecedented system to elucidate the mechanisms of IM development and potentially providing a cell source for regenerative therapies of the kidney.

The nucleotide sequences of STE2 and STE3, cell type‐specific sterile genes of Saccharomyces cerevisiae, were determined; major open reading frames encode 431 and 470 amino acids, respectively. STE2 and STE3 proteins seem to be folded in a similar fashion and are likely to be membrane‐bound. Both consist of seven hydrophobic segments in each NH2‐terminal region with a long hydrophilic domain in each COOH‐terminal region. However, the two putative gene products do not exhibit extensive sequence homology. The STE2 protein has no obvious hydrophobic signal peptide; the NH2 terminus of the STE3 protein might serve as a signal peptide. The STE2 transcript, 1.7 kb, was detected in MATa strains but not in MATα strains, while the STE3 transcript, also 1.7 kb, was detected only in MATα cells. In STE2, two canonical TATA sequences are located 18 and 27 bp upstream of the mRNA start site, which has been mapped 32 bp before the initiator ATG codon, while STE3 contains a similar sequence (TATAGA), which is preceded by a long AT sequence, 140 bp upstream of the initiator ATG codon. Transcription of STE2 in a cells seems to be enhanced by exogenous α‐factor.

The receptors for IL-3, GM-CSF, and IL-5 share a common beta subunit (beta c), and mice have an additional IL-3 beta subunit (beta IL3). We have independently generated mice carrying null mutations of each molecule. beta c mutant bone marrow showed no response to GM-CSF or IL-5, whereas IL-3 stimulation of beta c or beta IL3 mutant bone marrow was normal. beta c mutant mice showed lung pathology consisting of lymphocytic infiltration and areas resembling alveolar proteinosis, and also exhibited low basal numbers of eosinophils. Infection of beta c mutant mice by Nippostrongylus brasiliensis resulted in the absence of blood and lung eosinophilia. Animals repopulated with beta c mutant bone marrow cells showed slower leukocyte recovery and reduced eosinophil numbers. These data define the role of beta c in vivo, and show a phenotype that is likely to be the cumulative effect of loss of GM-CSF and IL-5 stimulation.

The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. Among the resulting cells, those expressing flk-1 and/or PDGFRα displayed chondrogenic activity in the presence of TGFβ3 and expressed cartilage-specific genes in 7 to 16 day pellet cultures. Depositions of cartilage matrix and type II collagen were detected by day 14. TGFβ-stimulated chondrogenesis was synergistically enhanced by PDGF-BB, resulting in a larger cartilage particle filled with a cartilaginous area containing type II collagen, with a surface cell layer expressing type I collagen. In contrast, noggin inhibited both the TGFβ-and TGFβ+PDGF-stimulated cartilage formation, suggesting that a BMPdependent pathway is involved. In fact, replacement of TGFβ3 with BMP4 on days 10 to 12 markedly elevated the cartilage matrix deposition during the following 7 to 8 days. Moreover, culture with TGFβ3 and PDGF-BB, followed by the incubation with BMP4 alone, resulted in a cartilage particle lacking type I collagen in the matrix and the surface layer, which suggests hyaline cartilage formation. Furthermore, such hyaline cartilage particles were mineralized. These studies indicate that the PDGFRα + and/or flk-1 + cells derived from embryonic stem cells possess the full developmental potential toward chondrocytes, in common with embryonic mesenchymal cells.

Directed specification and prospective isolation of chondrogenic paraxial mesoderm progeny from human pluripotent stem (PS) cells have not yet been achieved. Here we report the successful generation of KDR−PDGFRα+ progeny expressing paraxial mesoderm genes and the mesendoderm reporter MIXL1-GFP in a chemically defined medium containing the canonical WNT signaling activator, BMP-inhibitor, and the Nodal/Activin/TGFβ signaling controller. Isolated (GFP+)KDR−PDGFRα+ mesoderm cells were sensitive to sequential addition of the three chondrogenic factors PDGF, TGFβ and BMP. Under these conditions, the cells showed robust chondrogenic activity in micromass culture, and generated a hyaline-like translucent cartilage particle in serum-free medium. In contrast, both STRO1+ mesenchymal stem/stromal cells from adult human marrow and mesenchymal cells spontaneously arising from hPS cells showed a relatively weaker chondrogenic response in vitro, and formed more of the fibrotic cartilage particles. Thus, hPS cell-derived KDR−PDGFRα+ paraxial mesoderm-like cells have potential in engineered cartilage formation and cartilage repair.

Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dorsalizing the Xenopus embryo. A novel secreted protein, CHL (for chordin-like), with significant homology to chordin, was isolated from mouse bone marrow stromal cells. Injection of CHL RNA into Xenopus embryos induced a secondary axis. Recombinant CHL protein inhibited the BMP4-dependent differentiation of embryonic stem cells in vitro and interacted directly with BMPs, similar to chordin. However, CHL also weakly bound to TGFbetas. In situ hybridization revealed that the mouse CHL gene, located on the X chromosome, was expressed predominantly in mesenchyme-derived cell types: (1) the dermatome and limb bud mesenchyme and, later, the subdermal mesenchyme and the chondrocytes of the developing skeleton during embryogenesis and (2) a layer of fibroblasts/connective tissue cells in the gastrointestinal tract, the thick straight segments of kidney tubules, and the marrow stromal cells in adults. An exception was expression in the neural cells of the olfactory bulb and cerebellum. Interestingly, the spatiotemporal expression patterns of CHL were distinct from those of chordin in many areas examined. Thus, CHL may serve as an important BMP regulator for differentiating mesenchymal cells, especially during skeletogenesis, and for developing specific neurons.

The bcyl mutation makes the cdc33 start mutant arrest at random points in the cell cycle instead of only at Gl. We cloned and sequenced CDC33. This coding sequence is identical to that of the gene encoding the Saccharomyces cerevisiae 24-kilodalton mRNA cap-binding protein, eIF-4E. (3,8), whereas the CDC25 gene product appears to be involved in positive control of adenylate cyclase activity (4). Cyclic AMP (cAMP) exerts its effect by binding regulatory subunits (BCYJ gene product) of cAMPdependent protein kinase, thereby freeing active catalytic subunits (9). bcyl mutations render cAMP-dependent protein kinases cAMP independent. Thus bcyl can suppress the growth defect of cdc25 and cdc35 (cyrl) (4, 9). In addition, bcyl cells fail to arrest in the Gi phase of the cell cycle upon nutrient starvation (7). These findings demonstrate that cAMP-dependent protein phosphorylation is required for cell cycle initiation. To investigate the role of other genes involved in the control of start, we have chosen to study CDC33.The lengths of the Gl phase in cdc33-1 and CDC33+ strains were compared (Table 1). Since differences in the genetic backgrounds of strains have some effect on the kinetics of the cell cycle, such as the percentage of unbudded cells and doubling time, isogenic strains which differed only at the CDC33 locus were constructed. From the proportions of unbudded (Gl) and budded (S+G2+M) cells in exponentially growing cultures of cdc33-1 and CDC33+ cells, the length of the Gl phase of the total cell cycle time was calculated by the equation of Rivin and Fangman (14). The Gl period of exponentially growing cdc33-1 cells was approximately twice as long as that of CDC33+ cells. The duration of S+G2+M in cdc33-1 cells was slightly longer than that in CDC33+. These results indicate that even at a permissive temperature, the cdc33-1 mutation affected the Gl phase. To test the effect of bcyl on cdc33-J, we constructed the isogenic strain CB103 (bcyl:: URA3 cdc33-J). At the permissive temperature, the length of the Gl period in bcyl:: URA3 cdc33-1 cells was restored to that of CDC33+. * Corresponding author. However, bcyl:: URA3 cdc33-1 cells had the same length of S+G2+M as cdc33-1 cells. These results suggest that bcyl suppresses the defect of cdc33-1 within the Gl phase. When exponentially growing cultures of cdc33-1 and bcyl:: URA3 cdc33-1 cells were shifted from 25 to 35°C, the number of cells increased approximately twofold (data not shown). Upon cessation of growth, cdc33-1 cells were arrested in the Gl phase of the cell cycle, whereas bcyl::URA3 cdc33-1 cells stopped growth but the number of unbudded cells did not increase under the same conditions (Table 1). These results confirm that the bcyl mutation suppressed Gl arrest caused by cdc33-1 but did not suppress the growth defect of cdc33-1 at the restrictive temperature. These results suggest that CDC33 plays an essential role in the Gl phase that can be overcome by constitutive activation of cAMP-dependent protein kinase but that it has another function essen...