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1.
Clin Cancer Res ; 2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38864848

ABSTRACT

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 co-stimulatory domains (4-1BB and CD28). In vivo, the efficacy of local and systemic administration of GPC2 CAR T-cells were 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 co-stimulation (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 that led to significant tumor regression compared to 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.

2.
Res Sq ; 2024 Feb 16.
Article in English | MEDLINE | ID: mdl-38410461

ABSTRACT

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.

3.
J Vis Exp ; (198)2023 08 18.
Article in English | MEDLINE | ID: mdl-37607096

ABSTRACT

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.


Subject(s)
Receptors, Chimeric Antigen , Child , Humans , Receptors, Chimeric Antigen/genetics , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Cell Membrane , B-Lymphocytes
4.
Hum Gene Ther ; 30(2): 168-178, 2019 02.
Article in English | MEDLINE | ID: mdl-30024272

ABSTRACT

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.


Subject(s)
Adoptive Transfer , Electroporation , Leukemia , RNA, Messenger , Receptors, Chimeric Antigen , T-Lymphocytes/immunology , Animals , Antigens, CD19/genetics , Antigens, CD19/immunology , Cell Line, Tumor , Humans , Leukemia/genetics , Leukemia/immunology , Leukemia/pathology , Leukemia/therapy , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA, Messenger/isolation & purification , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/pathology , Xenograft Model Antitumor Assays
5.
Cytotherapy ; 20(1): 62-73, 2018 01.
Article in English | MEDLINE | ID: mdl-29107738

ABSTRACT

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.


Subject(s)
Bone Development , Extracellular Vesicles/metabolism , Mesenchymal Stem Cells/metabolism , Osteogenesis Imperfecta/pathology , Animals , Cell Proliferation , Child , Chondrocytes/cytology , Disease Models, Animal , Endopeptidase K/metabolism , Humans , Mice, Inbred C57BL , MicroRNAs/metabolism , Ribonucleases/metabolism , Solubility
6.
Cytotherapy ; 19(7): 867-880, 2017 07.
Article in English | MEDLINE | ID: mdl-28506444

ABSTRACT

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.


Subject(s)
Antigens, CD19/genetics , Interleukin-6/metabolism , Monocytes/metabolism , T-Lymphocytes/metabolism , Animals , Antigen-Presenting Cells/metabolism , Antigens, CD19/immunology , Antigens, CD19/metabolism , Cell Lineage , Coculture Techniques , Cytokines/metabolism , Humans , Lymphocyte Activation/immunology , Mice, Inbred NOD , Monocytes/immunology , Receptors, Antigen, T-Cell/immunology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Xenograft Model Antitumor Assays
7.
Bone ; 94: 1-9, 2017 01.
Article in English | MEDLINE | ID: mdl-27725318

ABSTRACT

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.


Subject(s)
Hematopoiesis , Osteoblasts/cytology , Osteoblasts/metabolism , Osteogenesis , Animals , Bone and Bones/cytology , Cell Differentiation/genetics , Cell Separation , Cells, Cultured , Crosses, Genetic , Female , Flow Cytometry , Fracture Healing , Gene Expression Regulation , Hematopoiesis/genetics , Integrases/metabolism , Leukocyte Common Antigens/metabolism , Male , Mice, Inbred C57BL , Mice, Transgenic , Ossification, Heterotopic/genetics , Ossification, Heterotopic/pathology , Osteogenesis/genetics
8.
J Clin Invest ; 126(10): 3814-3826, 2016 10 03.
Article in English | MEDLINE | ID: mdl-27571406

ABSTRACT

Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies.


Subject(s)
Antigens, CD19/metabolism , Antineoplastic Agents/administration & dosage , Interleukin-3 Receptor alpha Subunit/administration & dosage , Interleukin-3 Receptor alpha Subunit/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Receptors, Antigen, T-Cell/administration & dosage , Animals , Cell Line, Tumor , Drug Resistance, Neoplasm , Humans , Immunotherapy/methods , Mice, Inbred NOD , Mice, SCID , Neoplasm Recurrence, Local/prevention & control , Neoplastic Stem Cells/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/transplantation , Xenograft Model Antitumor Assays
9.
Cancer Discov ; 5(12): 1282-95, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26516065

ABSTRACT

UNLABELLED: The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE: CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.


