RESUMEN
Sesquizygotic multiple pregnancy is an exceptional intermediate between monozygotic and dizygotic twinning. We report a monochorionic twin pregnancy with fetal sex discordance. Genotyping of amniotic fluid from each sac showed that the twins were maternally identical but chimerically shared 78% of their paternal genome, which makes them genetically in between monozygotic and dizygotic; they are sesquizygotic. We observed no evidence of sesquizygosis in 968 dizygotic twin pairs whom we screened by means of pangenome single-nucleotide polymorphism genotyping. Data from published repositories also show that sesquizygosis is a rare event. Detailed genotyping implicates chimerism arising at the juncture of zygotic division, termed heterogonesis, as the likely initial step in the causation of sesquizygosis.
Asunto(s)
Quimera , Fertilización , Gemelos Monocigóticos/genética , Adulto , Alelos , Embolia Paradójica/complicaciones , Femenino , Genotipo , Humanos , Masculino , Polimorfismo de Nucleótido Simple , Embarazo , Embarazo Gemelar , Tromboembolia/etiología , Ultrasonografía Prenatal , Vena Cava InferiorRESUMEN
BACKGROUND: During adult life, blood vessel formation is thought to occur via angiogenic processes involving branching from existing vessels. An alternate proposal suggests that neovessels form from endothelial progenitors able to assemble the intimal layers. We here aimed to define vessel-resident endothelial progenitors in vivo in a variety of tissues in physiological and pathological situations such as normal aorta, lungs, and wound healing, tumors, and placenta, as well. METHODS: Based on protein expression levels of common endothelial markers using flow cytometry, 3 subpopulations of endothelial cells could be identified among VE-Cadherin+ and CD45- cells. RESULTS: Lineage tracing by using Cdh5creERt2/Rosa-YFP reporter strategy demonstrated that the CD31-/loVEGFR2lo/intracellular endothelial population was indeed an endovascular progenitor (EVP) of an intermediate CD31intVEGFR2lo/intracellular transit amplifying (TA) and a definitive differentiated (D) CD31hiVEGFR2hi/extracellular population. EVP cells arose from vascular-resident beds that could not be transferred by bone marrow transplantation. Furthermore, EVP displayed progenitor-like status with a high proportion of cells in a quiescent cell cycle phase as assessed in wounds, tumors, and aorta. Only EVP cells and not TA and D cells had self-renewal capacity as demonstrated by colony-forming capacity in limiting dilution and by transplantation in Matrigel plugs in recipient mice. RNA sequencing revealed prominent gene expression differences between EVP and D cells. In particular, EVP cells highly expressed genes related to progenitor function including Sox9, Il33, Egfr, and Pdfgrα. Conversely, D cells highly expressed genes related to differentiated endothelium including Ets1&2, Gata2, Cd31, Vwf, and Notch. The RNA sequencing also pointed to an essential role of the Sox18 transcription factor. The role of SOX18 in the differentiation process was validated by using lineage-tracing experiments based on Sox18CreERt2/Rosa-YFP mice. Besides, in the absence of functional SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced. CONCLUSIONS: Our findings support an entirely novel endothelial hierarchy, from EVP to TA to D, as defined by self-renewal, differentiation, and molecular profiling of an endothelial progenitor. This paradigm shift in our understanding of vascular-resident endothelial progenitors in tissue regeneration opens new avenues for better understanding of cardiovascular biology.
Asunto(s)
Células Endoteliales/metabolismo , Células Madre/metabolismo , Animales , Antígenos CD/metabolismo , Aorta/metabolismo , Aorta/patología , Trasplante de Médula Ósea , Cadherinas/metabolismo , Diferenciación Celular , Células Endoteliales/citología , Endotelio Vascular/citología , Femenino , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Placenta/metabolismo , Placenta/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Embarazo , Factores de Transcripción SOXF/metabolismo , Células Madre/citología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Heridas y Lesiones/patología , Heridas y Lesiones/terapiaRESUMEN
The prospect of using endothelial progenitors is currently hampered by their low engraftment upon transplantation. We report that mesenchymal stem/stromal cells (MSCs), independent of source and age, improve the engraftment of endothelial colony forming cells (ECFCs). MSC coculture altered ECFC appearance to an elongated mesenchymal morphology with reduced proliferation. ECFC primed via MSC contact had reduced self-renewal potential, but improved capacity to form tube structures in vitro and engraftment in vivo Primed ECFCs displayed major differences in transcriptome compared to ECFCs never exposed to MSCs, affecting genes involved in the cell cycle, up-regulating of genes influencing mesenchymal transition, adhesion, extracellular matrix. Inhibition of NOTCH signaling, a potential upstream regulator of mesenchymal transition, in large part modulated this gene expression pattern and functionally reversed the mesenchymal morphology of ECFCs. The collective results showed that primed ECFCs survive better and undergo a mesenchymal transition that is dependent on NOTCH signaling, resulting in significantly increased vasculogenic potential.-Shafiee, A., Patel, J., Wong, H. Y., Donovan, P., Hutmacher, D. W., Fisk, N. M., Khosrotehrani, K. Priming of endothelial colony-forming cells in a mesenchymal niche improves engraftment and vasculogenic potential by initiating mesenchymal transition orchestrated by NOTCH signaling.
