RESUMEN
The mouse hematopoietic system has served as a paradigm for analysis of developmental fate decisions in tissue homeostasis and regeneration. However, multiple immunophenotypic definitions of, and sometimes divergent nomenclatures used to classify, murine multipotent progenitors (MPPs) have emerged in the field over time. This has created significant confusion and inconsistency in the hematology field. To facilitate easier comparison of murine MPP phenotypes between research laboratories, a working group of four International Society for Experimental Hematology (ISEH) members with extensive experience studying the functional activities associated with different MPP phenotypic definitions reviewed the current state of the field with the goal of developing a position statement toward a simplified and unified immunophenotypic definition of MPP populations. In November of 2020, this position statement was presented as a webinar to the ISEH community for discussion and feedback. Hence, the Simplified MPP Identification Scheme presented here is the result of curation of existing literature, consultation with leaders in the field, and crowdsourcing from the wider experimental hematology community. Adoption of a unified definition and nomenclature, while still leaving room for individual investigator customization, will benefit scientists at all levels trying to compare these populations between experimental settings.
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Citometría de Flujo/métodos , Ratones , Células Madre Multipotentes/citología , Animales , Antígenos CD/análisis , Citometría de Flujo/economía , Citometría de Flujo/instrumentación , Hematopoyesis , Células Madre Hematopoyéticas/química , Células Madre Hematopoyéticas/citología , Ratones/metabolismo , Células Madre Multipotentes/químicaRESUMEN
The formation of the cerebellum is highly coordinated to obtain its characteristic morphology and all cerebellar cell types. During mouse postnatal development, cerebellar progenitors with astroglial-like characteristics generate mainly astrocytes and oligodendrocytes. However, a subset of astroglial-like progenitors found in the prospective white matter (PWM) produces astroglia and interneurons. Characterizing these cerebellar astroglia-like progenitors and distinguishing their developmental fates is still elusive. Here, we reveal that astrocyte cell surface antigen-2 (ACSA-2), lately identified as ATPase, Na+/K+ transporting, beta 2 polypeptide, is expressed by glial precursors throughout postnatal cerebellar development. In contrast to common astrocyte markers, ACSA-2 appears on PWM cells but is absent on Bergmann glia (BG) precursors. In the adult cerebellum, ACSA-2 is broadly expressed extending to velate astrocytes in the granular layer, white matter astrocytes, and to a lesser extent to BG. Cell transplantation and transcriptomic analysis revealed that marker staining discriminates two postnatal progenitor pools. One subset is defined by the co-expression of ACSA-2 and GLAST and the expression of markers typical of parenchymal astrocytes. These are PWM precursors that are exclusively gliogenic. They produce predominantly white matter and granular layer astrocytes. Another subset is constituted by GLAST positive/ACSA-2 negative precursors that express neurogenic and BG-like progenitor genes. This population displays multipotency and gives rise to interneurons besides all glial types, including BG. In conclusion, this work reports about ACSA-2, a marker that in combination with GLAST enables for the discrimination and isolation of multipotent and glia-committed progenitors, which generate different types of cerebellar astrocytes.
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Antígenos de Superficie/análisis , Cerebelo/química , Cerebelo/citología , Transportador 1 de Aminoácidos Excitadores/análisis , Células Madre Multipotentes/química , Neuroglía/química , Animales , Animales Recién Nacidos , Femenino , Separación Inmunomagnética/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroglía/clasificación , Análisis de Secuencia de ARN/métodosRESUMEN
To reduce the increasing need for corneal transplantation, attempts are currently aiming to restore corneal clarity, one potent source of cells are multipotent adult progenitor cells (MAPC®). These cells release a powerful cocktail of paracrine factors that can guide wound healing and tissue regeneration. However, their role in corneal regeneration has been overlooked. Thus, we sought to explore the potential of combining the cytoprotective storage feature of alginate, with MAPC to generate a storable cell-laden gel for corneal wound healing. 72 hours following hypothermic storage, alginate encapsulation was shown to maintain MAPC viability at either 4 or 15°C. Encapsulated MAPC (2 x106 cells/mL) stored at 15°C presented the optimum temperature that allowed for cell recovery. These cells had the ability to reattach to tissue culture plastic whilst exhibiting normal phenotype and this was maintained in serum-free and xenobiotic-free medium. Furthermore, corneal stromal cells presented a significant decrease in scratch-wounds in the presence of alginate encapsulated MAPC compared to a no-cell control (p = 0.018). This study shows that immobilization of MAPC within an alginate hydrogel does not hinder their ability to affect a secondary cell population via soluble factors and that these effects are successfully retained following hypothermic storage.
