RESUMEN
: Subendothelial cells (pericytes) are the clonogenic, multipotent and self-renewing skeletal stem cells (SSCs) found in bone marrow (BM) stroma. They express genes maintaining hematopoietic stem cell (HMC) niche identity and, transplanted in immunocompromised mice, organize the hematopoietic microenvironment (HME) generating humanized bone/BM ossicles. To create a mouse model of hematogenous metastasis of human prostate cancer (PC) cells to human bone/BM, we injected PC cells in the blood circulatory system of Severe Combined Immunodeficiency (SCID)/beige mice bearing heterotopic ossicles. Results indicate that PC cells could efficiently home to mice-implanted extraskeletal BM ossicles, but were not able to colonize mice skeletal segments. In humanized bone/BM ossicles, early foci of PC cells occupied a perisinusoidal position, in close contact with perivascular stromal cells. These findings demonstrate the importance of the SSC compartment in recreating a suitable environment to metastatic PC cells. Our data support the hypothesis that BM SSCs committed to a pericyte fate can specify for homing niches of PC cells, suggesting an involvement of specific interactions with subendothelial stromal cells in extravasation of circulating metastatic PC cells to BM.
RESUMEN
Recent observation identifies subendothelial (mural) cells expressing MCAM, a specific system of clonogenic, self-renewing, osteoprogenitors (a.k.a, "mesenchymal stem cells") in the microvascular compartment of post-natal human bone marrow (BM). In this study, we used MCAM/CD146, as a marker to localize, isolate and assay subendothelial clonogenic cells from the microvasculature of postnatal human skeletal muscle. We show here that these cells share with their BM counterpart, anatomic position (subendothelial/adventitial) and ex vivo clonogenicity (CFU-Fs). When assayed under the stringent conditions, these cells display a high spontaneous myogenic potential (independent of co-culture with myoblasts or of in vivo fusion with local myoblasts), which is otherwise only attained in cultures of satellite cells. These muscle-derived mural cells activated a myogenic program in culture. Cultured CD146+ cells expressed the myogenic factors (Pax7, Pax3 and Myf5), NCAM/CD56, desmin as well as proteins characteristic of more advanced myogenic differentiation, such as myosin heavy chain. In vivo, these cells spontaneously generate myotubes and myofibrils. These data identify the anatomy and phenotype of a novel class of committed myogenic progenitor in human post-natal skeletal muscle of subendothelial cells associated with the abluminal surface of microvascular compartment distinct from satellite cells.
Asunto(s)
Microvasos/metabolismo , Músculo Esquelético/citología , Antígeno CD146/metabolismo , Técnicas de Cocultivo , Humanos , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/metabolismoRESUMEN
A widely shared view reads that mesenchymal stem/stromal cells ("MSCs") are ubiquitous in human connective tissues, can be defined by a common in vitro phenotype, share a skeletogenic potential as assessed by in vitro differentiation assays, and coincide with ubiquitous pericytes. Using stringent in vivo differentiation assays and transcriptome analysis, we show that human cell populations from different anatomical sources, regarded as "MSCs" based on these criteria and assumptions, actually differ widely in their transcriptomic signature and in vivo differentiation potential. In contrast, they share the capacity to guide the assembly of functional microvessels in vivo, regardless of their anatomical source, or in situ identity as perivascular or circulating cells. This analysis reveals that muscle pericytes, which are not spontaneously osteochondrogenic as previously claimed, may indeed coincide with an ectopic perivascular subset of committed myogenic cells similar to satellite cells. Cord blood-derived stromal cells, on the other hand, display the unique capacity to form cartilage in vivo spontaneously, in addition to an assayable osteogenic capacity. These data suggest the need to revise current misconceptions on the origin and function of so-called "MSCs," with important applicative implications. The data also support the view that rather than a uniform class of "MSCs," different mesoderm derivatives include distinct classes of tissue-specific committed progenitors, possibly of different developmental origin.
