RESUMEN
BACKGROUND: Systemic infusion of mesenchymal stromal cells (MSCs) has been shown to induce acute acceleration of growth velocity in children with osteogenesis imperfecta (OI) despite minimal engraftment of infused MSCs in bones. Using an animal model of OI we have previously shown that MSC infusion stimulates chondrocyte proliferation in the growth plate and that this enhanced proliferation is also observed with infusion of MSC conditioned medium in lieu of MSCs, suggesting that bone growth is due to trophic effects of MSCs. Here we sought to identify the trophic factor secreted by MSCs that mediates this therapeutic activity. METHODS: To examine whether extracellular vesicles (EVs) released from MSCs have therapeutic activity, EVs were isolated from MSC conditioned medium by ultracentrifugation. To further characterize the trophic factor, RNA or microRNA (miRNA) within EVs was depleted by either ribonuclease (RNase) treatment or suppressing miRNA biogenesis in MSCs. The functional activity of these modified EVs was evaluated using an in vitro chondrocyte proliferation assay. Finally, bone growth was evaluated in an animal model of OI treated with EVs. RESULTS: We found that infusion of MSC-derived EVs stimulated chondrocyte proliferation in the growth plate, resulting in improved bone growth in a mouse model of OI. However, infusion of neither RNase-treated EVs nor miRNA-depleted EVs enhanced chondrocyte proliferation. CONCLUSION: MSCs exert therapeutic effects in OI by secreting EVs containing miRNA, and EV therapy has the potential to become a novel cell-free therapy for OI that will overcome some of the current limitations in MSC therapy.
Asunto(s)
Desarrollo Óseo , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteogénesis Imperfecta/patología , Animales , Proliferación Celular , Niño , Condrocitos/citología , Modelos Animales de Enfermedad , Endopeptidasa K/metabolismo , Humanos , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Ribonucleasas/metabolismo , SolubilidadRESUMEN
BACKGROUND AIMS: Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has demonstrated remarkable success in targeting B-cell malignancies but is often complicated by serious systemic toxicity in the form of cytokine release syndrome (CRS). CRS symptoms are primarily mediated by interleukin 6 (IL-6), and clinical management has focused on inhibition of IL-6 signaling. The cellular source and function of IL-6 in CRS remain unknown. METHODS: Using co-culture assays and data from patients on our clinical CAR T-cell trials, we investigated the cellular source of IL-6, as well as other CRS-associated cytokines, during CAR T-cell activation. We also explored the effect that IL-6 has on T-cell function. RESULTS: We demonstrated that IL-6 is secreted by monocyte-lineage cells in response to CAR T-cell activation in a contact-independent mechanism upon T-cell engagement of target leukemia. We observed that the presence of antigen-presenting cell-derived IL-6 has no impact on CAR T-cell transcriptional profiles or cytotoxicity. Finally, we confirm that CAR T cells do not secrete IL-6 in vivo during clinical CRS. DISCUSSION: These findings suggest that IL-6 blockade will not affect CD19 CAR T-cell-driven anti-leukemic cytotoxicity, permitting enhanced control of CRS while maintaining CAR T-cell efficacy.
