Keelin O'Donoghue

Microchimerism in female bone marrow and bone decades after fetal mesenchymal stem-cell trafficking in pregnancy

Fetal cells enter maternal blood during pregnancy and persist in women with autoimmune disease. The frequency of subsequent fetomaternal microchimerism in healthy women and its cell type is unknown. To test the hypothesis that fetal mesenchymal stem cells persist in maternal organs, we studied female bone marrow and ribs. Male cells were identified by XY fluorescence in-situ hybridisation in marrow-derived mesenchymal stem cells and in rib sections from all women with male pregnancies, but not in controls (9/9 vs 0/5, p=0.0005). We conclude that fetal stem cells transferred into maternal blood engraft in marrow, where they remain throughout life. This finding has implications for normal pregnancy, for obstetric complications that increase fetomaternal trafficking, and for graft survival after transplantation.

Human Fetal Mesenchymal Stem Cells as Vehicles for Gene Delivery

First‐trimester fetal blood contains a readily expandable population of stem cells, human fetal mesenchymal stem cells (hfMSCs), which might be exploited for autologous intrauterine gene therapy. We investigated the self‐renewal and differentiation of hfMSCs after transduction with onco‐retroviral and lentiviral vectors. After transduction with either a MoMuLV retrovirus or an HIV‐1‐based lentiviral vector carrying the β‐galactosidase and green fluorescent reporter gene, respectively, transgene expression, self‐renewal, and differentiation capabilities were assessed 2 and 14 weeks later. Transduction with the lentiviral vector resulted in higher efficiencies than with the MoMuLV‐based vector (mean, 97.7 ± 1.4% versus 80.2 ± 5.4%; p = .02). Transgene expression was maintained with lentiviral‐transduced cells (94.6 ± 2.6%) but decreased over 14 weeks in culture with onco‐retroviral‐transduced cells (48.3 ± 3.9%). The self‐renewal capability of these cells and their ability to undergo osteogenic, adipogenic, and myogenic differentiation was unimpaired after transduction with either vector. Finally, clonal expansion of lentivirally modified cells was expanded over 20 population doublings with maintenance of multiline age differentiation capacity. These results suggest that hfMSCs may be suitable targets for ex vivo genetic manipulation with onco‐retroviral or lentiviral vectors without affecting their stem cell properties.

Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration

Cell therapy for degenerative muscle diseases such as the muscular dystrophies requires a source of cells with the capacity to participate in the formation of new muscle fibers. We investigated the myogenic potential of human fetal mesenchymal stem cells (hfMSCs) using a variety of stimuli. The use of 5‐azacytidine or steroids did not produce skeletal muscle differentiation, whereas myoblast‐conditioned medium resulted in only 1%–2% of hfMSCs undergoing muscle differentiation. However, in the presence of galectin‐1, 66.1% ± 5.7% of hfMSCs, but not adult bone marrow‐derived mesenchymal stem cells, assumed a muscle phenotype, forming long, multinucleated fibers expressing both desmin and sarcomeric myosin via activation of muscle regulatory factors. Continuous exposure to galectin‐1 resulted in more efficient muscle differentiation than pulsed exposure (62.3% vs. 39.1%; p < .001). When transplanted into regenerating murine muscle, galectin‐1‐exposed hfMSCs formed fourfold more human muscle fibers than nonstimulated hfMSCs (p = .008), with similar results obtained in a scid/mdx dystrophic mouse model. These data suggest that hfMSCs readily undergo muscle differentiation in response to galectin‐1 through a stepwise progression similar to that which occurs during embryonic myogenesis. The high degree of myogenic conversion achieved by this method has relevance for the development of therapies for muscular dystrophies.

Microchimeric fetal cells cluster at sites of tissue injury in lung decades after pregnancy

Fetal cells trafficking into maternal blood during pregnancy engraft tissues and persist for decades in marrow and bone. While persistent fetal cells were initially implicated in autoimmune disease, animal studies suggest that microchimeric fetal cells play a broader role in response to tissue injury. This study investigated whether fetal cells participate in tissue repair after human pregnancy. Specimens were obtained from women undergoing surgery for suspected lung cancer. Y-fluorescence in-situ hybridization was performed on paraffin-embedded sections, with the investigator blinded to medical histories. Male cells were identified in lung/thymus tissue from all women with known male pregnancies, but not in those without sons. The frequency of male microchimeric cells was seven-fold greater in lung/thymus tissues than marrow and was two-fold greater than normal bone from the same women. Nested-polymerase chain reaction for sex determining region Y confirmed male DNA in tissues. Male cells in lung were clustered in tumour rather than surrounding healthy tissues. In conclusion, male presumed-fetal cells were identified in pathological post-reproductive tissues, where they were more likely to be located in diseased tissues at several-fold higher frequency than normal tissues. It is suggested that fetal cells are present at sites of tissue injury and may be stem cells, either recruited from marrow or having proliferated locally.

Stage I twin–twin transfusion syndrome: rates of progression and regression in relation to outcome

Twin–twin transfusion syndrome (TTTS) results in high rates of perinatal mortality and neurological morbidity. Fetoscopic laser ablation of placental anastomoses is now established as the treatment of choice for advanced disease. However, there remains controversy about its use in early‐stage TTTS, in which laser‐related fetal losses need to be balanced against relatively favorable outcomes with more conservative approaches. We investigated rates of progression and regression in Stage I TTTS and determined factors influencing the course of the disease.

