[Show abstract][Hide abstract] ABSTRACT: Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.
Science translational medicine 04/2014; 6(232):232fs16. · 10.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease compared with bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models, and now ex vivo treatment of CB with fucosyltransferase (FT) VI before transplantation is under clinical evaluation (NCT01471067). However, FTVII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. The present study compared the ability of FTVI and FTVII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPCs) in vivo.
CD34-selected CB HSPCs were treated with recombinant FTVI, FTVII or mock control and then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment.
Both FTVI and FTVII fucosylated CB CD34(+) cells in vitro, and both led to enhanced rates and magnitudes of engraftment compared with untreated CB CD34(+) cells in vivo. Engraftment after treatment with either FT was robust at multiple time points and in multiple tissues with similar multi-lineage potential. In contrast, only FTVII was able to fucosylate T and B lymphocytes.
Although FTVI and FTVII were found to be similarly able to fucosylate and enhance the engraftment of CB CD34(+) cells, differences in their ability to fucosylate lymphocytes may modulate graft-versus-tumor or graft-versus-host effects and may allow further optimization of CB transplantation.
[Show abstract][Hide abstract] ABSTRACT: This study assessed the potential of highly purified (Stro-1(+)) human mesenchymal precursor cells (hMPCs) in combination with the anti-scarring protein decorin to repair the injured spinal cord (SC). Donor hMPCs isolated from spinal cord injury (SCI) patients were transplanted into athymic rats as a suspension graft, alone or after previous treatment with, core (decorin(core)) and proteoglycan (decorin(pro)) isoforms of purified human recombinant decorin. Decorin was delivered via mini-osmotic pumps for 14 days following sub-acute (7 day) or chronic (1 month) SCI. hMPCs were delivered to the spinal cord at 3 weeks or 6 weeks after the initial injury at T9 level. Behavioral and anatomical analysis in this study showed statistically significant improvement in functional recovery, tissue sparing and cyst volume reduction following hMPCs therapy. The combination of decorin infusion followed by hMPCs therapy did not improve these measured outcomes over the use of cell therapy alone, in either sub-acute or chronic SCI regimes. However, decorin infusion did improve tissue sparing, reduce spinal tissue cavitation and increase transplanted cell survivability as compared to controls. Immunohistochemical analysis of spinal cord sections revealed differences in glial, neuronal and extracellular matrix molecule expression within each experimental group. hMPCs transplanted spinal cords showed the increased presence of serotonergic (5-HT) and sensory (CGRP) axonal growth within and surrounding transplanted hMPCs for up to 2 months; however, no evidence of hMPCs transdifferentiation into neuronal or glial phenotypes. The number of hMPCs was dramatically reduced overall, and no transplanted cells were detected at 8 weeks post-injection using lentiviral GFP labeling and human nuclear antigen antibody labeling. The presence of recombinant decorin in the cell transplantation regimes delayed in part the loss of donor cells, with small numbers remaining at 2 months after transplantation. In vitro co-culture experiments with embryonic dorsal root ganglion explants revealed the growth promoting properties of hMPCs. Decorin did not increase axonal outgrowth from that achieved by hMPCs. We provide evidence for the first time that (Stro-1(+)) hMPCs provide: i) an advantageous source of allografts for stem cell transplantation for sub-acute and chronic spinal cord therapy, and (ii) a positive host microenvironment that promotes tissue sparing/repair that subsequently improves behavioral outcomes after SCI. This was not measurably improved by recombinant decorin treatment, but does provide important information for the future development and potential use of decorin in contusive SCI therapy.
