RESUMO
The benefits of current treatments for depression are limited by low response rates, delayed therapeutic effects, and multiple side effects. Antidepressants affect a variety of neurotransmitter systems in different areas of the brain, and the mechanisms underlying their convergent effects on behavior have been unclear. Here we identify hippocampal bone morphogenetic protein (BMP) signaling as a common downstream pathway that mediates the behavioral effects of five different antidepressant classes (fluoxetine, bupropion, duloxetine, vilazodone, trazodone) and of electroconvulsive therapy. All of these therapies decrease BMP signaling and enhance neurogenesis in the hippocampus. Preventing the decrease in BMP signaling blocks the effect of antidepressant treatment on behavioral phenotypes. Further, inhibition of BMP signaling in hippocampal newborn neurons is sufficient to produce an antidepressant effect, while chemogenetic silencing of newborn neurons prevents the antidepressant effect. Thus, inhibition of hippocampal BMP signaling is both necessary and sufficient to mediate the effects of multiple classes of antidepressants.
Assuntos
Antidepressivos/farmacologia , Proteínas Morfogenéticas Ósseas/metabolismo , Hipocampo/metabolismo , Transdução de Sinais , Envelhecimento/patologia , Animais , Ansiolíticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Giro Denteado/efeitos dos fármacos , Giro Denteado/metabolismo , Cloridrato de Duloxetina/farmacologia , Eletroconvulsoterapia , Fluoxetina/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Hipocampo/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse Psicológico/complicações , Trazodona/farmacologia , Cloridrato de Vilazodona/farmacologiaRESUMO
Astrocytes perform a wide array of physiological functions, including structural support, ion exchange, and neurotransmitter uptake. Despite this diversity, molecular markers that label subpopulations of astrocytes are limited, and mechanisms that generate distinct astrocyte subtypes remain unclear. Here we identified serine protease high temperature requirement A 1 (HtrA1), a bone morphogenetic protein 4 signaling regulated protein, as a novel marker of forebrain astrocytes, but not of neural stem cells, in adult mice of both sexes. Genetic deletion of HtrA1 during gliogenesis accelerates astrocyte differentiation. In addition, ablation of HtrA1 in cultured astrocytes leads to altered chondroitin sulfate proteoglycan expression and inhibition of neurite extension, along with elevated levels of transforming growth factor-ß family proteins. Brain injury induces HtrA1 expression in reactive astrocytes, and loss of HtrA1 leads to an impairment in wound closure accompanied by increased proliferation of endothelial and immune cells. Our findings demonstrate that HtrA1 is differentially expressed in adult mouse forebrain astrocytes, and that HtrA1 plays important roles in astrocytic development and injury response.SIGNIFICANCE STATEMENT Astrocytes, an abundant cell type in the brain, perform a wide array of physiological functions. Although characterized as morphologically and functionally diverse, molecular markers that label astrocyte subtypes or signaling pathways that lead to their diversity remain limited. Here, after examining the expression profile of astrocytes generated in response to bone morphogenetic protein signaling, we identify high temperature requirement A 1 (HtrA1) as an astrocyte-specific marker that is differentially expressed in distinct adult mouse brain regions. HtrA1 is a serine protease that has been linked to cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, a small blood vessel disease in humans. Understanding the role of HtrA1 during development and after injury will provide insights into how distinct astrocyte populations are generated and their unique roles in injury and disease.
Assuntos
Astrócitos/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Neurogênese , Cicatrização , Animais , Astrócitos/citologia , Proliferação de Células , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Feminino , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prosencéfalo/citologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Gliosis and fibrosis after spinal cord injury (SCI) lead to formation of a scar that is an impediment to axonal regeneration. Fibrotic scarring is characterized by the accumulation of fibronectin, collagen, and fibroblasts at the lesion site. The mechanisms regulating fibrotic scarring after SCI and its effects on axonal elongation and functional recovery are not well understood. In this study, we examined the effects of eliminating an isoform of fibronectin containing the Extra Domain A domain (FnEDA) on both fibrosis and on functional recovery after contusion SCI using male and female FnEDA-null mice. Eliminating FnEDA did not reduce the acute fibrotic response but markedly diminished chronic fibrotic scarring after SCI. Glial scarring was unchanged after SCI in FnEDA-null mice. We found that FnEDA was important for the long-term stability of the assembled fibronectin matrix during both the subacute and chronic phases of SCI. Motor functional recovery was significantly improved, and there were increased numbers of axons in the lesion site compared to wildtype mice, suggesting that the chronic fibrotic response is detrimental to recovery. Our data provide insight into the mechanisms of fibrosis after SCI and suggest that disruption of fibronectin matrix stability by targeting FnEDA represents a potential therapeutic strategy for promoting recovery after SCI.