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Células Madre Adultas/metabolismo , Alginatos/química , Sustancia Propia/fisiología , Células Madre Multipotentes/metabolismo , Células del Estroma/fisiología , Adulto , Células Madre Adultas/química , Supervivencia Celular/fisiología , Células Cultivadas , Sustancia Propia/citología , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Madre Multipotentes/química , Comunicación Paracrina/fisiología , SolubilidadRESUMEN
The mammalian ovary is a highly dynamic organ, in which proliferation and differentiation occur constantly during the entire life span, particularly in camels that are characterized by a follicular wave pattern and induced ovulation. Granulosa cells are the main cells of mature follicles. Two distinct cell types, namely, the mural and cumulus granulosa cells are distinguished on the basis of antral fluid increase. The multipotency of follicular fluid and the luteinizing cell were recently demonstrated. However, reports regarding the plasticity of cumulus cells are lacking. We obtained cumulus cells from cumulus-oocyte complexes and showed that camel cumulus cells expressed stem cell mRNA transcripts (POU5A1, KLF4, SOX2, and MYC) and were able to differentiate into other non-ovarian follicular cell types in vitro, such as neurons, osteoblasts, and adipocytes. In contrast, removal of the ooplasm (oocytectemy) showed no effect on cumulus cell proliferation and differentiation. This is the first report to identify an invaluable source of multipotent stem cells, which is routinely discarded during in vitro embryo production. The plasticity and transdifferentiation capability of camel cumulus cells definitely requires attention as it provides a cheap biological experimental model for basic research in stem cells and for understanding ovarian differentiation, both of which are relevant for use in regenerative medicine and tissue engineering in humans and animals.
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Camelus , Células del Cúmulo/fisiología , Células Madre Multipotentes/fisiología , Folículo Ovárico/citología , Animales , Diferenciación Celular , Plasticidad de la Célula/genética , Proliferación Celular , Células Cultivadas , Femenino , Genes myc/genética , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Células Madre Multipotentes/química , Factor 3 de Transcripción de Unión a Octámeros/genética , ARN Mensajero/análisis , Factores de Transcripción SOXB1/genéticaRESUMEN
Primary cardiac neoplasms are rare and are usually benign myxomas and rhabdomyomas. Cardiac leiomyomas are usually seen as a part of the spectrum of intravenous leiomyomatosis or benign metastasizing leiomyoma. De novo occurrence of primary intracardiac leiomyomas (PICL) is a rarity. Herein we describe a 14-year-old boy presenting with intermittent dyspnea for 2 years, with a large right ventricular mass suggestive of myxoma on transthoracic echocardiography, without any extracardiac lesions. Histology and immunohistochemistry of the tumor excised under cardiopulmonary bypass confirmed a PICL arising at the cardiomyocyte-smooth muscle septal interface. A review of existing literature highlights an increased incidence in young patients and an overwhelming right ventricular anatomical predilection. Abnormalities in the multipotent cardiac progenitor cells of the second heart field may provide a potential microenvironment for the histogenesis of PICL.
