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1.  Prospective Surface Marker-Based Isolation and Expansion of Fetal Endothelial Colony-Forming Cells From Human Term Placenta 
Stem Cells Translational Medicine  2013;2(11):839-847.
A robust strategy was developed to isolate fetal endothelial colony-forming cells (ECFCs) from human term placentas, yielding much larger quantities of ECFCs than umbilical cord blood. The cells were comparable in immunophenotype, gene expression, and in vivo functional ability. The use of this placental source for ECFCs is expected to enhance progress toward clinical translation and future bio-banking applications.
The term placenta is a highly vascularized tissue and is usually discarded upon birth. Our objective was to isolate clinically relevant quantities of fetal endothelial colony-forming cells (ECFCs) from human term placenta and to compare them to the well-established donor-matched umbilical cord blood (UCB)-derived ECFCs. A sorting strategy was devised to enrich for CD45−CD34+CD31Lo cells prior to primary plating to obtain pure placental ECFCs (PL-ECFCs) upon culture. UCB-ECFCs were derived using a well-described assay. PL-ECFCs were fetal in origin and expressed the same cell surface markers as UCB-ECFCs. Most importantly, a single term placenta could yield as many ECFCs as 27 UCB donors. PL-ECFCs and UCB-ECFCs had similar in vitro and in vivo vessel forming capacities and restored mouse hind limb ischemia in similar proportions. Gene expression profiles were only minimally divergent between PL-ECFCs and UCB-ECFCs, probably reflecting a vascular source versus a circulating source. Finally, PL-ECFCs and UCB-ECFCs displayed similar hierarchies between high and low proliferative colonies. We report a robust strategy to isolate ECFCs from human term placentas based on their cell surface expression. This yielded much larger quantities of ECFCs than UCB, but the cells were comparable in immunophenotype, gene expression, and in vivo functional ability. We conclude that PL-ECFCs have significant bio-banking and clinical translatability potential.
PMCID: PMC3808199  PMID: 24106336
Endothelial cell; Placenta; Progenitor cells; Angiogenesis
2.  Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymal stem/stromal cells 
BMC Cell Biology  2014;15:15.
Fetal mesenchymal stem/stromal cells (MSC) represent a developmentally-advantageous cell type with translational potential.
To enhance adult MSC migration, studies have focussed on the role of the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12), but more recent work implicates an intricate system of CXCR4 receptor dimerization, intracellular localization, multiple ligands, splice variants and nuclear accumulation. We investigated the intracellular localization of CXCR4 in fetal bone marrow-derived MSC and role of intracellular trafficking in CXCR4 surface expression and function.
We found that up to 4% of human fetal MSC have detectable surface-localized CXCR4. In the majority of cells, CXCR4 is located not at the cell surface, as would be required for ‘sensing’ migratory cues, but intracellularly. CXCR4 was identified in early endosomes, recycling endosomes, and lysosomes, indicating only a small percentage of CXCR4 travelling to the plasma membrane. Notably CXCR4 was also found in and around the nucleus, as detected with an anti-CXCR4 antibody directed specifically against CXCR4 isoform 2 differing only in N-terminal sequence. After demonstrating that endocytosis of CXCR4 is largely independent of endogenously-produced SDF-1, we next applied the cytoskeletal inhibitors blebbistatin and dynasore to inhibit endocytotic recycling. These increased the number of cells expressing surface CXCR4 by 10 and 5 fold respectively, and enhanced the number of cells migrating to SDF1 in vitro (up to 2.6 fold). These molecules had a transient effect on cell morphology and adhesion, which abated after the removal of the inhibitors, and did not alter functional stem cell properties.
We conclude that constitutive endocytosis is implicated in the regulation of CXCR4 membrane expression, and suggest a novel pharmacological strategy to enhance migration of systemically-transplanted cells.
PMCID: PMC4065074  PMID: 24885150
Fetal mesenchymal stromal cells; Bone marrow; MSC; CXCR4; Chemokine receptor; Migration; Small molecule; Endocytosis
3.  Fetal Microchimeric Cells in a Fetus-Treats-Its-Mother Paradigm Do Not Contribute to Dystrophin Production in Serially Parous mdx Females 
Stem Cells and Development  2012;21(15):2809-2816.