Asunto(s)
Antígenos CD19/genética , Interleucina-6/metabolismo , Monocitos/metabolismo , Linfocitos T/metabolismo , Animales , Células Presentadoras de Antígenos/metabolismo , Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Linaje de la Célula , Técnicas de Cocultivo , Citocinas/metabolismo , Humanos , Activación de Linfocitos/inmunología , Ratones Endogámicos NOD , Monocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Successful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC. We show that TBI-induced migration of megakaryocytes to the endosteal niche depends on thrombopoietin signaling through the c-MPL receptor on megakaryocytes, as well as CD41 integrin-mediated adhesion. Moreover, niche osteoblast proliferation post-TBI required megakaryocyte-secreted platelet-derived growth factor-BB. Furthermore, blockade of c-MPL-dependent megakaryocyte migration and function after TBI resulted in a significant decrease in donor HSC engraftment in primary and competitive secondary transplantation assays. Finally, we administered thrombopoietin to mice beginning 5 days before marrow radioablation and ending 24 hours before transplant to enhance megakaryocyte function post-TBI, and found that this strategy significantly enhanced donor HSC engraftment, providing a rationale for improving hematopoietic recovery and perhaps overall outcome after clinical HSC transplantation.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Megacariocitos/citología , Osteoblastos/citología , Receptores de Trombopoyetina/fisiología , Nicho de Células Madre/fisiología , Irradiación Corporal Total , Animales , Becaplermina , Movimiento Celular/fisiología , Movimiento Celular/efectos de la radiación , Proliferación Celular , Endotelio Vascular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Supervivencia de Injerto , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/efectos de la radiación , Megacariocitos/metabolismo , Megacariocitos/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteoblastos/efectos de la radiación , Proteínas Proto-Oncogénicas c-sis/metabolismo , Transducción de Señal , Trombopoyetina/metabolismoRESUMEN
To study the cellular mechanism of the tendon repair process, we used a mouse Achilles tendon injury model to focus on the cells recruited to the injured site. The cells isolated from injured tendon 1 week after the surgery and uninjured tendons contained the connective tissue progenitor populations as determined by colony-forming capacity, cell surface markers, and multipotency. When the injured tendon-derived progenitor cells (inTPCs) were transplanted into injured Achilles tendons, they were not only integrated in the regenerating area expressing tenogenic phenotype but also trans-differentiated into chondrogenic cells in the degenerative lesion that underwent ectopic endochondral ossification. Surprisingly, the micromass culture of the inTPCs rapidly underwent chondrogenic differentiation even in the absence of exogenous bone morphogenetic proteins or TGFßs. The cells isolated from human ruptured tendon tissues also showed connective tissue progenitor properties and exhibited stronger chondrogenic ability than bone marrow stromal cells. The mouse inTPCs contained two subpopulations one positive and one negative for CD105, a coreceptor of the TGFß superfamily. The CD105-negative cells showed superior chondrogenic potential in vitro and induced larger chondroid degenerative lesions in mice as compared to the CD105-positive cells. These findings indicate that tendon progenitor cells are recruited to the injured site of tendons and have a strong chondrogenic potential and that the CD105-negative population of these cells would be the cause for chondroid degeneration in injured tendons. The newly identified cells recruited to the injured tendon may provide novel targets to develop therapeutic strategies to facilitate tendon repair.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/citología , Células Madre/citología , Tendones/citología , Animales , Células Cultivadas , Condrogénesis/fisiología , Endoglina , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Osteogénesis/fisiología , Tendones/metabolismoRESUMEN
BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have been applied to patients in cell therapy for various diseases. Recently, we introduced a novel MSC separation filter device which could yield approximately 2.5-fold more MSCs from bone marrow in a closed system compared with the conventional open density gradient centrifugation method. MSCs isolated with these two methods were phenotypically similar and met the criteria defining human MSC proposed by the International Society for Cellular Therapy. However, these criteria do not reflect the functional capacity of MSCs. It has been shown that the donor, source, isolation method, culture condition and cryopreservation of MSCs have potential to alter their therapeutic efficacy. To determine the equivalency of MSCs isolated by these two methods, we compared their genomic profiles as an index of their biologic potential and evaluated their growth promoting potential as an index of function. METHODS: The gene expression profiles of human MSCs isolated from 5 healthy donors with two distinct methods were obtained from microarray analyses. The functional activity of freshly expanded/cryopreserved MSCs from these two isolation methods was evaluated using an in vitro chondrocyte proliferation assay. RESULTS: Freshly expanded MSCs isolated by these two methods were found to exhibit similar gene expression profiles and equivalent therapeutic effects, while freshly thawed, cryopreserved MSCs lacked all measureable therapeutic activity. CONCLUSIONS: The MSC separation device generates genomically and functionally equivalent MSCs compared with the conventionally isolated MSCs, although freshly thawed, cryopreserved MSCs, isolated by either method, are devoid of activity in our bioassay.