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Neoplasias Cardíacas/patología , Leiomioma/patología , Células Madre Multipotentes/patología , Células Madre Neoplásicas/patología , Tabique Interventricular/patología , Adolescente , Biomarcadores de Tumor/análisis , Biopsia , Ecocardiografía , Femenino , Neoplasias Cardíacas/química , Neoplasias Cardíacas/cirugía , Humanos , Inmunohistoquímica , Lactante , Leiomioma/química , Leiomioma/cirugía , Masculino , Persona de Mediana Edad , Células Madre Multipotentes/química , Células Madre Neoplásicas/química , Microambiente Tumoral , Tabique Interventricular/química , Tabique Interventricular/cirugía , Adulto JovenRESUMEN
The study investigated the effects of adipose tissue-derived stem cells (ADSCs) modified with penile neuronal nitric oxide synthase (PnNOS) gene on intracellular calcium concentration in rat corpus cavernosum smooth muscle cells (CCSMCs). ADSCs and CCSMCs of Sprague-Dawley (SD) rats were isolated and cultured in vitro respectively. The rat PnNOS gene was transferred into the ADSCs mediated by a recombinant adenovirus vector. The expression of the PnNOS gene was detected. At the same time, the concentration of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) was assayed. After coculturing with the CCSMCs of SD rats, which were isolated and expanded ex vivo, the cGMP and NO levels of ADSCs and CCSMCs were measured. Intracellular calcium concentration ([Ca(2+) ]i ) in rat CCSMCs was measured with Fluo-3/AM by flow cytometer after cocultured with ADSCs overexpressing PnNOS gene. The mRNA and protein expression of PnNOS gene mediated by recombinant adenovirus vector significantly overexpressed and lasted at least 2 weeks. Meanwhile, the concentration of NO and cGMP in ADSCs was greatly increased. The concentration of cGMP was significantly increased, and [Ca(2+) ]i was obviously decreased in CCSMCs compared with the control groups (P < 0.05) after cocultured with ADSCs for 3 days. These findings demonstrated that ADSCs overexpressing PnNOS gene might decrease [Ca(2+) ]i in CCSMCs by up-regulating NO-cGMP signalling pathway.
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Tejido Adiposo/citología , Calcio/análisis , Células Madre Multipotentes/metabolismo , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Pene/metabolismo , Tejido Adiposo/metabolismo , Animales , Western Blotting , Calcio/metabolismo , GMP Cíclico/análisis , Citometría de Flujo , Técnicas de Transferencia de Gen , Masculino , Células Madre Multipotentes/química , Miocitos del Músculo Liso/química , Óxido Nítrico Sintasa de Tipo I/biosíntesis , Óxido Nítrico Sintasa de Tipo I/genética , Pene/química , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is associated with a higher risk of cardiovascular disease (CVD). Previous studies have indicated that the reduction of bone marrow-derived multipotent progenitors (CD34+ cells) may lead to reduced vascular repair capacity and may help to identify patients that pose an increased cardiovascular risk. However, the relationship between CD34+cells and CVD risk in AECOPD remains unclear. The aim of the present study was to assess CD34+ cell counts and their relationship with classical adverse cardiac outcome predictors in AECOPD. METHODS: For our study, 27 patients with AECOPD (GOLD stage III, IV), 26 with stable COPD (GOLD stage III, IV), and 24 healthy controls were enrolled. CD34+ cells were enumerated, and plasma concentrations of N-terminal pro-B-type natriuretic peptide (NT-proBNP), a systemic inflammation marker (high-sensitivity C-reactive protein, hsCRP) and mobilisation marker (matrix metalloproteinase-9, MMP-9), were measured. Echocardiography was performed to evaluate cardiac dysfunction and pulmonary hypertension. RESULTS: Compared with healthy controls, AECOPD patients had a significantly decreased CD34+ cell count (5.1 ± 2.6 versus 9.4 ± 3.6 × 10³/ml), especially in patients with a prior history of acute exacerbation. For patients with AECOPD, the CD34+ cell count was inversely correlated with NT-proBNP levels, pulmonary artery systolic pressure (PASP) and resting heart rate, and positively correlated with left ventricular ejection fraction (LVEF). In all three groups, CD34+ cell count was negatively correlated with hsCRP. CONCLUSIONS: The circulating CD34+ cell count was decreased and correlated with cardiac dysfunction in AECOPD patients, and thus may account for the increased cardiovascular risk in this population.