Throughout every pregnancy, genetically distinct fetal microchimeric stem/progenitor cells (FMCs) engraft in the mother, persist long after delivery, and may home to damaged maternal tissues. Phenotypically normal fetal lymphoid progenitors have been described to develop in immunodeficient mothers in a fetus-treats-its-mother paradigm. Since stem cells contribute to muscle repair, we assessed this paradigm in the mdx mouse model of Duchenne muscular dystrophy. mdx females were bred serially to either ROSAeGFP males or mdx males to obtain postpartum microchimeras that received either wild-type FMCs or dystrophin-deficient FMCs through serial gestations. To enhance regeneration, notexin was injected into the tibialis anterior of postpartum mice. FMCs were detected by qPCR at a higher frequency in injected compared to noninjected side muscle (P=0.02). However, the number of dystrophin-positive fibers was similar in mothers delivering wild-type compared to mdx pups. In addition, there was no correlation between FMC detection and percentage dystrophin, and no GFP+ve FMCs were identified that expressed dystrophin. In 10/11 animals, GFP+ve FMCs were detected by immunohistochemistry, of which 60% expressed CD45 with 96% outside the basal lamina defining myofiber contours. Finally we confirmed lack of FMC contribution to statellite cells in postpartum mdx females mated with Myf5-LacZ males. We conclude that the FMC contribution to regenerating muscles is insufficient to have a functional impact.
PMCID: PMC3464073  PMID: 22731493
4.  Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells 
The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs.
PMCID: PMC3659681  PMID: 23197756
Mesenchymal stem cells; Pluripotent stem cells; Differentiation; Induced pluripotent stem cells; Embryonic stem cells
5.  Upregulating CXCR4 in Human Fetal Mesenchymal Stem Cells Enhances Engraftment and Bone Mechanics in a Mouse Model of Osteogenesis Imperfecta 
Stem cells have considerable potential to repair damaged organs and tissues. We previously showed that prenatal transplantation of human first trimester fetal blood mesenchymal stem cells (hfMSCs) in a mouse model of osteogenesis imperfecta (oim mice) led to a phenotypic improvement, with a marked decrease in fracture rate. Donor cells differentiated into mature osteoblasts, producing bone proteins and minerals, including collagen type Iα2, which is absent in nontransplanted mice. This led to modifications of the bone matrix and subsequent decrease of bone brittleness, indicating that grafted cells directly contribute to improvement of bone mechanical properties. Nevertheless, the therapeutic effect was incomplete, attributing to the limited level of engraftment in bone. In this study, we show that although migration of hfMSCs to bone and bone marrow is CXCR4-SDF1 (SDF1 is stromal-derived factor) dependent, only a small number of cells present CXCR4 on the cell surface despite high levels of internal CXCR4. Priming with SDF1, however, upregulates CXCR4 to increase the CXCR4+ cell fraction, improving chemotaxis in vitro and enhancing engraftment in vivo at least threefold in both oim and wild-type bone and bone marrow. Higher engraftment in oim bones was associated with decreased bone brittleness. This strategy represents a step to improve the therapeutic benefits of fetal cell therapy toward being curative.
PMCID: PMC3727689  PMID: 23197643
CXCR4; Stem cell transplantation; Mesenchymal stem cells; SDF1; Fetal stem cells; Osteogenesis imperfecta
6.  Valproic Acid Confers Functional Pluripotency to Human Amniotic Fluid Stem Cells in a Transgene-free Approach 
Molecular Therapy  2012;20(10):1953-1967.
Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling.
PMCID: PMC3464631  PMID: 22760542
7.  Ontological Differences in First Compared to Third Trimester Human Fetal Placental Chorionic Stem Cells 
PLoS ONE  2012;7(9):e43395.