Asunto(s)
Células Endoteliales/metabolismo , Células Madre Mesenquimatosas/fisiología , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Técnicas de Cocultivo , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Placenta , Embarazo , Receptores Notch/genéticaRESUMEN
Since the discovery of endothelial colony forming cells (ECFC), there has been significant interest in their therapeutic potential to treat vascular injuries. ECFC cultures display significant heterogeneity and a hierarchy among cells able to give rise to high proliferative versus low proliferative colonies. Here we aimed to define molecularly this in vitro hierarchy. Based on flow cytometry, CD34 expression levels distinguished two populations. Only CD34 + ECFC had the capacity to reproduce high proliferative potential (HPP) colonies on replating, whereas CD34- ECFCs formed only small clusters. CD34 + ECFCs were the only ones to self-renew in stringent single-cell cultures and gave rise to both CD34 + and CD34- cells. Upon replating, CD34 + ECFCs were always found at the centre of HPP colonies and were more likely in G0/1 phase of cell cycling. Functionally, CD34 + ECFC were superior at restoring perfusion and better engrafted when injected into ischemic hind limbs. Transcriptomic analysis identified cyclin-dependent kinase (CDK) cell cycle inhibiting genes (p16, p21, and p57), the Notch signaling pathway (dll1, dll4, hes1, and hey1), and the endothelial cytokine il33 as highly expressed in CD34 + ECFC. Blocking the Notch pathway using a γ-secretase inhibitor (DAPT) led to reduced expression of cell cycle inhibitors, increased cell proliferation followed by a loss of self-renewal, and HPP colony formation capacity reflecting progenitor exhaustion. Similarly shRNA knockdown of p57 strongly affected self-renewal of ECFC colonies. ECFC hierarchy is defined by Notch signalling driving cell cycle regulators, progenitor quiescence and self-renewal potential.
Asunto(s)
Antígenos CD34/metabolismo , Células Progenitoras Endoteliales/trasplante , Neovascularización Fisiológica/genética , Receptores Notch/genética , Lesiones del Sistema Vascular/terapia , Animales , Linaje de la Célula/genética , Proliferación Celular/genética , Autorrenovación de las Células/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Dipéptidos/administración & dosificación , Células Progenitoras Endoteliales/metabolismo , Citometría de Flujo , Miembro Posterior/patología , Miembro Posterior/trasplante , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Receptores Notch/antagonistas & inhibidores , Receptores Notch/biosíntesis , Medicina Regenerativa , Transducción de Señal/efectos de los fármacos , Lesiones del Sistema Vascular/metabolismo , Lesiones del Sistema Vascular/patologíaRESUMEN
Given their heterogeneity and lack of defining markers, it is surprising that multipotent mesenchymal stem/stromal cells (MSCs) have attracted so much translational attention, especially as increasing evidence points to their predominant effect being not by donor differentiation but via paracrine mediators and exosomes. Achieving long-term MSC donor chimerism for treatment of chronic disease remains a challenge, requiring enhanced MSC homing/engraftment properties and manipulation of niches to direct MSC behaviour. Meanwhile advances in nanoparticle technology are furthering the development of MSCs as vehicles for targeted drug delivery. For treatment of acute injuries, systemic cell-free exosome delivery may ultimately displace current emphasis on empiric donor-cell transplantation for anti-inflammatory, immunomodulatory and repair-promoting effects. Exploration of potential clinical sources of MSCs has led to increased utilisation of perinatal MSCs in allogeneic clinical trials, reflecting their ease of collection and developmentally advantageous properties.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Animales , Ensayos Clínicos como Asunto , Exosomas/metabolismo , Humanos , Comunicación Paracrina , Cicatrización de HeridasRESUMEN
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
Asunto(s)
Endocitosis , Feto/citología , Células Madre Mesenquimatosas/citología , Receptores CXCR4/análisis , Receptores CXCR4/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Movimiento Celular , Núcleo Celular/metabolismo , Células Cultivadas , Endosomas/metabolismo , Femenino , Humanos , Lisosomas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Transporte de ProteínasRESUMEN