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Hipertensión Pulmonar/sangre , Células Madre Multipotentes , Enfermedad Pulmonar Obstructiva Crónica/sangre , Anciano , Antígenos CD34/análisis , Proteína C-Reactiva/metabolismo , Estudios de Casos y Controles , Recuento de Células , Progresión de la Enfermedad , Ecocardiografía , Femenino , Frecuencia Cardíaca , Humanos , Hipertensión Pulmonar/fisiopatología , Masculino , Metaloproteinasa 9 de la Matriz/sangre , Persona de Mediana Edad , Células Madre Multipotentes/química , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Volumen Sistólico , Disfunción Ventricular Izquierda/diagnóstico por imagen , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
Fourier transform infrared (FTIR) microspectroscopy shows potential as a benign, objective and rapid tool to screen pluripotent and multipotent stem cells for clinical use. It offers a new experimental approach that provides a holistic measurement of macromolecular composition such that a signature representing the internal cellular phenotype is obtained. The use of this technique therefore contributes information that is complementary to that acquired by conventional genetic and immunohistochemical methods.
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Células Madre Multipotentes/química , Células Madre Pluripotentes/química , Diferenciación Celular , Análisis por Conglomerados , Análisis Discriminante , Humanos , Análisis de los Mínimos Cuadrados , Células Madre Multipotentes/citología , Células Madre Pluripotentes/citología , Análisis de Componente Principal , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, cartilage damage, and cardiac diseases. Despite remarkable progress in clinical trials, most applications still use traditional culture media containing fetal bovine serum or serum-free media that contain serum albumin, insulin, and transferrin. The ill-defined and variable nature of traditional culture media remains a challenge and has created a need for better defined xeno-free culture media to meet the regulatory and long-term safety requirements for cell-based therapies. We developed and tested a serum-free and xeno-free culture medium (SFM-XF) using human bone marrow- and adipose-derived MSCs by investigating primary cell isolation, multiple passage expansion, mesoderm differentiation, cellular phenotype, and gene expression analysis, which are critical for complying with translation to cell therapy. Human MSCs expanded in SFM-XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of MSCs expanded in traditional serum-containing culture medium (SCM). To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded in SFM-XF and SCM were compared, revealing relatively similar expression profiles. In addition, the SFM-XF supported the isolation and propagation of human MSCs from primary human marrow aspirates, ensuring that these methods and reagents are compatible for translation to therapy. The SFM-XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to serum-containing media for the production of large scale, functionally competent MSCs for future clinical applications.
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Técnicas de Cultivo de Célula/normas , Diferenciación Celular , Medio de Cultivo Libre de Suero/química , Células Madre Mesenquimatosas/citología , Células Madre Multipotentes/citología , Tejido Adiposo/química , Tejido Adiposo/citología , Médula Ósea/química , Técnicas de Cultivo de Célula/métodos , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Condrogénesis , Medio de Cultivo Libre de Suero/normas , Citometría de Flujo , Inestabilidad Genómica , Humanos , Inmunofenotipificación , Cariotipificación , Células Madre Mesenquimatosas/química , Células Madre Multipotentes/química , Fenotipo , TranscriptomaRESUMEN
Normal multipotent tissue stem cells (SCs) are the driving force behind tissue turnover and repair. The cancer stem cell theory holds that tumors also contain stem-like cells that drive tumor growth and metastasis formation. However, very little is known about the regulation of SC maintenance pathways in cancer and how these are affected by cancer-specific genetic alterations and by treatment. Proteomics is emerging as a powerful tool to identify the signaling complexes and pathways that control multi- and pluri-potency and SC differentiation. Here, the authors review the novel insights that these studies have provided and present a comprehensive strategy for the use of proteomics in studying cancer SC biology.