[Show abstract][Hide abstract] ABSTRACT: Liposarcomas are tumors arising in white adipose tissue (WAT) with avidity for local recurrence. Aggressive dedifferentiated liposarcomas (DDLS) may arise from well-differentiated subtypes (WDLS) upon disease progression, however, this key issue is unresolved due in large part to knowledge gaps about liposarcoma cellular composition. Here, we wished to improve insights into liposarcoma cellular hierarchy. Tumor section analysis indicated that the populations, distinguishable based on the expression of CD34 (a marker of adipocyte progenitors) and CD36 (a marker of adipocyte differentiation), occupy distinct intra-tumoral locations in both WDLS and DDLS. Taking advantage of these markers, we separated cells from a panel of fresh human surgical specimens by fluorescence-activated cell sorting (FACS). Based on chromosome analysis and the culture phenotypes of the composing populations, we demonstrate that malignant cells comprise four mesenchymal populations distinguished by the expression of CD34 and CD36, while vascular (CD31+) and hematopoietic (CD45+) components are non-neoplastic. Finally, we show that mouse xenografts are derivable from both CD36-negative and CD36-positive DDLS cells, and that each population recreates the heterogeneity of CD36 expression in vivo. Combined, our results show that malignant cells in WDLS and DDLS can be classified according to distinct stages of adipogenesis and indicate immunophenotypic plasticity of malignant liposarcoma cells.
Stem Cell Research 05/2013; 11(2):772-781. · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are the focus of intensive efforts worldwide directed not only at elucidating their nature and unique properties but also developing cell-based therapies for a diverse range of diseases. More than three decades have passed since the original formulation of the concept, revolutionary at the time, that multiple connective tissues could emanate from a common progenitor or stem cell retained in the postnatal bone marrow. Despite the many important advances made since that time, substantial ambiguities still plague the field regarding the nature, identity, function, mode of isolation and experimental handling of MSCs. These uncertainties have a major impact on their envisioned therapeutic use.
Nature medicine 01/2013; 19(1):35-42. · 27.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background Poor engraftment due to low cell doses restricts the usefulness of umbilical-cord-blood transplantation. We hypothesized that engraftment would be improved by transplanting cord blood that was expanded ex vivo with mesenchymal stromal cells. Methods We studied engraftment results in 31 adults with hematologic cancers who received transplants of 2 cord-blood units, 1 of which contained cord blood that was expanded ex vivo in cocultures with allogeneic mesenchymal stromal cells. The results in these patients were compared with those in 80 historical controls who received 2 units of unmanipulated cord blood. Results Coculture with mesenchymal stromal cells led to an expansion of total nucleated cells by a median factor of 12.2 and of CD34+ cells by a median factor of 30.1. With transplantation of 1 unit each of expanded and unmanipulated cord blood, patients received a median of 8.34×10(7) total nucleated cells per kilogram of body weight and 1.81×10(6) CD34+ cells per kilogram - doses higher than in our previous transplantations of 2 units of unmanipulated cord blood. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 15 days in the recipients of expanded cord blood, as compared with 24 days in controls who received unmanipulated cord blood only (P<0.001); the median time to platelet engraftment was 42 days and 49 days, respectively (P=0.03). On day 26, the cumulative incidence of neutrophil engraftment was 88% with expansion versus 53% without expansion (P<0.001); on day 60, the cumulative incidence of platelet engraftment was 71% and 31%, respectively (P<0.001). Conclusions Transplantation of cord-blood cells expanded with mesenchymal stromal cells appeared to be safe and effective. Expanded cord blood in combination with unmanipulated cord blood significantly improved engraftment, as compared with unmanipulated cord blood only. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00498316 .).
New England Journal of Medicine 12/2012; 367(24):2305-2315. · 54.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study aimed to determine the potential of purified (Stro-1⁺) human mesenchymal precursor cells (hMPCs) to repair the injured spinal cord (SC) after transplantation into Tcell deficient athymic RNU nude rats following acute moderate contusive spinal cord injury (SCI). hMPCs were isolated from the bone marrow (BM) stroma of SCI patients and transplanted as a suspension graft in medium (with or without immunosuppression using cyclosporin A (CsA)). Extensive anatomical analysis shows statistically significant improvement in functional recovery, tissue sparing and cyst reduction. We provide quantitative assessment of supraspinal projections in combination with functional outcomes. hMPC transplanted animals consistently achieved mean BBB scores of 15 at 8 weeks postinjury. Quantitative histological staining revealed that graft recipient animals possessed more intact spinal tissue and reduced cyst formation than controls. Fluorogold (FG) retrograde tracing revealed sparing/regeneration of supraspinal and local propriospinal axonal pathways, but no statistical differences were observed compared to controls. Immunohistochemical analysis revealed increased serotonergic (5-HT) and sensory (CGRP) axonal growth within and surrounding transplanted donor hMPCs 2 weeks posttransplantation, but no evidence of hMPC transdifferentiation was seen. Although hMPCs initially survive at 2 weeks post-transplantation, their numbers were dramatically reduced and no cells were detected at 8 weeks post-transplantation using retroviral/lentiviral GFP labeling and a human nuclear antigen (HNA) antibody. Additional immunosuppression with CsA did not improve hMPC survival, or their ability to promote tissue sparing or functional recovery. We propose Stro-1⁺-selected hMPCs provide (i) a reproducible source for stem cell transplantation for SC therapy, and (ii) a positive host microenvironment resulting in the promotion of tissue sparing/repair that subsequently improves behavioral outcomes after SCI. Our results provide a new candidate for consideration as a stem cell therapy for the repair of traumatic CNS injury.