Assuntos
Cicatriz/metabolismo , Cicatriz/patologia , Fibronectinas/deficiência , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Animais , Feminino , Fibronectinas/genética , Fibrose/metabolismo , Fibrose/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Recuperação de Função Fisiológica/fisiologiaRESUMO
Intravenously infused synthetic 500nm nanoparticles composed of poly(lactide-co-glycolide) are taken up by blood-borne inflammatory monocytes via a macrophage scavenger receptor (macrophage receptor with collagenous structure), and the monocytes no longer traffic to sites of inflammation. Intravenous administration of the nanoparticles after experimental spinal cord injury in mice safely and selectively limited infiltration of hematogenous monocytes into the injury site. The nanoparticles did not bind to resident microglia, and did not change the number of microglia in the injured spinal cord. Nanoparticle administration reduced M1 macrophage polarization and microglia activation, reduced levels of inflammatory cytokines, and markedly reduced fibrotic scar formation without altering glial scarring. These findings thus implicate early-infiltrating hematogenous monocytes as highly selective contributors to fibrosis that do not play an indispensable role in gliosis after SCI. Further, the nanoparticle treatment reduced accumulation of chondroitin sulfate proteoglycans, increased axon density inside and caudal to the lesion site, and significantly improved functional recovery after both moderate and severe injuries to the spinal cord. These data provide further evidence that hematogenous monocytes contribute to inflammatory damage and fibrotic scar formation after spinal cord injury in mice. Further, since the nanoparticles are simple to administer intravenously, immunologically inert, stable at room temperature, composed of an FDA-approved material, and have no known toxicity, these findings suggest that the nanoparticles potentially offer a practical treatment for human spinal cord injury.
Assuntos
Fatores Imunológicos/administração & dosagem , Nanopartículas/administração & dosagem , Poliglactina 910/administração & dosagem , Traumatismos da Medula Espinal/tratamento farmacológico , Administração Intravenosa , Animais , Axônios/efeitos dos fármacos , Axônios/imunologia , Axônios/patologia , Tamanho Celular , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cicatriz/tratamento farmacológico , Cicatriz/imunologia , Cicatriz/patologia , Modelos Animais de Doenças , Feminino , Fibrose/tratamento farmacológico , Fibrose/imunologia , Fibrose/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/imunologia , Microglia/patologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/patologia , Atividade Motora/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Medula Espinal/imunologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/patologiaRESUMO
Astrogliosis after spinal cord injury (SCI) is a major impediment to functional recovery. More than half of new astrocytes generated after SCI are derived from ependymal zone stem cells (EZCs). We demonstrate that expression of ß1-integrin increases in EZCs following SCI in mice. Conditional knock-out of ß1-integrin increases GFAP expression and astrocytic differentiation by cultured EZCs without altering oligodendroglial or neuronal differentiation. Ablation of ß1-integrin from EZCs in vivo reduced the number of EZC progeny that continued to express stem cell markers after SCI, increased the proportion of EZC progeny that differentiated into GFAP+ astrocytes, and diminished functional recovery. Loss of ß1-integrin increased SMAD1/5/8 and p38 signaling, suggesting activation of BMP signaling. Coimmunoprecipitation studies demonstrated that ß1-integrin directly interacts with the bone morphogenetic protein receptor subunits BMPR1a and BMPR1b. Ablation of ß1-integrin reduced overall levels of BMP receptors but significantly increased partitioning of BMPR1b into lipid rafts with increased SMAD1/5/8 and p38 signaling. Thus ß1-integrin expression by EZCs reduces movement of BMPR1b into lipid rafts, thereby limiting the known deleterious effects of BMPR1b signaling on glial scar formation after SCI.