Asunto(s)
Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/citología , Microvasos/citología , Pericitos/citología , Células Satélite del Músculo Esquelético/citología , Transcriptoma , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Linaje de la Célula/genética , Condrogénesis/genética , Sangre Fetal/citología , Sangre Fetal/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Ratones , Microvasos/metabolismo , Osteogénesis/genética , Pericitos/metabolismo , Fenotipo , Células Satélite del Músculo Esquelético/metabolismo , Trasplante HeterólogoRESUMEN
AIM: Sinus augmentation procedures commonly employ osteoconductive scaffolding materials to stimulate and support bone formation. The aim of this study was to develop a simple screening methodology for the evaluation of the osteoconductive potential of various bone graft materials prior to clinical use. MATERIALS AND METHODS: Materials tested were Bio-Oss, Bi-Ostetic, OraGraft, and ProOsteon. These Simple and composite bone substitutes were embedded with osteoprogenitor cells derived from either the human maxillary sinus schneiderian membrane (hMSSM) or from maxillary tuberosity bone marrow and then monitored both in vitro and in vivo. RESULTS: Cell adherence and proliferation was most pronounced in OraGraft, followed by ProOsteon. In vivo bone formation, within the bone graft, was also observed, with most marked results in OraGraft and ProOsteon grafts. CONCLUSIONS: The proposed osteoconductivity testing method proved simple, informative, and reliable for the purpose of screening candidate biomaterials for sinus lifting or sinus augmentation.
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Sustitutos de Huesos/farmacología , Hidroxiapatitas/farmacología , Mucosa Nasal/cirugía , Osteogénesis/efectos de los fármacos , Elevación del Piso del Seno Maxilar/métodos , Adolescente , Adulto , Animales , Células de la Médula Ósea/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Ratones , Microscopía Electrónica de Rastreo , Minerales/farmacologíaRESUMEN
Human skeletal progenitors were engineered to stably express R201C mutated, constitutively active Gs alpha using lentiviral vectors. Long-term transduced skeletal progenitors were characterized by an enhanced production of cAMP, indicating the transfer of the fundamental cellular phenotype caused by activating mutations of Gs alpha. Like skeletal progenitors isolated from natural fibrous dysplasia (FD) lesions, transduced cells could generate bone but not adipocytes or the hematopoietic microenvironment on in vivo transplantation. In vitro osteogenic differentiation was noted for the lack of mineral deposition, a blunted upregulation of osteocalcin, and enhanced upregulation of other osteogenic markers such as alkaline phosphatase (ALP) and bone sialoprotein (BSP) compared with controls. A very potent upregulation of RANKL expression was observed, which correlates with the pronounced osteoclastogenesis observed in FD lesions in vivo. Stable transduction resulted in a marked upregulation of selected phosphodiesterase (PDE) isoform mRNAs and a prominent increase in total PDE activity. This predicts an adaptive response in skeletal progenitors transduced with constitutively active, mutated Gs alpha. Indeed, like measurable cAMP levels, the differentiative responses of transduced skeletal progenitors were profoundly affected by inhibition of PDEs or lack thereof. Finally, using lentiviral vectors encoding short hairpin (sh) RNA interfering sequences, we demonstrated that selective silencing of the mutated allele is both feasible and effective in reverting the aberrant cAMP production brought about by the constitutively active Gs alpha and some of its effects on in vitro differentiation of skeletal progenitors.
Asunto(s)
Células de la Médula Ósea/metabolismo , Displasia Fibrosa Ósea/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Células Madre/fisiología , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Displasia Fibrosa Ósea/patología , Displasia Fibrosa Ósea/fisiopatología , Vectores Genéticos , Humanos , Lentivirus/genética , Osteogénesis/genética , Fenotipo , Hidrolasas Diéster Fosfóricas/metabolismo , Células del Estroma/metabolismo , Transducción GenéticaRESUMEN
Expression of "stemness" markers is widely used as a predictor of stem cell properties of mesenchymal stem cells (MSC). Here, we show that bone marrow-derived (BM)-MSC show stem cell-like behavior in vivo; that is, they form ossicles with formation of bone, formation of adipocytes, and establishment of the murine hematopoietic microenvironment. Multipotent umbilical vein-derived stromal cells (UVSC), on the other hand, do not form bone, nor do they give rise to adipocytes in vivo. Despite these differences in stem-cell-like behavior, BM-MSC and UVSC express the two transcripts variants of POU5F1 at a similar level. Also, we found that in BM-MSC and UVSC, POU5F1 is detectable. However, more than 89% of the POU5F1 transcripts correspond to the POU5F1P1, -P3, or -P4 pseudogene. Despite low-level expression of POU5F1, we were unable to precipitate POU5F1 protein in either BM-MSC or UVSC. These results demonstrate that MSC stemness does not correlate to expression of POU5F1 transcripts or its pseudogenes.