Fetal microchimeric cells (FMCs) enter the maternal circulation and persist in tissue for decades. They have capacity to home to injured maternal tissue and differentiate along that tissue's lineage. This raises the question of the origin(s) of cells transferred to the mother during pregnancy. FMCs with a mesenchymal phenotype have been documented in several studies, which makes mesenchymal stem cells an attractive explanation for their broad plasticity. Here we assessed the recruitment and mesenchymal lineage contribution of FMCs in response to acute kidney fibrosis induced by aristolochic acid injection. Serial in vivo bioluminescence imaging revealed a biphasic recruitment of active collagen-producing FMCs during the repair process of injured kidney in post-partum wild-type mothers that had delivered transgenic pups expressing luciferase under the collagen type I-promoter. The presence of FMCs long-term post injury (day 60) was associated with profibrotic molecules (TGF-ß/CTGF), serum urea levels, and collagen deposition. Immunostaining confirmed FMCs at short term (day 15) using post-partum wild-type mothers that had delivered green fluorescent protein-positive pups and suggested a mainly hematopoietic phenotype. We conclude that there is biphasic recruitment to, and activity of, FMCs at the injury site. Moreover, we identified five types of FMC, implicating them all in the reparative process at different stages of induced renal interstitial fibrosis.
Asunto(s)
Lesión Renal Aguda/patología , Quimerismo/embriología , Feto/citología , Riñón/patología , Animales , Movimiento Celular , Femenino , Fibrosis , Hematopoyesis , Células Madre Mesenquimatosas/fisiología , Ratones Endogámicos C57BLRESUMEN
Osteogenesis imperfecta (OI or brittle bone disease) is a disorder of connective tissues caused by mutations in the collagen genes. We previously showed that intrauterine transplantation of human blood fetal stem/stromal cells in OI mice (oim) resulted in a significant reduction of bone fracture. This work examines the cellular mechanisms and mechanical bone modifications underlying these therapeutic effects, particularly examining the direct effects of donor collagen expression on bone material properties. In this study, we found an 84% reduction in femoral fractures in transplanted oim mice. Fetal blood stem/stromal cells engrafted in bones, differentiated into mature osteoblasts, expressed osteocalcin, and produced COL1a2 protein, which is absent in oim mice. The presence of normal collagen decreased hydroxyproline content in bones, altered the apatite crystal structure, increased the bone matrix stiffness, and reduced bone brittleness. In conclusion, expression of normal collagen from mature osteoblast of donor origin significantly decreased bone brittleness by improving the mechanical integrity of the bone at the molecular, tissue, and whole bone levels.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Células Madre Fetales/trasplante , Fracturas Óseas/prevención & control , Osteogénesis Imperfecta/cirugía , Animales , Fenómenos Biomecánicos , Western Blotting , Diferenciación Celular , Colágeno/metabolismo , Colágeno Tipo I , Modelos Animales de Enfermedad , Femenino , Fracturas del Fémur/prevención & control , Fémur/metabolismo , Fémur/fisiopatología , Células Madre Fetales/citología , Células Madre Fetales/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis Imperfecta/genética , Embarazo , Análisis de Componente Principal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría Raman , Trasplante HeterólogoRESUMEN
Umbilical cord blood-derived endothelial colony-forming cells (UCB-ECFC) show utility in neovascularization, but their contribution to osteogenesis has not been defined. Cocultures of UCB-ECFC with human fetal-mesenchymal stem cells (hfMSC) resulted in earlier induction of alkaline phosphatase (ALP) (Day 7 vs. 10) and increased mineralization (1.9×; p < .001) compared to hfMSC monocultures. This effect was mediated through soluble factors in ECFC-conditioned media, leading to 1.8-2.2× higher ALP levels and a 1.4-1.5× increase in calcium deposition (p < .01) in a dose-dependent manner. Transcriptomic and protein array studies demonstrated high basal levels of osteogenic (BMPs and TGF-ßs) and angiogenic (VEGF and angiopoietins) regulators. Comparison of defined UCB and adult peripheral blood ECFC showed higher osteogenic and angiogenic gene expression in UCB-ECFC. Subcutaneous implantation of UCB-ECFC with hfMSC in immunodeficient mice resulted in the formation of chimeric human vessels, with a 2.2-fold increase in host neovascularization compared to hfMSC-only implants (p = .001). We conclude that this study shows that UCB-ECFC have potential in therapeutic angiogenesis and osteogenic applications in conjunction with MSC. We speculate that UCB-ECFC play an important role in skeletal and vascular development during perinatal development but less so in later life when expression of key osteogenesis and angiogenesis genes in ECFC is lower.