Asunto(s)
Separación Celular/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Criopreservación/métodos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Adulto , Animales , Médula Ósea , Células de la Médula Ósea/citología , Diferenciación Celular , Proliferación Celular , Separación Celular/instrumentación , Células Cultivadas , Condrocitos/citología , Femenino , Perfilación de la Expresión Génica , Genómica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Trasplante Heterólogo , Adulto JovenRESUMEN
Transplantation of whole bone marrow (BMT) as well as ex vivo-expanded mesenchymal stromal cells (MSCs) leads to striking clinical benefits in children with osteogenesis imperfecta (OI); however, the underlying mechanism of these cell therapies has not been elucidated. Here, we show that non-(plastic)-adherent bone marrow cells (NABMCs) are more potent osteoprogenitors than MSCs in mice. Translating these findings to the clinic, a T cell-depleted marrow mononuclear cell boost (> 99.99% NABMC) given to children with OI who had previously undergone BMT resulted in marked growth acceleration in a subset of patients, unambiguously indicating the therapeutic potential of bone marrow cells for these patients. Then, in a murine model of OI, we demonstrated that as the donor NABMCs differentiate to osteoblasts, they contribute normal collagen to the bone matrix. In contrast, MSCs do not substantially engraft in bone, but secrete a soluble mediator that indirectly stimulates growth, data which provide the underlying mechanism of our prior clinical trial of MSC therapy for children with OI. Collectively, our data indicate that both NABMCs and MSCs constitute effective cell therapy for OI, but exert their clinical impact by different, complementary mechanisms. The study is registered at www.clinicaltrials.gov as NCT00187018.
Asunto(s)
Trasplante de Médula Ósea/métodos , Leucocitos Mononucleares/trasplante , Trasplante de Células Madre Mesenquimatosas/métodos , Osteogénesis Imperfecta/cirugía , Animales , Estatura/fisiología , Peso Corporal/fisiología , Matriz Ósea/metabolismo , Células Cultivadas , Niño , Colágeno/genética , Colágeno/metabolismo , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Vértebras Lumbares/crecimiento & desarrollo , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/fisiopatología , Factores de TiempoRESUMEN
The efficiency of hematopoietic stem cell (HSC) engraftment after bone marrow (BM) transplantation depends largely on the capacity of the marrow microenvironment to accept the transplanted cells. While radioablation of BM damages osteoblastic stem cell niches, little is known about their restoration and mechanisms governing their receptivity to engraft transplanted HSCs. We previously reported rapid restoration and profound expansion of the marrow endosteal microenvironment in response to marrow radioablation. Here, we show that this reorganization represents proliferation of mature endosteal osteoblasts which seem to arise from a small subset of high-proliferative, relatively radio-resistant endosteal cells. Multiple layers of osteoblasts form along the endosteal surface within 48 hours after total body irradiation, concomitant with a peak in marrow cytokine expression. This niche reorganization fosters homing of the transplanted hematopoietic cells to the host marrow space and engraftment of long-term-HSC. Inhibition of insulin-like growth factor (IGF)-1-receptor tyrosine kinase signaling abrogates endosteal osteoblast proliferation and donor HSC engraftment, suggesting that the cytokine IGF-1 is a crucial mediator of endosteal niche reorganization and consequently donor HSC engraftment. Further understanding of this novel mechanism of IGF-1-dependent osteoblastic niche expansion and HSC engraftment may yield clinical applications for improving engraftment efficiency after clinical HSC transplantation.
Asunto(s)
Trasplante de Médula Ósea/métodos , Trasplante de Células Madre Hematopoyéticas , Factor I del Crecimiento Similar a la Insulina/fisiología , Nicho de Células Madre/fisiología , Animales , Huesos/citología , Movimiento Celular , Proliferación Celular , Supervivencia de Injerto , Hematopoyesis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoblastos/fisiología , Irradiación Corporal TotalRESUMEN
Bone marrow transplantation (BMT) can give rise to donor-derived osteopoiesis in mice and humans; however, the source of this activity, whether a primitive osteoprogenitor or a transplantable marrow cell with dual hematopoietic and osteogenic potential, has eluded detection. To address this issue, we fractionated whole BM from mice according to cell surface immunophenotype and assayed the hematopoietic and osteopoietic potentials of the transplanted cells. Here, we show that a donor marrow cell capable of robust osteopoiesis possesses a surface phenotype of c-Kit(+) Lin(-) Sca-1(+) CD34(-/lo), identical to that of the long-term repopulating hematopoietic stem cell (LTR-HSC). Secondary BMT studies demonstrated that a single marrow cell able to contribute to hematopoietic reconstitution in primary recipients also drives robust osteopoiesis and LT hematopoiesis in secondary recipients. These findings indicate that LTR-HSC can give rise to progeny that differentiate to osteoblasts after BMT, suggesting a mechanism for prompt restoration of the osteoblastic HSC niche following BM injury, such as that induced by clinical BMT preparative regimens. An understanding of the mechanisms that regulate this differentiation potential may lead to novel treatments for disorders of bone as well as methods for preserving the integrity of endosteal hematopoietic niches.