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Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/citología , Células Madre Multipotentes/citología , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Osteogénesis , Adipocitos/citología , Adipocitos/metabolismo , Antígenos de Diferenciación/metabolismo , Células de la Médula Ósea/metabolismo , Linaje de la Célula , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Multipotentes/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Seudogenes , Células del Estroma/citología , Células del Estroma/metabolismo , Venas Umbilicales/citologíaRESUMEN
The identity of cells that establish the hematopoietic microenvironment (HME) in human bone marrow (BM), and of clonogenic skeletal progenitors found in BM stroma, has long remained elusive. We show that MCAM/CD146-expressing, subendothelial cells in human BM stroma are capable of transferring, upon transplantation, the HME to heterotopic sites, coincident with the establishment of identical subendothelial cells within a miniature bone organ. Establishment of subendothelial stromal cells in developing heterotopic BM in vivo occurs via specific, dynamic interactions with developing sinusoids. Subendothelial stromal cells residing on the sinusoidal wall are major producers of Angiopoietin-1 (a pivotal molecule of the HSC "niche" involved in vascular remodeling). Our data reveal the functional relationships between establishment of the HME in vivo, establishment of skeletal progenitors in BM sinusoids, and angiogenesis.
Asunto(s)
Médula Ósea/irrigación sanguínea , Huesos/citología , Células Madre Hematopoyéticas/citología , Células Madre Mesenquimatosas/citología , Angiopoyetina 1/metabolismo , Animales , Huesos/metabolismo , Antígeno CD146/metabolismo , Diferenciación Celular , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Ratones , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
OBJECTIVE: Bone tissue engineering is a promising approach for bone reconstruction in oral-maxillofacial surgery. This study investigates the suitability of oral skeletal tissues as convenient and accessible sources of osteogenic progenitors as an alternative to the iliac crest bone marrow. STUDY DESIGN: Samples of maxilla tuberosity (MT) and maxillary and mandibular periosteum (MP) were obtained during routine oral surgery, and donor site morbidity was assessed using a "split-mouth" approach. Cells isolated from MT (bone marrow stromal cells; MT-BMSCs) and from MP (periosteal cells; M-PCs), were analyzed for clonogenicity, phenotype, expression of osteogenic markers, and ability to form bone in vivo. RESULTS: Both MT-BMSCs and M-PCs included clonogenic cells, showed comparable phenotypic profiles, and expressed early osteogenic markers. Most importantly, both cell populations formed bone upon ectopic in vivo transplantation. CONCLUSION: MT-BMSCs and M-PCs behaved as osteoprogenitor cells in vitro and in vivo. MT and MP may be considered as suitable sources of cells for bone tissue engineering in humans.
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Células de la Médula Ósea/citología , Mandíbula/citología , Maxilar/citología , Periostio/citología , Ingeniería de Tejidos/métodos , ADP-Ribosil Ciclasa/metabolismo , Adulto , Análisis de Varianza , Animales , Antígenos CD/metabolismo , Regeneración Ósea , Proliferación Celular , Ensayo de Unidades Formadoras de Colonias/métodos , Femenino , Citometría de Flujo/métodos , Proteínas Ligadas a GPI , Humanos , Masculino , Ratones , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/análisisRESUMEN
Activating mutations of the Gsalpha gene, encoded by the guanine nucleotide-binding protein, alpha stimulating (GNAS) locus located on chromosome 20q13, underlie different clinical phenotypes characterized by skeletal lesions [fibrous dysplasia (FD) of bone], extraskeletal diseases (mainly endocrine hyperfunction and skin hyperpigmentation) and variable combinations thereof [the McCune-Albright syndrome (MAS)]. This clinical heterogeneity is commonly assumed to reflect the post-zygotic origin of the mutation. However, the pattern of imprinting of the Gsalpha gene in some human post-natal tissues suggests that parental-dependent epigenetic mechanisms may also play a role in the phenotypic effect of the mutated GNAS genotype. FD lesions are generated by mutated clonogenic osteoprogenitors that reside, along with their normal counterparts, in FD bone marrow stroma. We analyzed the allelic expression pattern of Gsalpha and other GNAS alternative transcripts in the progeny of normal and mutated clonogenic stromal cells isolated in vitro from a series of informative FD/MAS patients. We report here for the first time that the two Gsalpha alleles are unequally expressed in both normal and FD-mutated stromal clones. However, in contrast to imprinting, the ratio of Gsalpha allelic expression is randomly established in different clones from the same patient. This result suggests that a parental-independent modulation of Gsalpha expression occurs in clonogenic osteoprogenitor cells and, at the single cell level, may impact on the severity of an FD lesion. Furthermore, we show that normal and mutated clonogenic stromal cells express GNAS alternative transcripts other than the common Gsalpha, some of which may be relevant to the development of FD.