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Biomarcadores de Tumor/análisis , Neoplasias/metabolismo , Células Madre Neoplásicas/química , Células Madre Neoplásicas/metabolismo , Proteómica/métodos , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Separación Celular/métodos , Citometría de Flujo/métodos , Humanos , Espectrometría de Masas/métodos , Células Madre Multipotentes/química , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Células Madre Neoplásicas/citología , Transducción de Señal/fisiologíaRESUMEN
Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.
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Quitosano/química , Colágeno/química , Hidrogeles/química , Células Madre Multipotentes/citología , Trasplante de Células Madre/métodos , Tejido Adiposo/citología , Animales , Técnicas de Cultivo de Célula/métodos , Microesferas , Células Madre Multipotentes/química , Ratas , Andamios del TejidoRESUMEN
BACKGROUND: Several studies have reported the generation of spermatogonia-derived pluripotent stem cells from human testes. The initial aim of the present study was the derivation of equivalent stem cells from an established and experimentally accessible non-human primate model, the common marmoset monkey (Callithrix jacchus). However, an essential prerequisite in the absence of transgenic reporters in primates and man is the availability of validated endogenous markers for the identification of specific cell types in vitro. METHODS AND RESULTS: We cultured marmoset testicular cells in a similar way to that described for human testis-derived pluripotent cells and set out to characterize these cultures under different conditions and in differentiation assays applying established marker panels. Importantly, the cells emerged as testicular multipotent stromal cells (TMSCs) instead of (pluripotent) germ cell-derived cells. TMSCs expressed many markers such as GFR-α, GPR125, THY-1 (CD90), ITGA6, SSEA4 and TRA-1-81, which were considered as spermatogonia specific and were previously used for the enrichment or characterization of spermatogonia. Proliferation of TMSCs was highly dependent on basic fibroblast growth factor, a growth factor routinely present in germ cell culture media. As reliable markers for the distinction between spermatogonia and TMSCs, we established VASA, in combination with the spermatogonia-expressed factors, MAGEA4, PLZF and SALL4. CONCLUSIONS: Marmoset monkey TMSCs and spermatogonia exhibit an overlap of markers, which may cause erroneous interpretations of experiments with testis-derived stem cells in vitro. We provide a marker panel for the unequivocal identification of spermatogonia providing a better basis for future studies on primate, including human, testis-derived stem cells.
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Biomarcadores/análisis , Callithrix , Células Madre Multipotentes/química , Espermatogonias/química , Testículo/citología , Animales , Células Cultivadas , Masculino , Células Madre Pluripotentes/química , Células del Estroma/químicaRESUMEN
In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 and CD44. On the contrary, they were unexpectedly negative to CD73. A small percentage of the cells, finally, showed the expression of STRO-1. This last result might be due to the existence of a small subpopulation of STRO-1+ cells or to a poor cross-reactivity of the antibody. A remarkable donor-to-donor consistency and reproducibility of these findings was demonstrated. The data presented herein support the idea that equine AdMSCs may be easily isolated and selected by adherence to tissue culture plastic and exhibit a surface profile characterized by some peculiar differences in comparison to those described in other species. Continued characterization of these cells will help to clarify several aspects of their biology and may ultimately enable the isolation of specific, purified subpopulations.