Assuntos
Astrócitos/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Epêndima/citologia , Gliose/tratamento farmacológico , Integrina beta1/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/etiologia , Gliose/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/patologiaRESUMO
INTRODUCTION: Previous studies found that neuron specific enolase promoter (Nse-BMP4) transgenic mice have increased expression of the nociceptive mediator, substance P and exaggerated local injury responses associated with heterotopic ossification (HO). It is of interest great to know the pain responses in these mice and how the opioid signaling is involved in the downstream events such as mast cell (MC) activation. MATERIALS AND METHODS: This study utilized a transgenic mouse model of HO in which BMP4 is expressed under the control of the Nse-BMP4. The tactile sensitivity and the cold sensitivity of the mice were measured in a classic inflammatory pain model (carrageenan solution injected into the plantar surface of the left hind paw). The MC activation and the expression profiles of different components in the opioid signaling were demonstrated through routine histology and immunohistochemistry and Western blotting, in the superficial and deep muscle injury models. RESULTS: We found that the pain responses in these mice were paradoxically attenuated or unchanged, and we also found increased expression of both Methionine Enkephalin (Met-Enk), and the µ-opioid receptor (MOR). Met-Enk and MOR both co-localized within activated MCs in limb tissues. Further, Nse-BMP4;MOR(-/-) double mutant mice showed attenuated MC activation and had a significant reduction in HO formation in response to injuries. CONCLUSIONS: These observations suggest that opioid signaling may play a key role in MC activation and the downstream inflammatory responses associated with HO. In addition to providing insight into the role of MC activation and associated injury responses in HO, these findings suggest opioid signaling as a potential therapeutic target in HO and possibly others disorders involving MC activation.
Assuntos
Encefalina Metionina/fisiologia , Mastócitos/fisiologia , Ossificação Heterotópica/fisiopatologia , Animais , Proteína Morfogenética Óssea 4/genética , Temperatura Baixa , Imuno-Histoquímica , Inflamação/complicações , Inflamação/patologia , Mastócitos/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Músculo Esquelético/lesões , Mutação/genética , Mutação/fisiologia , Nociceptividade/fisiologia , Ossificação Heterotópica/patologia , Medição da Dor , Fosfopiruvato Hidratase/genética , Estimulação Física , Receptores Opioides mu/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Astrogliosis following spinal cord injury (SCI) involves an early hypertrophic response that serves to repair damaged blood-brain barrier and a subsequent hyperplastic response that results in a dense scar that impedes axon regeneration. The mechanisms regulating these two phases of astrogliosis are beginning to be elucidated. In this study, we found that microRNA-21 (miR-21) increases in a time-dependent manner following SCI in mouse. Astrocytes adjacent to the lesion area express high levels of miR-21 whereas astrocytes in uninjured spinal cord express low levels of miR-21. To study the role of miR-21 in astrocytes after SCI, transgenic mice were generated that conditionally overexpress either the primary miR-21 transcript in astrocytes or a miRNA sponge designed to inhibit miR-21 function. Overexpression of miR-21 in astrocytes attenuated the hypertrophic response to SCI. Conversely, expression of the miR-21 sponge augmented the hypertrophic phenotype, even in chronic stages of SCI recovery when astrocytes have normally become smaller in size with fine processes. Inhibition of miR-21 function in astrocytes also resulted in increased axon density within the lesion site. These findings demonstrate a novel role for miR-21 in regulating astrocytic hypertrophy and glial scar progression after SCI, and suggest miR-21 as a potential therapeutic target for manipulating gliosis and enhancing functional outcome.