[Show abstract][Hide abstract] ABSTRACT: Platelet-rich plasma (PRP) was prepared from human adult peripheral blood and from human umbilical cord (uc) blood and the properties were compared in a series of in vitro bioassays. Quantification of growth factors in PRP and platelet-poor plasma (PPP) fractions revealed increased levels of mitogenic growth factors PDGF-AB, PDGF-BB, and FGF-2, the angiogenic agent VEGF and the chemokine RANTES in ucPRP compared to adult PRP (aPRP) and PPP. To compare the ability of the various PRP products to stimulate proliferation of human bone marrow (BM), rat BM and compact bone (CB)-derived mesenchymal stem cells (MSC), cells were cultured in serum-free media for 4 and 7 days with varying concentrations of PRP, PPP, or combinations of recombinant mitogens. It was found that while all forms of PRP and PPP were more mitogenic than fetal bovine serum, ucPRP resulted in significantly higher proliferation by 7 days than adult PRP and PPP. We observed that addition of as little as 0.1% ucPRP caused greater proliferation of MSC effects than the most potent combination of recombinant growth factors tested, namely PDGF-AB + PDGF-BB + FGF-2, each at 10 ng/mL. Similarly, in chemotaxis assays, ucPRP showed greater potency than adult PRP, PPP from either source, or indeed than combinations of either recombinant growth factors (PDGF, FGF, and TGF-β1) or chemokines previously shown to stimulate chemotactic migration of MSC. Lastly, we successfully demonstrated that PRP and PPP represented a viable alternative to FBS containing media for the cryo-preservation of MSC from human and rat BM.
[Show abstract][Hide abstract] ABSTRACT: Delayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγ(null) (NSG) mice. By isolating fucosylated and nonfucosylated CD34(+) cells from CB, we showed that only fucosylated CD34(+) cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34(+) cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34(+) cells, we hypothesized that increasing the proportion of CD34(+) cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34(+) fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting.
[Show abstract][Hide abstract] ABSTRACT: In this study, poly(dl-lactide-co-glycolide)/porous silicon (PLGA/pSi) composite microspheres, synthesized by a solid-in-oil-in-water (S/O/W) emulsion method, are developed for the long-term controlled delivery of biomolecules for orthopedic tissue engineering applications. Confocal and fluorescent microscopy, together with material analysis, show that each composite microsphere contained multiple pSi particles embedded within the PLGA matrix. The release profiles of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (FITC-BSA), loaded inside the pSi within the PLGA matrix, indicate that both PLGA and pSi contribute to the control of the release rate of the payload. Protein stability studies show that PLGA/pSi composite can protect BSA from degradation during the long term release. We find that during the degradation of the composite material, the presence of the pSi particles neutralizes the acidic pH due to the PLGA degradation by-products, thus minimizing the risk of inducing inflammatory responses in the exposed cells while stimulating the mineralization in osteogenic growth media. Confocal studies show that the cellular uptake of the composite microspheres is avoided, while the fluorescent payload is detectable intracellularly after 7 days of co-incubation. In conclusion, the PLGA/pSi composite microspheres offer an additional level of controlled release and could be ideal candidates as drug delivery vehicles for orthopedic tissue engineering applications.