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Tejido Adiposo/citología , Citometría de Flujo/veterinaria , Células Madre Multipotentes/fisiología , 5'-Nucleotidasa/análisis , Tejido Adiposo/inmunología , Tejido Adiposo/fisiología , Animales , Antígenos de Superficie/análisis , Células Cultivadas , Femenino , Caballos , Receptores de Hialuranos/análisis , Masculino , Células Madre Multipotentes/química , Células Madre Multipotentes/inmunología , Antígenos Thy-1/análisisRESUMEN
Over the last few years, there has been an increased interest in the study of stem cells in biomedicine for therapeutic use and as a source for healing diseased or injured organs/tissues. More recently, vibrational spectroscopy has been applied to study stem cell differentiation. In this study, we have used both synchrotron based FTIR and Raman microspectroscopies to assess possible differences between human pluripotent (embryonic) and multipotent (adult mesenchymal) stem cells, and how O(2) concentration in cell culture could affect the spectral signatures of these cells. Our work shows that infrared spectroscopy of embryonic (pluripotent) and adult mesenchymal (multipotent) stem cells have different spectral signatures based on the amount of lipids in their cytoplasm (confirmed with cytological staining). Furthermore, O(2) concentration in cell culture causes changes in both the FTIR and Raman spectra of embryonic stem cells. These results show that embryonic stem cells might be more sensitive to O(2) concentration when compared to mesenchymal stem cells. While vibrational spectroscopy could therefore be of potential use in identifying different populations of stem cells further work is required to better understand these differences.
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Células Madre Multipotentes/química , Células Madre Pluripotentes/química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Espectrometría Raman/métodos , Células Cultivadas , Humanos , Lípidos/análisis , Células Madre Multipotentes/citología , Células Madre Pluripotentes/citología , Análisis de Componente PrincipalRESUMEN
Putative stem cells have been isolated from various tissue fluids such as synovial fluid, amniotic fluid, menstrual blood, etc. Recently the presence of nestin positive putative mammary stem cells has been reported in human breast milk. However, it is not clear whether they demonstrate multipotent nature. Since human breast milk is a non-invasive source of mammary stem cells, we were interested in examining the nature of these stem cells. In this pursuit, we could succeed in isolating and expanding a mesenchymal stem cell-like population from human breast milk. These cultured cells were examined by immunofluorescent labeling and found positive for mesenchymal stem cell surface markers CD44, CD29, SCA-1 and negative for CD33, CD34, CD45, CD73 confirming their identity as mesenchymal stem cells. Cytoskeletal protein marker analysis revealed that these cells expressed mesenchymal stem cells markers, namely, nestin, vimentin, smooth muscle actin and also manifests presence of E-Cadherin, an epithelial to mesenchymal transition marker in their early passages. Further we tested the multipotent differentiation potential of these cells and found that they can differentiate into adipogenic, chondrogenic and oesteogenic lineage under the influence of specific differentiation cocktails. This means that these mesenchymal stem cells isolated from human breast milk could potentially be "reprogrammed" to form many types of human tissues. The presence of multipotent stem cells in human milk suggests that breast milk could be an alternative source of stem cells for autologous stem cell therapy although the significance of these cells needs to be determined.
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Células Madre Mesenquimatosas/citología , Leche Humana/citología , Células Madre Multipotentes/citología , 5'-Nucleotidasa/análisis , Antígenos CD/análisis , Antígenos CD34/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Cadherinas/análisis , Diferenciación Celular , Células Cultivadas , Femenino , Humanos , Receptores de Hialuranos/análisis , Integrina beta1/análisis , Proteínas de Filamentos Intermediarios/análisis , Antígenos Comunes de Leucocito/análisis , Células Madre Mesenquimatosas/química , Microscopía Fluorescente , Células Madre Multipotentes/química , Proteínas del Tejido Nervioso/análisis , Nestina , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Vimentina/análisisRESUMEN
Myoblast transfer therapy for Duchenne muscular dystrophy (DMD) largely fails due to cell death and inability of transplanted cells to engraft in diseased muscles. One method attempting to enrich for cell subpopulations is the Hoechst 33342 dye exclusion assay, yielding a side population (SP) thought to be progenitor enriched and a main population (MP). However, in vitro and transplant studies yielded inconsistent results relative to downstream progeny. Cell surface markers expressed by skeletal muscle-derived MP and SP cells have not been fully characterized directly ex vivo. Using flow cytometry, MP and SP cells were characterized based on their expression of several well-accepted progenitor cell antigens. Both the MP and SP populations are heterogeneous and overlapping in the cells they contain. The percentages of cells in each population vary with species and specific muscle examined. MP and SP populations contain both satellite and multipotent progenitor cells, based on expression of CD34, Sca-1, Pax7, and M-cadherin. Thus, isolation using this procedure cannot be used to predict downstream differentiation outcomes, and explains the conflicting literature on these cells. Hoechst dye also results in significant mortality of sorted cells. As defined subpopulations are easily obtained using flow cytometry, sorting immediately ex vivo based on accepted myogenic precursor cell markers will yield superior results in terms of cell homogeneity for transplantation therapy.