Asunto(s)
Diferenciación Celular , Células Madre Hematopoyéticas/citología , Osteoblastos/citología , Nicho de Células Madre , Animales , Trasplante de Médula Ósea , Hematopoyesis , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Inmunohistoquímica , Inmunofenotipificación , Hibridación Fluorescente in Situ , Ratones , Osteoblastos/metabolismo , Fenotipo , Proyectos Piloto , Proteínas Proto-Oncogénicas c-kit/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The vitamin D binding protein, the GC protein, is a multifunctional protein that binds circulating vitamin D and also increases macrophage killing of tumor cells. Injecting exogenous GC protein concurrent with experimental tumor implant decreases tumor engraftment rate. Until now serum abundance of this protein was thought to be controlled by estrogen, glucocorticoids and inflammatory cytokines, but, not by vitamin D itself(1, 2). Nonetheless, increasing dietary vitamin D is thought to increase serum vitamin D, which is 98% bound by the GC protein. Based on the protection that excess GC protein offers we sought to determine if decreased GC protein abundance might decrease tumor immunity. Relatedly, we theorized, by contrast to the current model, that dietary vitamin D might affect serum abundance of GC protein. If exogenous vitamin D alters available GC levels, then this effect might indicate a novel pathway by which vitamin D enhances immunity. To examine these possibilities, we examined the effect of GC protein absence on tumor persistence or engraftment on two different and common tumor types (prostate cancer and breast cancer). We further examined the relationship between dietary vitamin D and serum GC abundance. We found that absence of GC protein allowed significantly more engraftment of breast tumor cells in female mice and of prostate tumor cells in male mice. Further, we found a U-shaped response of serum GC protein to dietary vitamin D dosage as well as to serum vitamin D, indicating the potential benefit of high exogenous doses to enhance immunity and reduce tumor burden.
RESUMEN
PURPOSE: Retinoblastoma is the most common intraocular malignancy in children. Although new chemotherapeutic approaches have improved ocular salvage rates, novel therapies are required for patients with refractory intraocular and metastatic disease. Chimeric antigen receptor (CAR) T cells targeting glypican-2 (GPC2) are a potential new therapeutic strategy. EXPERIMENTAL DESIGN: GPC2 expression and its regulation by the E2F1 transcription factor were studied in retinoblastoma patient samples and cellular models. In vitro, we performed functional studies comparing GPC2 CAR T cells with different costimulatory domains (4-1BB and CD28). In vivo, the efficacy of local and systemic administration of GPC2 CAR T cells was evaluated in intraocular and leptomeningeal human retinoblastoma xenograft models. RESULTS: Retinoblastoma tumors, but not healthy retinal tissues, expressed cell surface GPC2, and this tumor-specific expression was driven by E2F1. GPC2-directed CARs with 4-1BB costimulation (GPC2.BBz) were superior to CARs with CD28 stimulatory domains (GPC2.28z), efficiently inducing retinoblastoma cell cytotoxicity and enhancing T-cell proliferation and polyfunctionality. In vivo, GPC2.BBz CARs had enhanced persistence, which led to significant tumor regression compared with either control CD19 or GPC2.28z CARs. In intraocular models, GPC2.BBz CAR T cells efficiently trafficked to tumor-bearing eyes after intravitreal or systemic infusions, significantly prolonging ocular survival. In central nervous system (CNS) retinoblastoma models, intraventricular or systemically administered GPC2.BBz CAR T cells were activated in retinoblastoma-involved CNS tissues, resulting in robust tumor regression with substantially extended overall mouse survival. CONCLUSIONS: GPC2-directed CAR T cells are effective against intraocular and CNS metastatic retinoblastomas.