Subject(s)
Alternative Splicing , Antigens, CD19/genetics , Immunotherapy , Mutation , Receptors, Antigen, T-Cell/genetics , Recombinant Fusion Proteins/genetics , Antigens, CD19/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Membrane/metabolism , Epitopes/immunology , Exons , Humans , Immunotherapy/methods , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Protein Binding , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins/metabolism , Recurrence , Sequence Analysis, DNA , Serine-Arginine Splicing Factors , Transcription, Genetic
10.
Cytotherapy ; 17(3): 262-70, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25659640

ABSTRACT

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.


Subject(s)
Cell Separation/methods , Cell- and Tissue-Based Therapy/methods , Cryopreservation/methods , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Adult , Animals , Bone Marrow , Bone Marrow Cells/cytology , Cell Differentiation , Cell Proliferation , Cell Separation/instrumentation , Cells, Cultured , Chondrocytes/cytology , Female , Gene Expression Profiling , Genomics , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Transplantation, Heterologous , Young Adult
11.
Stem Cells ; 32(12): 3266-77, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25220576

ABSTRACT

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.


Subject(s)
Cell Differentiation/physiology , Mesenchymal Stem Cells/cytology , Stem Cells/cytology , Tendons/cytology , Animals , Cells, Cultured , Chondrogenesis/physiology , Endoglin , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Osteogenesis/physiology , Tendons/metabolism
12.
Biol Blood Marrow Transplant ; 19(11): 1566-73, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23916672

ABSTRACT

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.


Subject(s)
Bone Marrow Transplantation/methods , Hematopoietic Stem Cell Transplantation/methods , Osteoblasts/cytology , Animals , Bone Marrow Cells/cytology , Mice , Osteogenesis , Transplantation, Autologous
13.
Stem Cells ; 31(10): 2193-204, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23818291

ABSTRACT

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.


Subject(s)
Bone Marrow Transplantation/methods , Hematopoietic Stem Cell Transplantation , Insulin-Like Growth Factor I/physiology , Stem Cell Niche/physiology , Animals , Bone and Bones/cytology , Cell Movement , Cell Proliferation , Graft Survival , Hematopoiesis , Mice , Mice, Inbred C57BL , Mice, Transgenic , Osteoblasts/physiology , Whole-Body Irradiation
14.
Blood ; 121(26): 5238-49, 2013 Jun 27.
Article in English | MEDLINE | ID: mdl-23667055

ABSTRACT

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.


Subject(s)
Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Megakaryocytes/cytology , Osteoblasts/cytology , Receptors, Thrombopoietin/physiology , Stem Cell Niche/physiology , Whole-Body Irradiation , Animals , Becaplermin , Cell Movement/physiology , Cell Movement/radiation effects , Cell Proliferation , Endothelium, Vascular , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Graft Survival , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/radiation effects , Megakaryocytes/metabolism , Megakaryocytes/radiation effects , Mice , Mice, Inbred C57BL , Osteoblasts/metabolism , Osteoblasts/radiation effects , Proto-Oncogene Proteins c-sis/metabolism , Signal Transduction , Thrombopoietin/metabolism
15.
Mol Ther ; 21(6): 1224-31, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23587920

ABSTRACT

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.


Subject(s)
Cell Differentiation , Hematopoietic Stem Cells/cytology , Osteoblasts/cytology , Stem Cell Niche , Animals , Bone Marrow Transplantation , Hematopoiesis , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Immunohistochemistry , Immunophenotyping , In Situ Hybridization, Fluorescence , Mice , Osteoblasts/metabolism , Phenotype , Pilot Projects , Proto-Oncogene Proteins c-kit/metabolism , Reverse Transcriptase Polymerase Chain Reaction
16.
Cytotherapy ; 15(2): 146-53, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23321326

ABSTRACT

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.