Asunto(s)
Alelos , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Mioblastos Esqueléticos/metabolismo , Adolescente , Adulto , Secuencia de Bases , Médula Ósea/metabolismo , Niño , Cromograninas , Femenino , Displasia Fibrosa Ósea/genética , Displasia Fibrosa Ósea/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Mioblastos Esqueléticos/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
Mouse bone marrow cells cultured with human breast cancer MCF-7 cell-conditioned media showed osteoclastogenesis with an increment of bone resorption, although conditioned media from an adriamycin-selected MCF-7 clone (MCF-7ADR) had no effect. Consistently, MCF-7 cells induced 5-fold more in vivo experimental osteolytic bone metastases, with no soft tissue lesions, compared to MCF-7ADR cells. Paracrine factors stimulating (interleukin (IL)-6, IL-1beta, tumor necrosis factor-alpha (TNF-alpha)) or inhibiting (IL-12, IL-18, granulocyte macrophage-colony stimulating factor (GM-CSF)) osteoclastogenesis were significantly increased in MCF-7ADR relative to MCF-7 cells, suggesting that the inhibitory cytokines could selectively overwhelm the effects of the stimulatory ones. Treatment of osteoblast primary cultures with MCF-7-conditioned medium induced a selective upregulation of IL-6 expression, suggesting an indirect stimulation of osteoclastogenesis via the osteoblasts. MCF-7 and MCF-7ADR showed no difference in proliferation rate. However, a higher ability to migrate and invade gelatin and matrigel was observed in MCF-7ADR. Enhanced invasiveness might result from increased metalloproteinase (MMP) activity and cytoskeleton rearrangement. MCF-7ADR cells expressed higher levels of c-Src, focal adhesion kinase (FAK), and protein tyrosine kinase 2 (PYK2) involved in cell adhesion and motility. MCF-7 and MCF-7ADR expressed high and faint levels of functional estrogen receptor alpha (ERalpha), respectively. MCF-7ADR also showed significantly higher levels of the protein kinase C (PKC) alpha and beta2 and a selective activation of PKC compared to MCF-7, where the most abundant isoforms were beta1 and delta. Heat shock protein 27 (Hsp27) was more abundant in MCF-7 cells, but failed to translocate to the nucleus in response to heat shock. In conclusion, we have demonstrated that despite the fact that MCF-7ADR cells showed a more invasive phenotype relative to MCF-7, they have low potential to induce osteolytic bone lesions and stimulate osteoclastogenesis and osteoclast activity. Therefore, we believe that reduced aggressiveness of breast carcinomas could correlate with a greater osteolytic activity featuring their bone metastases.
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Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Osteoclastos/patología , Animales , Neoplasias Óseas/enzimología , Neoplasias Óseas/metabolismo , Resorción Ósea/patología , Diferenciación Celular , División Celular , Movimiento Celular , Tamaño de la Célula , Células Cultivadas , Citocinas/genética , Citoesqueleto/patología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Quinasa 1 de Adhesión Focal , Quinasa 2 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Metaloproteasas/metabolismo , Ratones , Invasividad Neoplásica , Osteoclastos/enzimología , Osteoclastos/metabolismo , Fenotipo , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Inhibidores Tisulares de Metaloproteinasas/genética , Transcripción GenéticaRESUMEN
We employed potent and selective c-Src inhibitors to investigate the functional and molecular consequences of inhibited c-Src tyrosine kinase activity in osteoclasts. These pyrrolopyrimidine derivatives reduced osteoclast numbers and induced osteoclast disruption in vivo. In vitro, they inhibited resorption pit formation and osteoclastogenesis, impaired adhesion ability and actin ring organization, and induced programmed cell death in mature osteoclasts. The cell death receptor Fas and p53 were insensitive to c-Src modulation. The expression of the cyclin-dependent kinase (CDK)-inhibitor p21WAF1/CIP1 was markedly reduced, but neither Bcl-2 nor Bcl-xL or Bax were modulated by c-Src inhibition. Caspase-9, and to a lesser extent caspase-3, but not caspase-8, were transiently cleaved (activated) by treatment with the c-Src inhibitors. c-Src inhibition stabilized p38 mitogen-activated protein kinase (MAPK), whereas the c-Jun N-terminal kinase (JNK) pathway did not appear to be modulated by our compounds. Most interestingly, transient extracellular signal regulated kinase (ERK1/2) dephosphorylation followed by sustained remarkable rephosphorylation overwhelming control levels was observed in response to c-Src inhibition. Blockade of ERK1/2 rephosphorylation by PD98059 reduced osteoclast nuclear disruption, suggesting the involvement of this pathway in apoptosis. Collectively, these data demonstrate that small pyrrolopyrimidine derivatives impair osteoclast function and induce cell damage suggestive of apoptosis in vivo and in vitro, with mechanisms presumably involving selective sustained ERK1/2 phosphorylation.