Jerry Chan

Human first-trimester fetal MSC express pluripotency markers and grow faster and have longer telomeres than adult MSC.

The biological properties of stem cells are key to the success of cell therapy, for which MSC are promising candidates. Although most therapeutic applications to date have used adult bone marrow MSC, increasing evidence suggests that MSC from neonatal and mid‐gestational fetal tissues are more plastic and grow faster. Fetal stem cells have been isolated earlier in development, from first‐trimester blood and hemopoietic organs, raising the question of whether they are biologically closer to embryonic stem cells and thus have advantages over adult bone marrow MSC. In this study, we show that human first‐trimester fetal blood, liver, and bone marrow MSC but not adult MSC express the pluripotency stem cell markers Oct‐4, Nanog, Rex‐1, SSEA‐3, SSEA‐4, Tra‐1‐60, and Tra‐1‐81. In addition, fetal MSC, irrespective of source, had longer telomeres (p < .001), had greater telomerase activity (p < .01), and expressed more human telomerase reverse transcriptase (p < .01). Fetal MSC were also more readily expandable and senesced later in culture than their adult counterparts (p < .01). Compared with adult MSC, first‐trimester fetal tissues constitute a source of MSC with characteristics that appear advantageous for cell therapy.

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.

Murine but not human mesenchymal stem cells generate osteosarcoma-like lesions in the lung.

Murine mesenchymal stem cells are capable of differentiation into multiple cell types both in vitro and in vivo and may be good candidates to use as cell therapy for diseased or damaged organs. We have previously reported a method of enriching a population of murine MSCs that demonstrated a diverse differentiation potential both in vitro and in vivo. In this study, we show that this enriched population of murine mesenchymal stem cells embolize within lung capillaries following systemic injection and then rapidly expand within, and invade into, the lung parenchyma, forming tumor nodules. These lesions rarely contain cells bearing the immunohistochemical characteristics of lung epithelium, but they do show the characteristics of immature bone and cartilage that resembles exuberant fracture callus or well‐differentiated osteosarcoma. Our findings indicate that murine mesenchymal stem cells can behave in a manner similar to tumor cells, with dysregulated growth and aberrant differentiation within the alveolar microenvironment after four passages. We demonstrate that unlike human MSCs, MSCs from different mouse strains can acquire chromosomal abnormalities after only a few in vitro passages. Moreover, other parameters, such as mouse strain used, might also play a role in the induction of these tumors. These findings might be clinically relevant for future stem cell therapy studies.

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.

First trimester embryo-fetoscopic and ultrasound-guided fetal blood sampling for ex vivo viral transduction of cultured human fetal mesenchymal stem cells

BACKGROUND Intrauterine stem cell transplantation is a promising approach for early onset genetic diseases. However, its utility is limited by the development of the fetal immune system after 14 weeks gestation. An ex vivo gene therapy approach targeting autologous first trimester stem cells to replace the missing or defective gene product should overcome this barrier. We investigated the feasibility of harvesting circulating first trimester human fetal mesenchymal stem cells (hfMSCs) for ex vivo gene therapy. METHODS Thin-gauge embryofetoscopic-directed or ultrasound-guided blood sampling (FBS) was performed in 18 pre-termination fetuses at a mean of 10(+0) (range 7(+2) to 13(+4)) weeks gestation through extra-fetal vessels. Harvested blood was plated for isolation of hfMSC and transduced by lentiviruses. RESULTS FBS was successful in 12/18 procedures (67%). Success rates were comparable in fetoscopic (4/6) and ultrasound-guided (8/12) procedures, but procedural time was shorter in the ultrasound-guided arm (P = 0.01). Fetal bradycardia occurred post-FBS in 33% and 25% of fetoscopic and ultrasound cases, respectively, 5 min post-procedure. hfMSCs were isolated in two-thirds of cases, with high efficiency lentiviral transduction achieved without affecting short-term cell renewal. CONCLUSIONS This phase-one study demonstrates the feasibility of the ex vivo fetal gene therapy approach, in which harvested hfMSCs are genetically manipulated prior to infusion back into the fetus where they should engraft and home to injured tissues. The fetal ex vivo gene therapy paradigm is also of relevance to haemopoietic stem cells to treat inherited haematological diseases. Optimization of stem cell harvest and longer-term safety is required before translation into clinical trials in ongoing pregnancies.

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.