Assuntos
Astrócitos/metabolismo , MicroRNAs/metabolismo , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Animais , Modelos Animais de Doenças , Células HEK293 , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Traumatismos da Medula Espinal/patologiaRESUMO
Stem cell replacement holds the potential for sensorineural hearing loss (SNHL) treatment. However, its translation into clinical practice requires strategies for improving stem cell survival following intracochlear transplantation. Considering recent findings showing that the inner ear contains a resident population of immune cells, we hypothesized that immune evasion would improve the survival and residence time of transplanted stem cells in the cochlea, potentially leading to better outcomes. To test this, we leveraged genetic engineering techniques to develop hypoimmunogenic human-induced pluripotent stem cells (hi-iPSC), which lack human leukocyte antigen expression. We found that gene editing does not affect the biological properties of hi-iPSCs, including their capacity to differentiate into otic neural progenitors (ONPs). Compared to wild-type ONPs, more hypoimmunogenic ONPs (derived from hi-iPSCs) were found in the inner ear of immunocompetent mice ten days following cochlear xenotransplantation. This approach may open a new avenue for experimental and clinical SNHL treatments.
Assuntos
Perda Auditiva , Células-Tronco Pluripotentes Induzidas , Camundongos , Humanos , Animais , Transplante Heterólogo , Diferenciação Celular , Perda Auditiva/metabolismo , Transplante de Células-Tronco/métodos , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
Ketamine treatment decreases depressive symptoms within hours, but the mechanisms mediating these rapid antidepressant effects are unclear. Here, we demonstrate that activity of adult-born immature granule neurons (ABINs) in the mouse hippocampal dentate gyrus is both necessary and sufficient for the rapid antidepressant effects of ketamine. Ketamine treatment activates ABINs in parallel with its behavioral effects in both stressed and unstressed mice. Chemogenetic inhibition of ABIN activity blocks the antidepressant effects of ketamine, indicating that this activity is necessary for the behavioral effects. Conversely, chemogenetic activation of ABINs without any change in neuron numbers mimics both the cellular and the behavioral effects of ketamine, indicating that increased activity of ABINs is sufficient for rapid antidepressant effects. These findings thus identify a specific cell population that mediates the antidepressant actions of ketamine, indicating that ABINs can potentially be targeted to limit ketamine's side effects while preserving its therapeutic efficacy.
Assuntos
Ketamina , Animais , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Depressão/tratamento farmacológico , Hipocampo , Ketamina/farmacologia , Ketamina/uso terapêutico , Camundongos , NeurôniosRESUMO
Astrogliosis following spinal cord injury (SCI) involves an early hypertrophic response that is beneficial and a subsequent formation of a dense scar. We investigated the role of bone morphogenetic protein (BMP) signaling in gliosis after SCI and find that BMPR1a and BMPR1b signaling exerts opposing effects on hypertrophy. Conditional ablation of BMPR1a from glial fibrillary acidic protein (GFAP)-expressing cells leads to defective astrocytic hypertrophy, increased infiltration by inflammatory cells, and reduced axon density. BMPR1b-null mice conversely develop "hyperactive" reactive astrocytes and consequently have smaller lesion volumes. The effects of ablation of either receptor are reversed in the double knock-out animals. These findings indicate that BMPR1a and BMPR1b exert directly opposing effects on the initial reactive astrocytic hypertrophy. Also, BMPR1b knock-out mice have an attenuated glial scar in the chronic stages following injury, suggesting that it has a greater role in glial scar progression. To elucidate the differing roles of the two receptors in astrocytes, we examined the effects of ablation of either receptor in serum-derived astrocytes in vitro. We find that the two receptors exert opposing effects on the posttranscriptional regulation of astrocytic microRNA-21. Further, overexpression of microRNA-21 in wild-type serum-derived astrocytes causes a dramatic reduction in cell size accompanied by reduction in GFAP levels. Hence, regulation of microRNA-21 by BMP signaling provides a novel mechanism for regulation of astrocytic size. Targeting specific BMPR subunits for therapeutic purposes may thus provide an approach for manipulating gliosis and enhancing functional outcomes after SCI.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Gliose/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Astrócitos/metabolismo , Axônios/ultraestrutura , Células Cultivadas , Feminino , Gliose/patologia , Hiperplasia/patologia , Hiperplasia/fisiopatologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Smad/metabolismo , Traumatismos da Medula Espinal/patologia , Regulação para CimaRESUMO