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Separación Celular/métodos , Citometría de Flujo , Células Madre Multipotentes/química , Músculo Esquelético/química , Células Satélite del Músculo Esquelético/química , Animales , Antígenos CD34/análisis , Antígenos Ly/análisis , Bencimidazoles/toxicidad , Biomarcadores/análisis , Cadherinas/análisis , Diferenciación Celular , Linaje de la Célula , Supervivencia Celular/efectos de los fármacos , Colorantes Fluorescentes/toxicidad , Antígenos Comunes de Leucocito/análisis , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Músculo Esquelético/citología , Factor de Transcripción PAX7/análisis , Fenotipo , ConejosRESUMEN
Human dental pulp derived from exfoliated deciduous teeth has been described as a promising alternative source of multipotent stem cells. While these cells share certain similarities with mesenchymal stem-like cells (MSC) isolated from other tissues, basically they are still poorly characterized. In this study, for the first time, a proteomic map of abundantly expressed proteins in stromal cells derived from the dental pulp of human exfoliated deciduous teeth (SHED) was established. We also analyzed proteomic signatures of 2 clonal strains derived from SHEDs by single-cell cloning. The SHEDs were established from enzyme-disaggregated deciduous dental pulp from 6-year-old children. They had typical fibroblastoid morphology and high colony-forming efficiency index (16.4%). Cloning was performed at the second passage using limiting dilution in a 96-well plate (0.3 cell/well). Differentiation assessment revealed strong osteogenic but no adipogenic potential of the SHEDs in either clonal strain. The cells expressed characteristic antigens of MSC-like cells, including CD73, CD90, CD105, CD146, and did not express hematopoietic markers CD14, CD34, and CD45, as assessed with FACS analysis. For proteomic studies, cytosolic and nuclear proteins were analyzed with 2-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ionization (MALDI)-time of fl ight (TOF)-mass spectrometry (MS). All proteins were identified with high level of confidence (the lowest sequence coverage was 27%). Identification of highly expressed proteins in SHEDs revealed proteomic profiles very similar to that of MSC-like cells derived from other tissues. We also found a high degree of similarity between proteomic signatures of primary SHEDs and clonal cell strains. Thus, our data confirm a close resemblance between SHEDs and MSC-like cells from other tissues and may serve as starting point for creating-comprehensive proteomic maps.
Asunto(s)
Pulpa Dental/citología , Células Madre Multipotentes/química , Proteoma/análisis , Células del Estroma/química , Diente Primario/citología , Adipogénesis/fisiología , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Separación Celular/métodos , Células Cultivadas , Niño , Electroforesis en Gel Bidimensional , Citometría de Flujo , Humanos , Procesamiento de Imagen Asistido por Computador , Datos de Secuencia Molecular , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Osteogénesis/fisiología , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
Spermatogonial stem cells isolated from the adult mouse testis acquire under certain culture conditions pluripotency and become so-called multipotent adult germline stem cells (maGSCs). They can be differentiated into somatic cells of the three germ layers. We investigated a subset of the maGSCs and ESCs proteomes using cell lines derived from two different mouse strains, narrow range immobilized pH gradients to favor the detection of less abundant proteins, and DIGE to ensure confident comparison between the two cell types. 2-D reference maps of maGSCs and ESCs in the pI ranges 3-6 and 5-8 were created, and protein entities were further processed for protein identification. By peptide mass fingerprinting and tandem mass spectrometry combined with searches of protein sequence databases, a set of 409 proteins was identified, corresponding to a library of 166 nonredundant stem cell-associated proteins. The identified proteins were classified according to their main known/postulated functions using bioinformatics. Furthermore, we used DIGE to highlight the ESC-like nature of maGSCs on the proteome scale. We concluded that the proteome of maGSCs is highly similar to that of ESCs as we could identify only a small subset of 18 proteins to be differentially expressed between the two cell types. Moreover, comparative analysis of the cell line proteomes from two different mouse strains showed that the interindividual differences in maGSCs proteomes are minimal. With our study, we created for the first time a proteomic map for maGSCs and compared it to the ESCs proteome from the same mouse. We confirmed on the proteome level the ESC-like nature of maGSCs.