Human mesenchymal stromal/stem cells (MSC) isolated from fetal tissues hold promise for use in tissue engineering applications and cell-based therapies, but their collection is restricted ethically and technically. In contrast, the placenta is a potential source of readily-obtainable stem cells throughout pregnancy. In fetal tissues, early gestational stem cells are known to have advantageous characteristics over neonatal and adult stem cells. Accordingly, we investigated whether early fetal placental chorionic stem cells (e-CSC) were physiologically superior to their late gestation fetal chorionic counterparts (l-CSC). We showed that e-CSC shared a common phenotype with l-CSC, differentiating down the osteogenic, adipogenic and neurogenic pathways, and containing a subset of cells endogenously expressing NANOG, SOX2, c-MYC, and KLF4, as well as an array of genes expressed in pluripotent stem cells and primordial germ cells, including CD24, NANOG, SSEA4, SSEA3, TRA-1-60, TRA-1-81, STELLA, FRAGILIS, NANOS3, DAZL and SSEA1. However, we showed that e-CSC have characteristics of an earlier state of stemness compared to l-CSC, such as smaller size, faster kinetics, uniquely expressing OCT4A variant 1 and showing higher levels of expression of NANOG, SOX2, c-MYC and KLF4 than l-CSC. Furthermore e-CSC, but not l-CSC, formed embryoid bodies containing cells from the three germ layer lineages. Finally, we showed that e-CSC demonstrate higher tissue repair in vivo; when transplanted in the osteogenesis imperfecta mice, e-CSC, but not l-CSC increased bone quality and plasticity; and when applied to a skin wound, e-CSC, but not l-CSC, accelerated healing compared to controls. Our results provide insight into the ontogeny of the stemness phenotype during fetal development and suggest that the more primitive characteristics of early compared to late gestation fetal chorionic stem cells may be translationally advantageous.
PMCID: PMC3433473  PMID: 22962584
8.  The effects of culture on genomic imprinting profiles in human embryonic and fetal mesenchymal stem cells 
Epigenetics  2011;6(1):52-62.
Human embryonic stem (hES) cells and fetal mesenchymal stem cells (fMSC) offer great potential for regenerative therapy strategies. It is therefore important to characterize the properties of these cells in vitro. One major way the environment impacts on cellular physiology is through changes to epigenetic mechanisms. Genes subject to epigenetic regulation via genomic imprinting have been characterized extensively. The integrity of imprinted gene expression therefore provides a measurable index for epigenetic stability. Allelic expression of 26 imprinted genes and DNA methylation at associated differentially methylated regions (DMRs) was measured in fMSC and hES cell lines. Both cell types exhibited monoallelic expression of 13 imprinted genes, biallelic expression of six imprinted genes, and there were seven genes that differed in allelic expression between cell lines. fMSC s exhibited the differential DNA methylation patterns associated with imprinted expression. This was unexpected given that gene expression of several imprinted genes was biallelic. However, in hES cells, differential methylation was perturbed. These atypical methylation patterns did not correlate with allelic expression. Our results suggest that regardless of stem cell origin, in vitro culture affects the integrity of imprinted gene expression in human cells. We identify biallelic and variably expressed genes that may inform on overall epigenetic stability. As differential methylation did not correlate with imprinted expression changes we propose that other epigenetic effectors are adversely influenced by the in vitro environment. Since DMR integrity was maintained in fMSC but not hES cells, we postulate that specific hES cell derivation and culturing practices result in changes in methylation at DMRs.
PMCID: PMC3052914  PMID: 20864803
genomic imprinting; embryonic stem cells; mesenchymal stem cells; differentiation; methylation; epigenetic stability
9.  Public-private partnership in cord blood banking 
BMJ : British Medical Journal  2008;336(7645):642-644.
Demand for stem cells from cord blood is greater than supply. Nicholas Fisk and Rifat Atun examine the potential of Virgin’s combination of personal and public banking to increase storage
PMCID: PMC2270950  PMID: 18356233
10.  Folic Acid Supplementation and Spontaneous Preterm Birth: Adding Grist to the Mill? 
PLoS Medicine  2009;6(5):e1000077.
Nicholas Fisk and colleagues discuss a new study reporting that additional voluntary folic acid supplementation was associated with a major reduction in very preterm births.
PMCID: PMC2671593  PMID: 19434229
11.  The Effect of Polyhydramnios on Cervical Length in Twins: A Controlled Intervention Study in Complicated Monochorionic Pregnancies 
PLoS ONE  2008;3(12):e3834.
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.
Pre-procedural cervical length correlated with AFI (linear fit = 5.07 -0.04x, R2 = 0.17, P = 0.03) in patients with polyhydramnios (n = 28). 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.