Heterotopic ossification (HO) is a disabling condition associated with neurologic injury, inflammation, and overactive bone morphogenetic protein (BMP) signaling. The inductive factors involved in lesion formation are unknown. We found that the expression of the neuro-inflammatory factor Substance P (SP) is dramatically increased in early lesional tissue in patients who have either fibrodysplasia ossificans progressiva (FOP) or acquired HO, and in three independent mouse models of HO. In Nse-BMP4, a mouse model of HO, robust HO forms in response to tissue injury; however, null mutations of the preprotachykinin (PPT) gene encoding SP prevent HO. Importantly, ablation of SP(+) sensory neurons, treatment with an antagonist of SP receptor NK1r, deletion of NK1r gene, or genetic down-regulation of NK1r-expressing mast cells also profoundly inhibit injury-induced HO. These observations establish a potent neuro-inflammatory induction and amplification circuit for BMP-dependent HO lesion formation, and identify novel molecular targets for prevention of HO.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Ossificação Heterotópica/metabolismo , Substância P/metabolismo , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Feminino , Humanos , Imuno-Histoquímica , Isoindóis/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Miosite Ossificante/genética , Miosite Ossificante/metabolismo , Antagonistas dos Receptores de Neurocinina-1 , Ossificação Heterotópica/genética , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Receptores da Neurocinina-1/metabolismo , Células Receptoras Sensoriais/metabolismo , Taquicininas/genética , Taquicininas/metabolismoRESUMO
BACKGROUND: Skin stem cells contribute to all three major lineages of epidermal appendages, i.e., the epidermis, the hair follicle, and the sebaceous gland. In hair follicles, highly proliferative committed progenitor cells, called matrix cells, are located at the base of the follicle in the hair bulb. The differentiation of these early progenitor cells leads to specification of a central hair shaft surrounded by an inner root sheath (IRS) and a companion layer. Multiple signaling molecules, including bone morphogenetic proteins (BMPs), have been implicated in this process. METHODS: To further probe the contribution of BMP signaling to hair follicle development and maintenance we employed a transgenic mouse that expresses the BMP inhibitor, Noggin, to disrupt BMP signaling specifically in subset of hair follicle progenitors under the control of neuron specific enolase (Nse) promoter. We then studied the skin tumor phenotypes of the transgenic mice through histology, immunohistochemistry and Western Blotting to delineate the underlying mechanisms. Double transgenic mice expressing BMP as well as noggin under control of the Nse promoter were used to rescue the skin tumor phenotypes. RESULTS: We found that the transgene is expressed specifically in a subpopulation of P-cadherin positive progenitor cells in Nse-Noggin mice. Blocking BMP signaling in this cell population led to benign hair follicle-derived neoplasias resembling human trichofolliculomas, associated with down-regulation of E-cadherin expression and dynamic regulation of CD44. CONCLUSIONS: These observations further define a critical role for BMP signaling in maintaining the homeostasis of hair follicles, and suggest that dysregulation of BMP signaling in hair follicle progenitors may contribute to human trichofolliculoma.
Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Caderinas/genética , Cisto Folicular/metabolismo , Doenças do Cabelo/metabolismo , Folículo Piloso/metabolismo , Neoplasia de Células Basais/metabolismo , Neoplasias Cutâneas/metabolismo , Células-Tronco/patologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Caderinas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Cisto Folicular/patologia , Doenças do Cabelo/patologia , Folículo Piloso/patologia , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasia de Células Basais/patologia , Transdução de Sinais , Neoplasias Cutâneas/patologia , Células-Tronco/metabolismoRESUMO
Stem cell-replacement therapies have been proposed as a potential tool to treat sensorineural hearing loss by aiding the regeneration of spiral ganglion neurons (SGNs) in the inner ear. However, transplantation procedures have yet to be explored thoroughly to ensure proper cell differentiation and optimal transplant procedures. We hypothesized that the aggregation of human embryonic stem cell (hESC)-derived otic neuronal progenitor (ONP) cells into a multicellular form would improve their function and their survival in vivo post-transplantation. We generated hESC-derived ONP spheroids-an aggregate form conducive to differentiation, transplantation, and prolonged cell survival-to optimize conditions for their transplantation. Our findings indicate that these cell spheroids maintain the molecular and functional characteristics similar to those of ONP cells, which are upstream in the SGN lineage. Moreover, our phenotypical, electrophysiological, and mechanical data suggest an optimal spheroid transplantation point after 7 days of in vitro three-dimensional (3D) culture. We have also developed a feasible transplantation protocol for these spheroids using a micropipette aided by a digital microinjection system. In summary, the present work demonstrates that the transplantation of ONP cells in spheroid form into the inner ear through micropipette 7 days after seeding for 3D spheroid culture is an expedient and viable method for stem cell replacement therapies in the inner ear.