Differentiation of human fetal mesenchymal stem cells into cells with an oligodendrocyte phenotype

The potential of mesenchymal stem cells (MSC) to differentiate into neural lineages has raised the possibility of autologous cell transplantation as a therapy for neurodegenerative diseases. We have identified a population of circulating human fetal mesenchymal stem cells (hfMSC) that are highly proliferative and can readily differentiate into mesodermal lineages such as bone, cartilage, fat and muscle. Here, we demonstrate for the first time that primary hfMSC can differentiate into cells with an oligodendrocyte phenotype both in vitro and in vivo. By exposing hfMSC to neuronal conditioned medium or by introducing the pro-oligodendrocyte gene, Olig-2, hfMSC adopted an oligodendrocyte-like morphology, expressed oligodendrocyte markers and appeared to mature appropriately in culture. Importantly we also demonstrate the differentiation of a clonal population of hfMSC into both mesodermal (bone) and ectodermal (oligodendrocyte) lineages. In the developing murine brain transplanted hfMSC integrated into the parenchyma but oligodendrocyte differentiation of these naïve hfMSC was very low. However, the proportion of cells expressing oligodendrocyte markers increased significantly (from 0.2% to 4%) by pre-exposing the cells to differentiation medium in vitro prior to transplantation. Importantly, the process of in vivo differentiation occurred without cell fusion. These findings suggest that hfMSC may provide a potential source of oligodendrocytes for study and potential therapy.

Interstitial laser therapy for fetal reduction in monochorionic multiple pregnancy: loss rate and association with aplasia cutis congenita.

To evaluate experience with interstitial laser therapy for intrafetal vascular ablation in monochorionic (MC) multiple pregnancy.

Fetal cells in the maternal appendix: a marker of inflammation or fetal tissue repair?

BACKGROUNDnFetal microchimeric cells that have trafficked into the maternal circulation persist in maternal tissues for years after pregnancy, but their biological role is unclear. We investigated whether fetal cells participate in maternal tissue repair during human pregnancy.nnnMETHODSnAppendix specimens were acquired from women undergoing appendicectomy during (n = 8) or after (n = 1) pregnancy. Fluorescence in situ hybridization (FISH) determined the presence of male presumed-fetal cells, and immunostaining indicated the fetal cell phenotype.nnnRESULTSnMale cells were identified in appendiceal tissues from all women with known present or past male pregnancies (n = 7) and from a woman with a previous spontaneous abortion of undetermined gender (n = 1), but not in one woman with three daughters. One woman was only 6 weeks pregnant at appendicectomy. Male cells were evenly distributed through appendix tissues, in larger numbers where there was a greater degree of inflammation and when the current pregnancy was male. Combined immunostaining and Y-FISH demonstrated male desmin+ muscle cells and CD3+ lymphocytes, suggesting fetal cells had differentiated.nnnCONCLUSIONSnMale-presumed fetal cells of haematopoietic and mesenchymal origin were identified in the appendix of all pregnant women who had sons. We suggest that fetal cells are present at sites of maternal tissue injury during pregnancy, and may participate in tissue repair.

Identification of candidate surface antigens for non-invasive prenatal diagnosis by comparative global gene expression on human fetal mesenchymal stem cells

Transplacental passage of circulating first-trimester fetal mesenchymal stem cells (fMSC) raises the prospect of harvesting fetal cells in maternal blood. Despite high sensitivity in model systems, negative selection and culture strategies yield fMSC only rarely in post-termination maternal blood. The different adhesion molecule profile of fMSC to competitor maternal cell types suggests that improved positive selection strategies may facilitate non-invasive prenatal diagnosis. We aimed to identify surface antigens specific to fMSC and not maternal peripheral blood lymphocytes (PBL), using genome-wide analysis of actively expressed transcripts. Maternal PBL and fMSC cultured from first-trimester blood, liver and bone marrow were assessed for global gene expression by Affymetrix U133Plus2.0 arrays. Data were analysed using Affymetrix GCOS01.2. Transcripts present in all fMSC (n = 9) but absent in all PBL samples (n = 3) were selected for further analysis of cell-surface membrane molecules by RT-PCR and immunocytochemistry. Of 1544 genes expressed in fMSC and not maternal PBL, filtering for cell-surface molecules yielded 159 genes. Of these, 29 had a mean expression ratio of >300 (P < 0.001), which represented 18 unique genes, and their positive expression in all fMSC samples was confirmed by RT-PCR. Candidates for non-invasive prenatal diagnosis were chosen for further analysis by immunocytochemistry. Surface expression of OSMR and COL1 proteins on all fMSC, but no maternal PBL was confirmed. Identification of novel surface antigens on circulating human fMSC and not maternal PBL facilitates positive selection strategies for isolating fMSC for non-invasive prenatal diagnosis.

The effect of polyhydramnios on cervical length in twins: a controlled intervention study in complicated monochorionic pregnancies.

Objective To test the hypothesis that cervical shortening in polyhydramnios reflects the degree of excess amniotic fluid, and increases with normalisation of amniotic fluid volume. Study Design Prospective cohort study of 40 women with monochorionic twins undergoing interventional procedures between 16–26 weeks. Cervical length was assessed via transvaginal sonography pre-procedure, 1 and 24 hours post-procedure, and results compared between amnioreduction and control procedures. Amniotic fluid index (AFI) was measured pre- and post-procedure. Results Pre-procedural cervical length correlated with AFI (linear fitu200a=u200a5.07 -0.04x, R2u200a=u200a0.17, Pu200a=u200a0.03) in patients with polyhydramnios (nu200a=u200a28). Drainage of 2000ml fluid (range 700–3500ml), reduced AFI from 42cm to 21cm (P<0.001). Their pre-procedural cervical length did not change at one (mean Δ:−0.1cm, 95%CI, −0.4 to 0.2) or 24 hours (0.2cm, −0.1 to 0.6) after amnioreduction. There was no change in cervical length at control procedures. Conclusion Cervical shortening in twins with polyhydramnios does not appear to be an acute process; cervical length can be measured before or after therapeutic procedures.