Asunto(s)
Endotelio Vascular/citología , Sangre Fetal/citología , Células Madre Mesenquimatosas/citología , Osteogénesis/fisiología , Animales , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Técnicas de Cocultivo , Medios de Cultivo Condicionados , Sangre Fetal/metabolismo , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Análisis por MicromatricesRESUMEN
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.
Asunto(s)
Líquido Amniótico/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Ácido Valproico/farmacología , Líquido Amniótico/citología , Diferenciación Celular , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Genoma Humano , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipificación , Cinética , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fenotipo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma , Transgenes , Inactivación del Cromosoma X/efectos de los fármacosRESUMEN
Intrauterine gene transfer (IUGT) offers ontological advantages including immune naiveté mediating tolerance to the vector and transgenic products, and effecting a cure before development of irreversible pathology. Despite proof-of-principle in rodent models, expression efficacy with a therapeutic transgene has yet to be demonstrated in a preclinical nonhuman primate (NHP) model. We aimed to determine the efficacy of human Factor IX (hFIX) expression after adeno-associated-viral (AAV)-mediated IUGT in NHP. We injected 1.0-1.95 × 10(13) vector genomes (vg)/kg of self-complementary (sc) AAV5 and 8 with a LP1-driven hFIX transgene intravenously in 0.9G late gestation NHP fetuses, leading to widespread transduction with liver tropism. Liver-specific hFIX expression was stably maintained between 8 and 112% of normal activity in injected offspring followed up for 2-22 months. AAV8 induced higher hFIX expression (P = 0.005) and milder immune response than AAV5. Random hepatocellular integration was found with no hotspots. Transplacental spread led to low-level maternal tissue transduction, without evidence of immunotoxicity or germline transduction in maternal oocytes. A single intravenous injection of scAAV-LP1-hFIXco to NHP fetuses in late-gestation produced sustained clinically-relevant levels of hFIX with liver-specific expression and a non-neutralizing immune response. These data are encouraging for conditions where gene transfer has the potential to avert perinatal death and long-term irreversible sequelae.
Asunto(s)
Dependovirus/genética , Factor IX/genética , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/genética , Hemofilia B/terapia , Animales , Línea Celular , Dependovirus/inmunología , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/farmacocinética , Células HEK293 , Hemofilia B/genética , Humanos , Inyecciones , Macaca fascicularis , Placenta/metabolismo , Embarazo , Transducción Genética , Integración ViralRESUMEN
Maternal and perinatal disease accounts for nearly 10% of the global burden of disease, with only modest progress towards achievement of the Millennium Development Goals. Despite a favourable new global health landscape in research and development (R&D) to produce new drugs for neglected diseases, R&D investment in maternal/perinatal health remains small and non-strategic. Investment in obstetric R&D by industry or the not-for-profit sector has lagged behind other specialties, with the number of registered pipeline drugs only 1-5% that for other major disease areas. Using a Delphi exercise with maternal/perinatal experts in global and translational research, we estimate that equitable pharmaceutical R&D and public sector research funding over the next 10-20 years could avert 1.1% and 1.9% of the global disease burden, respectively. In contrast, optimal uptake of existing research would prevent 3.0%, justifying the current focus on health service provision. Although R&D predominantly occurs in high-income countries, more than 98% of the estimated reduction in disease burden in this field would be in developing countries. We conclude that better pharmaceutical and public sector R&D would prevent around 1/3 and 2/3, respectively, of the disease burden addressable by optimal uptake of existing research. Strengthening R&D may be an important complementary strategy to health service provision to address global maternal and perinatal disease burden.