Assuntos
Células-Tronco Embrionárias Humanas , Diferenciação Celular , Humanos , Neurônios , Esferoides Celulares , Gânglio Espiral da Cóclea , Transplante de Células-TroncoRESUMO
Heterotopic ossification (HO), the abnormal formation of true marrow-containing bone within extraskeletal soft tissues, is a serious bony disorder that may be either acquired or hereditary. We utilized an animal model of the genetic disorder fibrodysplasia ossificans progressiva to examine the cellular mechanisms underlying HO. We found that HO in these animals was triggered by soft tissue injuries and that the effects were mediated by macrophages. Spreading of HO beyond the initial injury site was mediated by an abnormal adaptive immune system. These observations suggest that dysregulation of local stem/progenitor cells could be a common cellular mechanism for typical HO irrespective of the signal initiating the bone formation.
Assuntos
Ossificação Heterotópica/patologia , Células-Tronco/patologia , Animais , Proteína Morfogenética Óssea 4/metabolismo , Linhagem da Célula , Sistema Imunitário/imunologia , Integrases/metabolismo , Macrófagos/imunologia , Camundongos , Modelos Biológicos , Músculos/patologia , Ossificação Heterotópica/imunologia , Fosfopiruvato Hidratase/metabolismo , Pele/patologiaRESUMO
Although the application of human embryonic stem cells (hESCs) in stem cell-replacement therapy remains promising, its potential is hindered by a low cell survival rate in post-transplantation within the inner ear. Here, we aim to enhance the in vitro and in vivo survival rate and neuronal differentiation of otic neuronal progenitors (ONPs) by generating an artificial stem cell niche consisting of three-dimensional (3D) hESC-derived ONP spheroids with a nanofibrillar cellulose hydrogel and a sustained-release brain-derivative neurotrophic factor delivery system. Our results demonstrated that the transplanted hESC-derived ONP spheroids survived and neuronally differentiated into otic neuronal lineages in vitro and in vivo and also extended neurites toward the bony wall of the cochlea 90 days after the transplantation without the use of immunosuppressant medication. Our data in vitro and in vivo presented here provide sufficient evidence that we have established a robust, reproducible protocol for in vivo transplantation of hESC-derived ONPs to the inner ear. Using our protocol to create an artificial stem cell niche in the inner ear, it is now possible to work on integrating transplanted hESC-derived ONPs further and also to work toward achieving functional auditory neurons generated from hESCs. Our findings suggest that the provision of an artificial stem cell niche can be a future approach to stem cell-replacement therapy for inner-ear regeneration. STATEMENT OF SIGNIFICANCE: Inner ear regeneration utilizing human embryonic stem cell-derived otic neuronal progenitors (hESC-derived ONPs) has remarkable potential for treating sensorineural hearing loss. However, the local environment of the inner ear requires a suitable stem cell niche to allow hESC-derived ONP engraftment as well as neuronal differentiation. To overcome this obstacle, we utilized three-dimensional spheroid formation (direct contact), nanofibrillar cellulose hydrogel (extracellular matrix), and a neurotrophic factor delivery system to artificially create a stem cell niche in vitro and in vivo. Our in vitro and in vivo data presented here provide sufficient evidence that we have established a robust, reproducible protocol for in vivo transplantation of hESC-derived ONPs to the inner ear.