Cervical shortening in twins with polyhydramnios does not appear to be an acute process; cervical length can be measured before or after therapeutic procedures.
PMCID: PMC2584788  PMID: 19048106
12.  Market Failure and the Poverty of New Drugs in Maternal Health 
PLoS Medicine  2008;5(1):e22.
A new survey finds that only 17 drugs are under active development for maternal health indications, which is less than 3% of the pipeline in cardiovascular health (660 drugs).
PMCID: PMC2211556  PMID: 18215109
13.  High Risk of Unexpected Late Fetal Death in Monochorionic Twins Despite Intensive Ultrasound Surveillance: A Cohort Study 
PLoS Medicine  2005;2(6):e172.
The rationale for fetal surveillance in monochorionic twin pregnancies is timely intervention to prevent the increased fetal/perinatal morbidity and mortality attributed to twin–twin transfusion syndrome and intrauterine growth restriction. We investigated the residual risk of fetal death after viability in otherwise uncomplicated monochorionic diamniotic twin pregnancies.
Methods and Findings
We searched an electronic database of 480 completed monochorionic pregnancies that underwent fortnightly ultrasound surveillance in our tertiary referral fetal medicine service between 1992 and 2004. After excluding pregnancies with twin–twin transfusion syndrome, growth restriction, structural abnormalities, or twin reversed arterial perfusion sequence, and monoamniotic and high-order multiple pregnancies, we identified 151 uncomplicated monochorionic diamniotic twin pregnancies with normal growth, normal liquor volume, and normal Doppler studies on fortnightly ultrasound scans. Ten unexpected intrauterine deaths occurred in seven (4.6%) of 151 previously uncomplicated monochorionic diamniotic pregnancies, within 2 wk of a normal scan, at a median gestational age of 34+1 wk (weeks+days; range 28+0 to 36+3). Two of the five cases that underwent autopsy had features suggestive of acute late onset twin–twin transfusion syndrome, but no antenatal indicators of transfusional imbalance or growth restriction, either empirically or in a 1:3 gestation-matched case–control comparison. The prospective risk of unexpected antepartum stillbirth after 32 wk was 1/23 monochorionic diamniotic pregnancies (95% confidence interval 1/11 to 1/63).
Despite intensive fetal surveillance, structurally normal monochorionic diamniotic twin pregnancies without TTTS or IUGR are complicated by a high rate of unexpected intrauterine death. This prospective risk of fetal death in otherwise uncomplicated monochorionic diamniotic pregnancies after 32 wk of gestation might be obviated by a policy of elective preterm delivery, which now warrants evaluation.
Despite intensive fetal surveillance, structurally normal monochorionic diamniotic twin pregnancies were found to be complicated by a high rate of unexpected intrauterine death late in pregnancy.
PMCID: PMC1160580  PMID: 15971947
14.  Life before Birth 
BMJ : British Medical Journal  2005;330(7495):851.
Channel 4, 7 April at 9 pm
Rating: ★★
PMCID: PMC556092
15.  Can Routine Commercial Cord Blood Banking Be Scientifically and Ethically Justified? 
PLoS Medicine  2005;2(2):e44.
Background to the debate: Umbilical cord blood—the blood that remains in the placenta after birth—can be collected and stored frozen for years. A well-accepted use of cord blood is as an alternative to bone marrow as a source of hematopoietic stem cells for allogeneic transplantation to siblings or to unrelated recipients; women can donate cord blood for unrelated recipients to public banks. However, private banks are now open that offer expectant parents the option to pay a fee for the chance to store cord blood for possible future use by that same child (autologous transplantation.)
Private banks offer expectant parents the option to pay a fee for the chance to store cord blood for possible future use by the child. The practice is controversial, for scientific and ethical reasons
PMCID: PMC549592  PMID: 15737000
18.  Association between maternal anxiety in pregnancy and increased uterine artery resistance index: cohort based study 
BMJ : British Medical Journal  1999;318(7177):153-157.
To investigate whether maternal anxiety in the third trimester is associated with an increased uterine artery resistance index.
Cohort based study.
100 pregnant women, with a mean gestation of 32 weeks.