Asunto(s)
Investigación Biomédica/economía , Salud Global , Bienestar Materno , Países en Desarrollo , Femenino , Humanos , Recién Nacido , Atención Perinatal/normas , Mortalidad Perinatal , Embarazo , Apoyo a la Investigación como AsuntoRESUMEN
Fetal cells enter the maternal circulation from the early first trimester of pregnancy, where they persist in tissue decades later. We investigated in mice whether fetal microchimeric cells (FMCs) can be detected in maternal kidney, and whether they play a role in kidney homeostasis. FMCs were identified in vivo in two models: one an adaptive model following unilateral nephrectomy, the other an injury via unilateral renal ischaemia reperfusion. Both models were carried out in mothers that had been mated with transgenic mice expressing luciferase transgene under the control of collagen type I, and had given birth to either 1 or 3 litters. FMCs were detected by Y-probe fluorescent in situ hybridization (FISH) and bioluminescence, and the cell number quantified by real-time polymerase chain reaction. In the adaptive model, the remaining kidney showed more cells by all 3 parameters compared with the nephrectomized kidney, while ischaemia reperfusion resulted in higher levels of FMC participation in injured compared to contralateral kidneys. Bioluminescence showed that FMCs switch on collagen type I transcription implicating mesenchymal lineage cells. After injury, Y-probe in situ hydridization was found mainly in the tubular epithelial network. Finally, we compared FMCs with bone marrow cells and found similar dynamics but altered distribution within the kidney. We conclude that FMCs (1) are long-term sequelae of pregnancy and (2) are recruited to the kidney as a result of injury or adaptation, where they activate the transcriptional machinery of matrix proteins.
Asunto(s)
Movimiento Celular , Quimerismo/embriología , Colágeno Tipo I/genética , Feto/citología , Riñón/metabolismo , Riñón/patología , Transcripción Genética , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Recuento de Células , Movimiento Celular/genética , Colágeno Tipo I/metabolismo , Femenino , Hibridación Fluorescente in Situ , Luciferasas/metabolismo , Mediciones Luminiscentes , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transgenes/genética , Cicatrización de Heridas/genética , Cromosoma Y/genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
After four decades of study, the biological role of fetal microchimerism (FMC) remains elusive. Transfer of fetal cells to the mother begins soon after implantation, and increases with gestational age. FMC cells then decline after delivery, but remain detectable for years post-partum. These cells have been implicated in rheumatoid arthritis remission during pregnancy and the prevention of breast cancer by graft-versus-tumor-effects. However, any beneficial effects contrast with their suspected malevolence in triggering of systemic sclerosis after childrearing or their stromal support for tumor formation. Recent evidence that FMC cells participate in disease and tissue repair has stirred controversy on their origin. The detection of FMC cells during early embryogenesis together with the diversity of hematopoietic, mesenchymal and endothelial markers, and plasticity of morphology when integrated into various tissues, provides evidence for their stemness. However, proof of their phenotype in conventional stem cell differentiation assays has been beset with difficulty in isolating and expanding them in culture. Unraveling the function of FMC cells will provide insight into both their engagement in disease and their therapeutic potential.
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Quimerismo , Células Madre Fetales/citología , Células Madre Fetales/metabolismo , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/prevención & control , Femenino , Células Madre Fetales/fisiología , Humanos , Neoplasias/patología , Embarazo , Esclerosis/patologíaRESUMEN
Down syndrome (DS) children have a high frequency of acute megakaryoblastic leukemia (AMKL) in early childhood. At least 2 in utero genetic events are required, although not sufficient, for DS-AMKL: trisomy 21 (T21) and N-terminal-truncating GATA1 mutations. To investigate the role of T21 in DS-AMKL, we compared second trimester hemopoiesis in DS without GATA1 mutations to gestation-matched normal controls. In all DS fetal livers (FLs), but not marrows, megakaryocyte-erythroid progenitor frequency was increased (55.9% +/- 4% vs 17.1% +/- 3%, CD34(+)CD38(+) cells; P < .001) with common myeloid progenitors (19.6% +/- 2% vs 44.0% +/- 7%) and granulocyte-monocyte (GM) progenitors (15.8% +/- 4% vs 34.5% +/- 9%) commensurately reduced. Clonogenicity of DS-FL versus normal FL CD34(+) cells was markedly increased (78% +/- 7% vs 15% +/- 3%) affecting megakaryocyte-erythroid ( approximately 7-fold higher) and GM and colony-forming unit-granulocyte, erythrocyte macrophage, megakaryocyte (CFU-GEMM) progenitors. Replating efficiency of CFU-GEMM was also markedly increased. These data indicate that T21 itself profoundly disturbs FL hemopoiesis and they provide a testable hypothesis to explain the increased susceptibility to GATA1 mutations in DS-AMKL and DS-associated transient myeloproliferative disorder.