Asunto(s)
Células Madre Embrionarias/citología , Células Germinativas/citología , Proteoma/análisis , Espermatogonias/citología , Células Madre Adultas/química , Células Madre Adultas/citología , Animales , Línea Celular , Células Madre Embrionarias/química , Células Germinativas/química , Masculino , Ratones , Células Madre Multipotentes/química , Células Madre Multipotentes/citología , Proteómica/métodos , Especificidad de la Especie , Espermatogonias/químicaRESUMEN
Our previous study indicated that systemically transplanted dermal multipotent cells (DMCs) were recruited more frequently to bone morrow (BM) of rats with sublethal irradiation than that of normal rats, and the interactions between stromal-derived factor (SDF-1) and its receptor (CXC chemokine receptor 4, CXCR4) played an important role in this process. In the present study, we aimed to investigate whether CXCR4 gene transfer could promote the distribution of DMCs into irradiated BM and accelerate its function recovery. Firstly, adenovirus vector of CXCR4 (Adv-CXCR4) and green fluorescent protein (Adv-GFP) were constructed. Then male DMCs infected by Adv-CXCR4 (group A), or infected by Adv-GFP (group B), and non-infected DMCs (group C) were transplanted into irradiated female rats, and real-time polymerase chain reaction for the sex-determining region of Y chromosome was employed to determined the amount of DMCs in BM. The functional recovery of BM was examined by hematopoietic progenitor colonies assay. The results showed that the amount of DMCs in BM of group A was greater than that in group B and group C from day 5 after injury (P < 0.05), and the amount of CFU-F, CFU-E and CFU-GM were greater than that in group B and group C from day 14 after injury (P < 0.05). These findings suggest that DMCs infected by Adv-CXCR4 distributed more frequently to the bone marrow of sublethally irradiated rats and could accelerate hematopoiesis function recovery.
Asunto(s)
Células de la Médula Ósea/química , Dermis , Células Madre Multipotentes/química , Receptores CXCR4/genética , Adenoviridae/genética , Animales , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Masculino , Ratas , Ratas Wistar , Receptores CXCR4/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Injection of endothelial progenitor cells (EPC) expanded ex vivo has been shown to increase neovascularization in preclinical models of ischemia and in adult patients, but the precise origin and identity of the cell population responsible for these clinical benefits are controversial. Given the potential usefulness of EPC as a cell therapy product, their thorough characterization is of major importance. This review describes the two cell populations currently called EPC and the means to find differential phenotypic markers. We have shown that BMP2/4 are specific markers of late EPC and play a key role in EPC commitment and outgrowth during neovascularization. Several authors have attempted to expand EPC ex vivo in order to obtain a homogeneous cell therapy product. One possible mean of expanding EPC ex vivo is to activate the thrombin receptor PAR-1 with the specific peptide SFLLRN. Indeed, PAR-1 activation increases angiogenic properties of EPC through activation of SDF-1, angiopoietin and IL-8 pathways. This review summarizes the characterization of EPC and different methods of ex vivo expansion.