Subject(s)
Bone Marrow Cells/cytology , Cell Culture Techniques , Cell Differentiation , Mesenchymal Stem Cells/cytology , Cell Proliferation , Cell- and Tissue-Based Therapy , Humans , Mesenchymal Stem Cell Transplantation
17.
Blood ; 120(9): 1933-41, 2012 Aug 30.
Article in English | MEDLINE | ID: mdl-22829629

ABSTRACT

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.


Subject(s)
Bone Marrow Transplantation/methods , Leukocytes, Mononuclear/transplantation , Mesenchymal Stem Cell Transplantation/methods , Osteogenesis Imperfecta/surgery , Animals , Body Height/physiology , Body Weight/physiology , Bone Matrix/metabolism , Cells, Cultured , Child , Collagen/genetics , Collagen/metabolism , Female , Flow Cytometry , Gene Expression , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Lumbar Vertebrae/growth & development , Male , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mice, Transgenic , Osteoblasts/cytology , Osteoblasts/metabolism , Osteogenesis , Osteogenesis Imperfecta/genetics , Osteogenesis Imperfecta/physiopathology , Time Factors
18.
Blood ; 118(8): 2358-61, 2011 Aug 25.
Article in English | MEDLINE | ID: mdl-21715305

ABSTRACT

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.


Subject(s)
Bone Marrow Transplantation/pathology , Cytokines/pharmacology , Osteogenesis/drug effects , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Cell Differentiation/drug effects , Cell Differentiation/physiology , Granulocyte Colony-Stimulating Factor/pharmacology , Growth Hormone/pharmacology , Mice , Mice, Transgenic , Osteoblasts/cytology , Osteoblasts/drug effects , Osteocytes/cytology , Osteocytes/drug effects , Osteogenesis/physiology , Parathyroid Hormone/pharmacology , Recombinant Proteins , Stem Cell Factor/pharmacology , Transplantation Chimera
19.
Exp Hematol ; 38(9): 836-44, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20447443

ABSTRACT

OBJECTIVE: Transplantable osteoprogenitors, as well as hematopoietic progenitors, reside in bone marrow. We previously reported the first clinical trial of bone marrow transplantation (BMT) for a genetic disorder of bone, osteogenesis imperfecta. Although the patients demonstrated striking clinical benefits after transplantation, measured osteopoietic engraftment was low and did not seem to be durable. Therefore, we sought an animal model, which closely reflects the clinical experience, to facilitate development of strategies to improve the efficiency of osteoprogenitor engraftment after BMT. MATERIALS AND METHODS: We transplanted unfractionated bone marrow cells from green fluorescent protein-transgenic mice into lethally irradiated recipients in four combinations of inbred mouse strains: from C57BL/6 into C57BL/6 (C-C), from C57BL/6 into FVB/N (C-F), from FVB/N into C57BL/6 (F-C), and from FVB/N into FVB/N (F-F). At 2 weeks after transplantation, we assessed donor hematopoietic and osteopoietic engraftment by flow cytometry, using a novel mean fluorescence assay, and by immunohistochemical staining for green fluorescent protein. RESULTS: Hematopoietic reconstitution by donor cells was complete in all four combinations. Although osteopoietic engraftment of the transplanted cells was also documented in all the four groups, the magnitude of osteopoietic engraftment differed markedly among the strains where F-F > C-F > F-C > C-C. CONCLUSION: Our findings indicate that the genetic background of inbred mouse strains affects efficiency of osteopoietic engraftment after BMT. Thus, the murine strain must be considered when comparing experimental outcomes. Moreover, comparing the genetic variation among murine strains may lend insight into the factors governing osteopoietic differentiation of transplanted marrow cells.


Subject(s)
Bone Marrow Transplantation , Hematopoietic Stem Cells , Models, Biological , Osteogenesis , Animals , Humans , Mice , Mice, Transgenic , Species Specificity , Transplantation, Homologous
20.
Cytotherapy ; 12(8): 1035-43, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20429790

ABSTRACT

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.


Subject(s)
Chimerism , DNA Mutational Analysis , Hematopoietic Stem Cell Transplantation , Adolescent , Adult , Child , Child, Preschool , Female , Genes, Reporter/genetics , Histocompatibility/genetics , Humans , Infant , Infant, Newborn , Male , Middle Aged , Polymorphism, Single Nucleotide , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity
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