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Anomalías Congénitas/genética , Síndrome de Down/complicaciones , Síndrome de Down/genética , Factor de Transcripción GATA1/genética , Predisposición Genética a la Enfermedad/etiología , Hepatopatías/genética , Células Progenitoras Mieloides/patología , Antígenos CD34/metabolismo , Recuento de Células , Anomalías Congénitas/patología , Femenino , Predisposición Genética a la Enfermedad/embriología , Humanos , Leucemia Megacarioblástica Aguda/complicaciones , Leucemia Megacarioblástica Aguda/embriología , Leucemia Megacarioblástica Aguda/genética , Hepatopatías/complicaciones , Hepatopatías/embriología , Mutación/fisiología , Células Progenitoras Mieloides/metabolismo , Embarazo , Segundo Trimestre del Embarazo , Factores de TiempoRESUMEN
Mesenchymal stem cells (MSCs) from human adult bone marrow (haMSCs) represent a promising source for bone tissue engineering. However, their low frequencies and limited proliferation restrict their clinical utility. Alternative postnatal, perinatal, and fetal sources of MSCs appear to have different osteogenic capacities, but have not been systematically compared with haMSCs. We investigated the proliferative and osteogenic potential of MSCs from human fetal bone marrow (hfMSCs), human umbilical cord (hUCMSCs), and human adult adipose tissue (hATMSCs), and haMSCs, both in monolayer cultures and after loading into three-dimensional polycaprolactone-tricalcium-phosphate scaffolds.Although all MSCs had comparable immunophenotypes, only hfMSCs and hUCMSCs were positive for the embryonic pluripotency markers Oct-4 and Nanog. hfMSCs expressed the lowest HLA-I level (55% versus 95%-99%) and the highest Stro-1 level (51% versus 10%-27%), and had the greatest colony-forming unit-fibroblast capacity (1.6x-2.0x; p < .01) and fastest doubling time (32 versus 54-111 hours; p < .01). hfMSCs had the greatest osteogenic capacity, as assessed by von-Kossa staining, alkaline phosphatase activity (5.1x-12.4x; p < .01), calcium deposition (1.6x-2.7x in monolayer and 1.6x-5.0x in scaffold culture; p < .01), calcium visualized on micro-computed tomography (3.9x17.6x; p < .01) and scanning electron microscopy, and osteogenic gene induction. Two months after implantation of cellular scaffolds in immunodeficient mice, hfMSCs resulted in the most robust mineralization (1.8x-13.3x; p < .01).The ontological and anatomical origins of MSCs have profound influences on the proliferative and osteogenic capacity of MSCs. hfMSCs had the most proliferative and osteogenic capacity of the MSC sources, as well as being the least immunogenic, suggesting they are superior candidates for bone tissue engineering.
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Células Madre Adultas/citología , Huesos/fisiología , Feto/citología , Células Madre Mesenquimatosas/citología , Osteogénesis , Ingeniería de Tejidos , Tejido Adiposo/citología , Adulto , Células Madre Adultas/efectos de los fármacos , Animales , Huesos/efectos de los fármacos , Fosfatos de Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Implantes Experimentales , Lactante , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/ultraestructura , Ratones , Ratones SCID , Persona de Mediana Edad , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Poliésteres/farmacología , Andamios del Tejido , Cordón Umbilical/citologíaRESUMEN
Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC.
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Células Madre Adultas/citología , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Osteogénesis/genética , Transcripción Genética , Adulto , Células Madre Adultas/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Femenino , Feto/metabolismo , Expresión Génica , Humanos , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
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Técnicas de Cultivo de Célula/métodos , Sangre Fetal/citología , Células Madre Fetales/citología , Células Madre Mesenquimatosas/citología , Adolescente , Adulto , Células Cultivadas , Femenino , Sangre Fetal/metabolismo , Terapia Genética/métodos , Humanos , Embarazo , Primer Trimestre del Embarazo , Células Madre , Ultrasonografía/métodosRESUMEN
BACKGROUND: Fetal 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. METHODS: Appendix 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. RESULTS: Male 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. CONCLUSIONS: Male-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.