Asunto(s)
Glipicanos , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Retinoblastoma , Linfocitos T , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Retinoblastoma/inmunología , Retinoblastoma/patología , Retinoblastoma/terapia , Ratones , Receptores Quiméricos de Antígenos/inmunología , Glipicanos/inmunología , Glipicanos/antagonistas & inhibidores , Inmunoterapia Adoptiva/métodos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Línea Celular Tumoral , Neoplasias del Sistema Nervioso Central/terapia , Neoplasias del Sistema Nervioso Central/inmunología , Neoplasias del Sistema Nervioso Central/secundario , Neoplasias del Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , FemeninoRESUMEN
Novel chimeric antigen receptor (CAR) T-cell approaches are needed to improve therapeutic efficacy in solid tumors. High-risk neuroblastoma is an aggressive pediatric solid tumor that expresses cell-surface GPC2 and GD2 with a tumor microenvironment infiltrated by CD16a-expressing innate immune cells. Here we engineer T-cells to express a GPC2-directed CAR and simultaneously secrete a bispecific innate immune cell engager (BiCE) targeting both GD2 and CD16a. In vitro, GPC2.CAR-GD2.BiCE T-cells induce GPC2-dependent cytotoxicity and secrete GD2.BiCE that promotes GD2-dependent activation of antitumor innate immunity. In vivo, GPC2.CAR-GD2.BiCE T-cells locally deliver GD2.BiCE and increase intratumor retention of NK-cells. In mice bearing neuroblastoma patient-derived xenografts and reconstituted with human CD16a-expressing immune cells, GD2.BiCEs enhance GPC2.CAR antitumor efficacy. A CAR.BiCE strategy should be considered for tumor histologies where antigen escape limits CAR efficacy, especially for solid tumors like neuroblastoma that are infiltrated by innate immune cells.
Asunto(s)
Gangliósidos , Inmunidad Innata , Inmunoterapia Adoptiva , Células Asesinas Naturales , Neuroblastoma , Receptores Quiméricos de Antígenos , Linfocitos T , Neuroblastoma/inmunología , Neuroblastoma/terapia , Neuroblastoma/patología , Animales , Humanos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Ratones , Gangliósidos/inmunología , Línea Celular Tumoral , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Glipicanos/inmunología , Glipicanos/metabolismo , Microambiente Tumoral/inmunología , FemeninoRESUMEN
Transplantation of bone marrow cells leads to engraftment of osteopoietic and hematopoietic progenitors. We sought to determine whether the recently described transient expansion of the host osteoblastic niche after marrow radioablation promotes engraftment of both osteopoietic and hematopoietic progenitor cells. Mice infused with marrow cells 24 hours after total body irradiation (TBI) demonstrated significantly greater osteopoietic and hematopoietic progenitor chimerism than did mice infused at 30 minutes or 6 hours. Irradiated mice with a lead shield over 1 hind limb showed greater hematopoietic chimerism in the irradiated limb than in the shielded limb at both the 6- and 24-hour intervals. By contrast, the osteopoietic chimerism was essentially equal in the 2 limbs at each of these intervals, although it significantly increased when cells were infused 24 hours compared with 6 hours after TBI. Similarly, the number of donor phenotypic long-term hematopoietic stem cells was equivalent in the irradiated and shielded limbs after each irradiation-to-infusion interval but was significantly increased at the 24-hour interval. Our findings indicate that a 24-hour delay in marrow cell infusion after TBI facilitates expansion of the endosteal osteoblastic niche, leading to enhanced osteopoietic and hematopoietic engraftment.