Assuntos
Orelha Interna , Nicho de Células-Tronco , Diferenciação Celular , Celulose , Preparações de Ação Retardada , Humanos , Hidrogéis/farmacologia , Fatores de Crescimento NeuralRESUMO
Gliosis and fibrosis after spinal cord injury (SCI) lead to formation of a scar that is thought to present both molecular and mechanical barriers to neuronal regeneration. The scar consists of a meshwork of reactive glia and deposited, cross-linked, extracellular matrix (ECM) that has long been assumed to present a mechanically "stiff" blockade. However, remarkably little quantitative information is available about the rheological properties of chronically injured spinal tissue. In this study we utilize atomic force microscopy microindentation to provide quantitative evidence of chronic mechanical stiffening after SCI. Using the results of this tissue characterization, we assessed the sensitivity of both mouse and human astrocytes in vitro and determined that they are exquisitely mechanosensitive within the relevant range of substrate stiffness observed in the injured/uninjured spinal cord. We then utilized a novel immune modifying nanoparticle (IMP) treatment as a tool to reveal fibrotic scarring as one of the key drivers of mechanical stiffening after SCI in vivo. We also demonstrate that glial scar-forming astrocytes form a highly aligned, anisotropic network of glial fibers after SCI, and that IMP treatment mitigates this pathological alignment. Taken together, our results identify chronic mechanical stiffening as a critically important aspect of the complex lesion milieu after SCI that must be considered when assessing and developing potential clinical interventions for SCI.
Assuntos
Fenômenos Biomecânicos/fisiologia , Gliose/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Vértebras Torácicas/fisiopatologia , Animais , Astrócitos/química , Astrócitos/fisiologia , Células Cultivadas , Células-Tronco Embrionárias/química , Células-Tronco Embrionárias/fisiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica/métodos , Gravidez , Vértebras Torácicas/químicaRESUMO
BACKGROUND: Heterotopic ossification (HO), either acquired (aHO) or hereditary, such as fibrodysplasia ossificans progressiva (FOP), is a serious condition without effective treatment. Understanding of the core process of injury-induced HO is still severely limited. METHODS: Double-pulse thymidine analog labeling was used to explore the distinctive domains evolved in injury-induced lesions in an animal model of HO (Nse-BMP4). Histological studies were performed to see whether a similar zonal pattern is also consistently found in biopsies from patients with aHO and FOP. In vivo clonal analysis with Rainbow mice, genetic loss-of-function studies with diphtheria toxin A (DTA)-mediated depletion and lineage tracing with Zsgreen reporter mice were used to obtain further evidence that Tie2-cre-, Gli1-creERT-, and Glast-creERT-labeled cells contribute to HO as niche-dwelling progenitor/stem cells. Immunohistochemistry was used to test whether vasculature, neurites, macrophages, and mast cells are closely associated with the proposed niche and thus are possible candidate niche supportive cells. Similar methods also were employed to further understand the signaling pathways that regulate the niche and the resultant HO. RESULTS: We found that distinctive domains evolved in injury-induced lesions, including, from outside-in, a mesenchymal stem cell (MSC) niche, a transient domain and an inner differentiated core in an animal model of HO (Nse-BMP4). A similar zonal structure was found in patients with aHO and FOP. In vivo clonal analysis with Rainbow mice and genetic loss-of-function studies with DTA provided evidence that Tie2-cre-, Gli1-creERT-, and Glast-creERT-labeled cells contribute to HO as niche-dwelling progenitor/stem cells; consistently, vasculature, neurites, macrophages, and mast cells are closely associated with the proposed niche and thus are possible candidate niche supportive cells. Further mechanistic study found that BMP and hedgehog (Hh) signaling co-regulate the niche and the resultant HO. CONCLUSIONS: Available data provide evidence of a potential core mechanism in which multiple disease-specific cellular and extracellular molecular elements form a unique local microenvironment, i.e., an injury-induced stem cell niche, which regulates the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs). The implication for HO is that therapeutic approaches must consider several different disease specific factors as parts of a functional unit, instead of treating one factor at a time.
