Ectopic bone formation by mesenchymal stem cells derived from human term placenta and the decidua

Mesenchymal stem cells (MSCs) are one of the most attractive cell types for cell-based bone tissue repair applications. Fetal-derived MSCs and maternal-derived MSCs have been isolated from chorionic villi of human term placenta and the decidua basalis attached to the placenta following delivery, respectively. Chorionic-derived MSCs (CMSCs) and decidua-derived MSCs (DMSCs) generated in this study met the MSCs criteria set by International Society of Cellular Therapy. These criteria include: (i) adherence to plastic; (ii) >90% expression of CD73, CD105, CD90, CD146, CD44 and CD166 combined with <5% expression of CD45, CD19 and HLA-DR; and (iii) ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. In vivo subcutaneous implantation into SCID mice showed that both bromo-deoxyuridine (BrdU)-labelled CMSCs and DMSCs when implanted together with hydroxyapatite/tricalcium phosphate particles were capable of forming ectopic bone at 8-weeks post-transplantation. Histological assessment showed expression of bone markers, osteopontin (OPN), osteocalcin (OCN), biglycan (BGN), bone sialoprotein (BSP), and also a marker of vasculature, alpha-smooth muscle actin (α-SMA). This study provides evidence to support CMSCs and DMSCs as cellular candidates with potent bone forming capacity.Gina D. Kusuma, Danijela Menicanin, Stan Gronthos, Ursula Manuelpillai, Mohamed H. Abumaree, Mark D. Pertile, Shaun P. Brennecke, Bill Kalioni

Increased methylation and decreased expression of homeobox genes TLX1, HOXA10 and DLX5 in human placenta are associated with trophoblast differentiation

Homeobox genes regulate embryonic and placental development, and are widely expressed in the human placenta, but their regulatory control by DNA methylation is unclear. DNA methylation analysis was performed on human placentae from first, second and third trimesters to determine methylation patterns of homeobox gene promoters across gestation. Most homeobox genes were hypo-methylated throughout gestation, suggesting that DNA methylation is not the primary mechanism involved in regulating HOX genes expression in the placenta. Nevertheless, several genes showed variable methylation patterns across gestation, with a general trend towards an increase in methylation over gestation. Three genes (TLX1, HOXA10 and DLX5) showed inverse gains of methylation with decreasing mRNA expression throughout pregnancy, supporting a role for DNA methylation in their regulation. Proteins encoded by these genes were primarily localised to the syncytiotrophoblast layer, and showed decreased expression later in gestation. siRNA mediated downregulation of DLX5, TLX1 and HOXA10 in primary term villous cytotrophoblast resulted in decreased proliferation and increased expression of differentiation markers, including ERVW-1. Our data suggest that loss of DLX5, TLX1 and HOXA10 expression in late gestation is required for proper placental differentiation and function.Boris Novakovic, Thierry Fournier, Lynda K. Harris, Joanna James, Claire T. Roberts, Hannah E. J. Yong, Bill Kalionis, Danièle Evain-Brion, Peter R. Ebeling, Euan M. Wallace, Richard Saffery and Padma Murth

Control of gene expression in the P2-related template coliphages

The PstI fragment (65.5% to 77.4%) of coliphage 186, known genetically to encode the major control genes, has been sequenced, and an analysis performed to assess coding capacity, transcription-translation signals, and to identify any other significant features. Our analysis indicates that the region encodes: (1) seven genes, including the int and cI genes, which overlap, the late control gene B, and two genes, named CP75 and CP76, encoding potential DNA-binding proteins; (2) a promoter pB and terminator tB for the rightward transcription of the B gene, and we predict the existence of this transcript in a lysogen; (3) a promoter pL and terminator tL for leftward transcription that encodes the int and cI genes, and represents the presumed lysogenic transcript; (4) a promoter pR for rightward transcription to give the presumed (early) lytic transcript that is overlapping and convergent with the lysogenic transcript; and finally (5), a potential operator site for repressor binding in the region of the pR promoter. Preliminary evidence is presented to support this analysis.Bill Kalionis, Ian B. Dodd, J.Barry Ega