Asunto(s)
Trasplante de Médula Ósea/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Osteoblastos/citología , Animales , Células de la Médula Ósea/citología , Ratones , Osteogénesis , Trasplante AutólogoRESUMEN
Transplantation of whole bone marrow (BMT) leads to engraftment of both osteoprogenitor cells and hematopoietic cells; however, the robust osteopoietic chimerism seen early after BMT decreases with time. Using our established murine model, we demonstrate that a post-BMT regimen of either granulocyte-colony stimulating factor, growth hormone, parathyroid hormone, or stem cell factor each stimulates greater donor osteoblast chimerism at 4 months posttransplantation than saline-treated controls and approximates the robust osteopoietic chimerism seen early after BMT; however, only growth hormone led to significantly more donor-derived osteocytes than controls. Importantly, there were no adverse hematologic consequences of the different treatments. Our data demonstrate that these cytokines can stimulate the differentiation of transplanted donor marrow cells into the osteopoietic lineage after BMT. Post-BMT cytokine therapy may generate durable osteopoietic engraftment, which should lead to sustained clinical benefit and render BMT more applicable to bone disorders.
Asunto(s)
Trasplante de Médula Ósea/patología , Citocinas/farmacología , Osteogénesis/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Factor Estimulante de Colonias de Granulocitos/farmacología , Hormona del Crecimiento/farmacología , Ratones , Ratones Transgénicos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteocitos/citología , Osteocitos/efectos de los fármacos , Osteogénesis/fisiología , Hormona Paratiroidea/farmacología , Proteínas Recombinantes , Factor de Células Madre/farmacología , Quimera por TrasplanteRESUMEN
BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have been studied as cell therapy to treat a vast array of diseases. In clinical MSC production, the isolated cells must undergo extensive ex vivo expansion to obtain a sufficient dose of MSCs for the investigational treatment. However, extended tissue culture is fraught with potential hazards, including contamination and malignant transformation. Changes of gene expression with prolonged culture may alter the therapeutic potential of the cells. Increasing the recovery of MSCs from the freshly harvested bone marrow allowing for less ex vivo expansion would represent a major advance in MSC therapy. METHODS: Human bone marrow cells from eight healthy donors were processed using a marrow filter device and, in parallel, using buoyant density centrifugation by two independent investigators. The initial nucleated cell recovery and the final yield, immunophenotype and trilineage differentiation potential of second-passage MSCs were examined. RESULTS: The marrow filter device generated significantly greater initial cell recovery requiring less investigator time and resulted in approximately 2.5-fold more MSCs after the second passage. The immunophenotype and differentiation potential of MSCs isolated using the two methods were equivalent and consistent with the defining criteria. The two independent investigators generated comparable results. CONCLUSIONS: This novel filter device is a fast, efficient and reliable system to isolate MSCs and should greatly expedite pre-clinical and clinical investigations of MSC therapy.
Asunto(s)
Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Madre Mesenquimatosas/citología , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Trasplante de Células Madre MesenquimatosasRESUMEN
Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination. The CliniMACS Prodigy, an automated cell processor, allows for manufacturing cell therapy products at a clinical scale in a closed system, minimizing the risk of contamination. Processing occurs semi-automatically under the control of a computer and thus minimizes human involvement in the process, which saves time and reduces variability and errors. This manuscript and video describes the T cell transduction (TCT) process for manufacturing CAR-T cells using this processor. The TCT process involves CD4+/CD8+ T cell enrichment, activation, transduction with a viral vector, expansion, and harvest. Using the Activity Matrix, a functionality that allows ordering and timing of these steps, the TCT process can be customized extensively. We provide a walk-through of CAR-T cell manufacturing in compliance with current Good Manufacturing Practice (cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstrate the feasibility and discuss the advantages and disadvantages of using a semi-automatic process for clinical CAR-T cell manufacturing. Finally, we describe an ongoing investigator-initiated clinical trial that targets pediatric B-cell malignancies [NCT05480449] as an example of how this manufacturing process can be applied in a clinical setting.