Assuntos
Miosite Ossificante/genética , Ossificação Heterotópica/genética , Osteogênese/genética , Nicho de Células-Tronco/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Toxina Diftérica/genética , Modelos Animais de Doenças , Transportador 1 de Aminoácido Excitatório/genética , Humanos , Mutação com Perda de Função/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Miosite Ossificante/patologia , Miosite Ossificante/terapia , Ossificação Heterotópica/patologia , Ossificação Heterotópica/terapia , Fragmentos de Peptídeos/genética , Receptor TIE-2/genética , Transdução de Sinais/genética , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
Heterotopic ossification (HO), true bone formation in soft tissue, is closely associated with abnormal injury/immune responses. We hypothesized that a key underlying mechanism of HO might be injury-induced dysregulation of immune checkpoint proteins (ICs). We found that the earliest stages of HO are characterized by enhanced infiltration of polarized macrophages into sites of minor injuries in an animal model of HO. The non-specific immune suppressants, Rapamycin and Ebselen, prevented HO providing evidence of the central role of the immune responses. We examined the expression pattern of ICs and found that they are dysregulated in HO lesions. More importantly, loss of function of inhibitory ICs (including PD1, PD-L1, and CD152) markedly inhibited HO, whereas loss of function of stimulatory ICs (including CD40L and OX-40L) facilitated HO. These findings suggest that IC inhibitors may provide a therapeutic approach to prevent or limit the extent of HO.
RESUMO
Heterotopic ossification (HO), acquired or hereditary, endochondral or intramembranous, is the formation of true bone outside the normal skeleton. Since perivascular Gli1+ progenitors contribute to injury induced organ fibrosis, and CD133 is expressed by a variety of populations of adult stem cells, this study utilized Cre-lox based genetic lineage tracing to test the contribution to endochondral HO of adult stem/progenitor cells that expressed either Gli1 or CD133. We found that both lineages contributed broadly to different normal tissues with distinct patterns, but that only Gli1-creERT labeled stem/progenitor cells contributed to all stages of endochondral HO in a BMP dependent, injury induced, transgenic mouse model. Hedgehog (Hh) signaling was abnormal at endochondral HO lesion sites with increased signaling surrounding the lesion but diminished signaling within it. Thus, local dysregulation of Hh signaling participates in the pathophysiology of endochondral HO. However, unlike a previous report of intramembranous HO, systemic inhibition of Hh signaling was insufficient to prevent the initiation of the endochondral HO process or to treat the existing endochondral HO, suggesting that Hh participates in, but is not essential for endochondral HO in this model. This could potentially reflect the underlying difference between intramembranous and endochondral HO. Nevertheless, identification of this novel stem/precursor cell population as a HO-contributing cell population provides a potential drugable target.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Ossificação Heterotópica/metabolismo , Ossificação Heterotópica/patologia , Osteogênese/fisiologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Transgênicos , Osteogênese/genética , Pirimidinonas/farmacologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Tiofenos/farmacologiaRESUMO
The use of human embryonic stem cells (hESCs) for regeneration of the spiral ganglion will require techniques for promoting otic neuronal progenitor (ONP) differentiation, anchoring of cells to anatomically appropriate and specific niches, and long-term cell survival after transplantation. In this study, we used self-assembling peptide amphiphile (PA) molecules that display an IKVAV epitope (IKVAV-PA) to create a niche for hESC-derived ONPs that supported neuronal differentiation and survival both in vitro and in vivo after transplantation into rodent inner ears. A feature of the IKVAV-PA gel is its ability to form organized nanofibers that promote directed neurite growth. Culture of hESC-derived ONPs in IKVAV-PA gels did not alter cell proliferation or viability. However, the presence of IKVAV-PA gels increased the number of cells expressing the neuronal marker beta-III tubulin and improved neurite extension. The self-assembly properties of the IKVAV-PA gel allowed it to be injected as a liquid into the inner ear to create a biophysical niche for transplanted cells after gelation in vivo. Injection of ONPs combined with IKVAV-PA into the modiolus of X-SCID rats increased survival and localization of the cells around the injection site compared to controls. Human cadaveric temporal bone studies demonstrated the technical feasibility of a transmastoid surgical approach for clinical intracochlear injection of the IKVAV-PA/ONP combination. Combining stem cell transplantation with injection of self-assembling PA gels to create a supportive niche may improve clinical approaches to spiral ganglion regeneration.