Control of gene expression in the temperate coliphage 186 : VIII. Control of lysis and lysogeny by a transcriptional switch involving face-to-face promoters

The lysogenic and early lytic operons of the temperate coliphage 186 are transcribed divergently. Primer extension mapping of the 5' ends of these in vivo transcripts showed that the rightward lytic promoter, pR, and the leftward lysogenic promoter, pL, are arranged face-to-face, with their transcripts overlapping by 60 bases. We examined the control of transcription from pR and pL using galK as a reporter gene. The product of the lysogenic cI gene strongly repressed pR transcription while allowing pL transcription. The product of the lytic apl gene (formerly CP75) strongly repressed pL transcription while allowing pR transcription. Thus, the cI-pR-pL-apl region functioned as a transcriptional switch, determining whether transcription was lytic or lysogenic. Also, the cI gene product was able to stimulate pL, possibly by alleviating an inhibition of pL transcription caused by convergent transcription from pR. Other consequences of the face-to-face promoter arrangement are discussed.Ian B. Dodd, Bill Kalionis and J. Barry Ega

Reduced aldehyde dehydrogenase expression in preeclamptic decidual mesenchymal stem/stromal cells is restored by aldehyde dehydrogenase agonists

High resistance to oxidative stress is a common feature of mesenchymal stem/stromal cells (MSC) and is associated with higher cell survival and ability to respond to oxidative damage. Aldehyde dehydrogenase (ALDH) activity is a candidate "universal" marker for stem cells. ALDH expression was significantly lower in decidual MSC (DMSC) isolated from preeclamptic (PE) patients. ALDH gene knockdown by siRNA transfection was performed to create a cell culture model of the reduced ALDH expression detected in PE-DMSC. We showed that ALDH activity in DMSC is associated with resistance to hydrogen peroxide (H2O2)-induced toxicity. Our data provide evidence that ALDH expression in DMSC is required for cellular resistance to oxidative stress. Furthermore, candidate ALDH activators were screened and two of the compounds were effective in upregulating ALDH expression. This study provides a proof-of-principle that the restoration of ALDH activity in diseased MSC is a rational basis for a therapeutic strategy to improve MSC resistance to cytotoxic damage

Isolation and identification of homeobox genes from the human placenta including a novel member of the Distal-less family, DLX4

We have carried out a DNA binding site screen of a 32-week human placental cDNA library using a consensus homeodomain binding site as a probe. This study represents the first library screen carried out to isolate homeobox genes from the human placenta. We have shown that three homeobox genes known to be expressed in the embryo, HB24, GAX and MSX2 are also expressed in the placenta. We have also identified a novel homeobox gene, DLX4, that shows 85% sequence identity with the homeodomain encoded by the Drosophila Distal-less (Dll) gene. DLX4 therefore represents a new member of the Distal-less family of homeobox genes. This is the first evidence that members of the Distal-less family of homeobox genes are expressed in the placenta. Using fluorescence in situ hybridisation (FISH), DLX4 has been assigned to human chromosome 17q21-q22. This places DLX4 in the same region of chromosome 17 as another member of the Distal-less family, DLX3 (Scherer et al., 1995), and the HOX-B homeobox gene cluster (Acampora et al., 1989: Boncinelli et al., 1991). Members of the Distal-less family (DLX1 and DLX2; DLX5 and DLX6) are found as closely linked pairs on human chromosomes (Simeone et al., 1994). We predict that DLX3 and DLX4 are closely linked and have arisen through gene duplication and divergence from a common ancestral precursor.Leonie M Quinn, Brett V Johnson, Jillian Nicholl, Grant R Sutherland, Bill Kalioni