Asunto(s)
Receptores Quiméricos de Antígenos , Niño , Humanos , Receptores Quiméricos de Antígenos/genética , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Membrana Celular , Linfocitos BRESUMEN
Adequate recovery of hematopoietic stem cell (HSC) niches after cytotoxic conditioning regimens is essential to successful bone marrow transplantation. Yet, very little is known about the mechanisms that drive the restoration of these niches after bone marrow injury. Here we describe a profound disruption of the marrow microenvironment after lethal total body irradiation of mice that leads to the generation of osteoblasts restoring the HSC niche, followed by a transient, reversible expansion of this niche. Within 48 hours after irradiation, surviving host megakaryocytes were observed close to the endosteal surface of trabecular bone rather than in their normal parasinusoidal site concomitant with an increased stromal-derived factor-1 level. A subsequent increase in 2 megakaryocyte-derived growth factors, platelet-derived growth factor-beta and basic fibroblast growth factor, induces a 2-fold expansion of the population of N-cadherin-/osteopontin-positive osteoblasts, relative to the homeostatic osteoblast population, and hence, increases the number of potential niches for HSC engraftment. After donor cell engraftment, this expanded microenvironment reverts to its homeostatic state. Our results demonstrate the rapid recovery of osteoblastic stem cell niches after marrow radioablation, provide critical insights into the associated mechanisms, and suggest novel means to manipulate the bone marrow microenvironment to promote HSC engraftment.
Asunto(s)
Trasplante de Médula Ósea , Células Madre Hematopoyéticas/inmunología , Recuperación de la Función/inmunología , Acondicionamiento Pretrasplante , Irradiación Corporal Total , Animales , Cadherinas/inmunología , Quimiocina CXCL12/inmunología , Factor 2 de Crecimiento de Fibroblastos/inmunología , Megacariocitos/inmunología , Ratones , Osteoblastos , Osteopontina/inmunología , Proteínas Proto-Oncogénicas c-sis/inmunología , Recuperación de la Función/efectos de la radiación , Factores de TiempoRESUMEN
BACKGROUND AIMS: Chimerism is an important outcome measure in hematopoietic cell transplantation as well as somatic cell therapy. Commonly used methods to estimate chimerism are restricted by either gender or inefficient sensitivity. In principle, real-time polymerase chain reaction (PCR)-based assays can be used to assess single nucleotide polymorphisms (SNP), which are a vast resource of molecular markers, and such assays demonstrate a substantially higher sensitivity (0.001%), but the specificity is unclear because of a low-level signal from mismatched sequences. METHODS: In this study, we cloned 14 pairs of SNP selected from the SNP HapMap database and examined the specificity and sensitivity of their detection by real-time PCR using two primer/fluorescent probe pairs to allow genotyping of the two possible variant alleles. Clinical donor-recipient pairs from 18 families were used to explore the efficacy of using SNP assays to measure chimerism. RESULTS: We found that the polymorphic nucleotide influences the ability to distinguish the signal generated by the target and mismatched sequences. Moreover, the specific fluorescent reporter probe can affect the difference in signal intensity between the target and mismatched sequences. Real-time PCR SNP assays can attain a sensitivity of 0.1-0.5% with 100% specificity. When comparing possible clinical donor-recipient pairs, we found an average 3.3 out of 14 SNP were informative. CONCLUSIONS: By optimal selection of the polymorphic sequences and fluorescent reporter, the real-time PCR SNP assay is superior to the short-tandem repeat chimerism assay and broadly applicable. This strategy may be applied in future clinical trials of bone marrow cell therapy.
Asunto(s)
Quimerismo , Análisis Mutacional de ADN , Trasplante de Células Madre Hematopoyéticas , Adolescente , Adulto , Niño , Preescolar , Femenino , Genes Reporteros/genética , Histocompatibilidad/genética , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: Based on the recognition that marrow contains progenitors for bone as well as blood, we undertook the first trial of bone marrow transplantation (BMT) for a genetic disorder of bone, osteogenesis imperfecta. While we documented striking clinical benefit soon after transplantation, the measured level of osteopoietic engraftment was low. To improve the efficacy of BMT for bone disorders, we sought to gain insight into the cellular mechanism of engraftment of transplantable marrow osteoprogenitors. MATERIALS AND METHODS: We transplanted unfractionated bone marrow harvested from green fluorescent protein-transgenic FVB/N mice into lethally irradiated FVB/N recipients. At 3 weeks posttransplantation, we assessed hematopoietic engraftment by flow cytometry and osteopoietic engraftment by immunohistochemical staining for the green fluorescent protein. RESULTS: We show that engraftment of transplantable marrow osteoprogenitors is saturable with a maximal engraftment of about 15% of all bone cells in the epiphysis and metaphysis of the femur at 3 weeks after transplantation. The number of engrafting sites is not up- or downregulated in response to initial progenitor cell engraftment, and there is no evidence for clonal succession of osteopoietic differentiation of engrafted progenitors. CONCLUSIONS: Our findings indicate that the capacity for initial osteopoietic engraftment after BMT is limited and "megadose" stem cell transplantation is unlikely to enhance engraftment. Thus, novel strategies to foster osteopoietic chimerism must be developed.