Outcome measures
Self rating Spielberger questionnaire for state anxiety and trait anxiety, and uterine blood flow waveform patterns as assessed by colour Doppler ultrasound.
A significant association was found between uterine artery resistance index and scores for both Spielberger state anxiety and trait anxiety (rs=0.31, P<0.002 and 0.28 P<0.005 respectively). Women with state anxiety scores >40 (n=15) had a higher mean uterine resistance index than those with scores ⩽40 (mean difference with mean resistance index 24%, 95% confidence interval 12% to 38%; P<0.0001). Similarly, women with trait anxiety scores >40 (n=32) had a higher mean resistance index than those with scores ⩽40, although to a lesser extent. The presence of notches in the waveform pattern produced by uterine artery blood flow was found in 4/15 (27%) women with high state anxiety scores compared with 4/85 (5%) with low anxiety scores (P<0.02).
This study shows an association between maternal anxiety in pregnancy and increased uterine artery resistance index. It suggests a mechanism by which the psychological state of the mother may affect fetal development, and may explain epidemiological associations between maternal anxiety and low birth weight. The influence of maternal anxiety may be one mechanism by which the intrauterine environment contributes to later disease in offspring.
Key messagesWomen who were anxious during pregnancy had significantly abnormal patterns of blood flow through the uterine arteriesOf the most anxious group, 27% had an increased resistance index of clinical concern, compared with 4% in the less anxious groupThe study did not establish whether the impaired blood flow was predominantly linked with state anxiety or trait anxietyThe findings may help to explain previous studies that have linked stress or anxiety in pregnancy with small for gestational age babies
PMCID: PMC27690  PMID: 9888905
19.  Randomised trial of educational visits to enhance use of systematic reviews in 25 obstetric units 
BMJ : British Medical Journal  1998;317(7165):1041-1046.
Objective To evaluate the effectiveness of an educational visit to help obstetricians and midwives select and use evidence from a Cochrane database containing 600 systematic reviews.
Design Randomised single blind controlled trial with obstetric units allocated to an educational visit or control group.
Setting 25 of the 26 district general obstetric units in two former NHS regions.
Subjects The senior obstetrician and midwife from each intervention unit participated in educational visits. Clinical practices of all staff were assessed in 4508 pregnancies.
Intervention Single informal educational visit by a respected obstetrician including discussion of evidence based obstetrics, guidance on implementation, and donation of Cochrane database and other materials.
Main outcome measures Rates of perineal suturing with polyglycolic acid, ventouse delivery, prophylactic antibiotics in caesarean section, and steroids in preterm delivery, before and 9 months after visits, and concordance of guidelines with review evidence for same marker practices before and after visits.
Results Rates varied greatly, but the overall baseline mean of 43% (986/2312) increased to 54% (1189/2196) 9 months later. Rates of ventouse delivery increased significantly in intervention units but not in control units; there was no difference between the two types of units in uptake of other practices. Pooling rates from all 25 units, use of antibiotics in caesarean section and use of polyglycolic acid sutures increased significantly over the period, but use of steroids in preterm delivery was unchanged. Labour ward guidelines seldom agreed with evidence at baseline; this hardly improved after visits. Educational visits cost £860 each (at 1995 prices).
Conclusions There was considerable uptake of evidence into practice in both control and intervention units between 1994 and 1995. Our educational visits added little to this, despite the informal setting, targeting of senior staff from two disciplines, and donation of educational materials. Further work is needed to define cost effective methods to enhance the uptake of evidence from systematic reviews and to clarify leadership and roles of senior obstetric staff in implementing the evidence.
Key messagesThere was marked variation in four common obstetric practices known to improve patient outcomes in 25 district general obstetric units across south east England in both 1994 and 1995Labour ward guidelines in the 25 units showed little concordance with Cochrane review evidence in 1994 and 1995The gap between Cochrane review evidence and clinical practice narrowed in 1995, but 46% (1010) of 2196 pregnant women studied were still not managed according to current evidenceEducational visits to senior staff led to a significant but clinically modest uptake of evidence from systematic reviews in only one of the four practices studiedReducing practice variations and improving clinical knowledge management by helping clinicians to locate, select, and implement systematic review evidence remain important challenges for the NHS
PMCID: PMC28686  PMID: 9774287

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