Asunto(s)
Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Supervivencia de Injerto/inmunología , Osteoblastos/citología , Osteoblastos/trasplante , Animales , Células de la Médula Ósea/inmunología , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Inmunohistoquímica , Ratones , Ratones Transgénicos , Osteoblastos/inmunología , Sensibilidad y EspecificidadRESUMEN
T cells made with messenger RNA (mRNA) encoding chimeric antigen receptor (CAR) offer a safe alternative to those transduced with viral CARs by mitigating the side effects of constitutively active T cells. Previous studies have shown that mRNA CAR T cells are transiently effective but lack persistence and potency across tumor types. It was hypothesized that the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology, such as incorporating a modified nucleoside, 1-methylpseudouridine, into the mRNA and applying a novel purification method using RNase III to eliminate dsRNA contaminants. T cells electroporated with nucleoside-modified and purified mRNA encoding CD19 CAR showed an initial twofold increase in CAR surface expression, as well as a twofold improvement in cytotoxic killing of leukemia cells that persisted up to 5 days. T cells generated with nucleoside-modified and purified CAR mRNA also showed reduced expression of checkpoint regulators and a differential pattern of genetic activation compared to those made with conventional mRNA. In vivo studies using a leukemia mouse model revealed that the most robust 100-fold suppression of leukemic burden was achieved using T cells electroporated with purified mRNAs, regardless of their nucleoside modification. The results provide a novel approach to generate mRNA for clinical trials, and poise mRNA CAR T cells for increased efficacy during testing as new CAR targets emerge.
Asunto(s)
Traslado Adoptivo , Electroporación , Leucemia , ARN Mensajero , Receptores Quiméricos de Antígenos , Linfocitos T/inmunología , Animales , Antígenos CD19/genética , Antígenos CD19/inmunología , Línea Celular Tumoral , Humanos , Leucemia/genética , Leucemia/inmunología , Leucemia/patología , Leucemia/terapia , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , ARN Mensajero/genética , ARN Mensajero/inmunología , ARN Mensajero/aislamiento & purificación , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite years of extensive investigation, the cellular origin of heterotopic ossification (HO) has not been fully elucidated. We have previously shown that circulating bone marrow-derived osteoblast progenitor cells, characterized by the immunophenotype CD45-/CD44+/CXCR4+, contributed to the formation of heterotopic bone induced by bone morphogenetic protein (BMP)-2. In contrast, other reports have demonstrated the contribution of CD45+ hematopoietic derived cells to HO. Therefore, in this study, we developed a novel triple transgenic mouse strain that allows us to visualize CD45+ cells with red fluorescence and mature osteoblasts with green fluorescence. These mice were generated by crossing CD45-Cre mice with Z/RED mice that express DsRed, a variant of red fluorescent protein, after Cre-mediated recombination, and then crossing with Col2.3GFP mice that express green fluorescent protein (GFP) in mature osteoblasts. Utilizing this model, we were able to investigate if hematopoietic derived cells have the potential to give rise to mature osteoblasts. Analyses of this triple transgenic mouse model demonstrated that DsRed and GFP did not co-localize in either normal skeletogenesis, bone regeneration after fracture, or HO. This indicates that in these conditions hematopoietic derived cells do not differentiate into mature osteoblasts. Interestingly, we observed the presence of previously unidentified DsRed positive bone lining cells (red BLCs) which are derived from hematopoietic cells but lack CD45 expression. These red BLCs fail to produce GFP even under in vitro osteogenic conditions. These findings indicate that, even though both osteoblasts and hematopoietic cells are developmentally derived from mesoderm, hematopoietic derived cells do not contribute to osteogenesis in fracture healing or HO.