[Show abstract][Hide abstract] ABSTRACT: The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of mouse bone marrow stromal cells (BMSCs), a common problem being contamination with hematopoietic cells. Taking advantage of burgeoning evidence demonstrating the perivascular location of stromal cell stem/progenitors, we hypothesized that a potential reason for the low yield of mouse BMSCs is the flushing of the marrow used to remove single-cell suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of BMSCs physically associated with the abluminal surface of blood vessels. Herein, we describe a simple methodology based on preparation and enzymatic disaggregation of intact marrow plugs, which yields distinct populations of both stromal and endothelial cells. The recovery of CFU-F obtained by pooling the product of each digestion (1631.8 + 199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32 + 1.9) by at least 2 orders of magnitude (P < .001; N = 8) with an accompanying 113.95-fold enrichment of CFU-F frequency when plated at low oxygen (5%). Purified BMSC populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and from freshly prepared single-cell suspensions. Furthermore, this population demonstrates robust multilineage differentiation using standard in vivo and in vitro bioassays.
[Show abstract][Hide abstract] ABSTRACT: Directed specification and prospective isolation of chondrogenic paraxial mesoderm progeny from human pluripotent stem (PS) cells have not yet been achieved. Here we report the successful generation of KDR(-)PDGFRα(+) progeny expressing paraxial mesoderm genes and the mesendoderm reporter MIXL1-GFP in a chemically defined medium containing the canonical WNT signaling activator, BMP-inhibitor, and the Nodal/Activin/TGFβ signaling controller. Isolated (GFP(+))KDR(-)PDGFRα(+) mesoderm cells were sensitive to sequential addition of the three chondrogenic factors PDGF, TGFβ and BMP. Under these conditions, the cells showed robust chondrogenic activity in micromass culture, and generated a hyaline-like translucent cartilage particle in serum-free medium. In contrast, both STRO1(+) mesenchymal stem/stromal cells from adult human marrow and mesenchymal cells spontaneously arising from hPS cells showed a relatively weaker chondrogenic response in vitro, and formed more of the fibrotic cartilage particles. Thus, hPS cell-derived KDR(-)PDGFRα(+ )paraxial mesoderm-like cells have potential in engineered cartilage formation and cartilage repair.
[Show abstract][Hide abstract] ABSTRACT: Adipose stromal cells (ASCs) serve as mesenchymal progenitors in white adipose tissue (WAT). Intercellular interactions involving ASCs have remained obscure. By merging phage display technology with fluorescence-activated cell sorting (FACS), we screened a combinatorial library for peptides that target mouse ASCs in vivo. We isolated peptide CSWKYWFGEC that specifically homes to ASCs, used it as bait to purify the corresponding ASC surface receptor, and identified it as a previously unreported cleavage product of decorin (DCN) lacking the glycanation site (termed ΔDCN). We demonstrate that ΔDCN is differentially expressed on ASC surface. In a screen for ΔDCN-binding proteins, we identified resistin, an adipokine for which the receptor has been unknown. Expression of ΔDCN in 3T3-L1 cells promoted proliferation and migration but suppressed lipid accumulation upon adipogenesis induction, which was resistin dependent. We conclude that ΔDCN serves as a functional receptor of resistin in adipocyte progenitors and may regulate WAT expansion.
[Show abstract][Hide abstract] ABSTRACT: Umbilical cord blood (CB) is becoming an important source of haematopoietic support for transplant patients lacking human leukocyte antigen matched donors. The ethnic diversity, relative ease of collection, ready availability as cryopreserved units from CB banks, reduced incidence and severity of graft versus host disease and tolerance of higher degrees of HLA disparity between donor and recipient, are positive attributes when compared to bone marrow or cytokine-mobilized peripheral blood. However, CB transplantation is associated with significantly delayed neutrophil and platelet engraftment and an elevated risk of graft failure. These hurdles are thought to be due, at least in part, to low total nucleated cell and CD34(+) cell doses transplanted. Here, current strategies directed at improving TNC and CD34(+) cell doses at transplant are discussed, with particular attention paid to the use of a mesenchymal stem cell (MSC)/CB mononuclear cell ex vivo co-culture expansion system.
Best practice & research. Clinical haematology 03/2011; 24(1):83-92. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Obesity complicates a number of diseases through mechanisms that are poorly defined. Mobilization and recruitment of progenitor cells to pathological sites is an important factor in disease progression. Here, we analyzed the influence of obesity on the systemic circulation of CD34(+) cell populations and correlated frequencies of cells displaying previously established cell marker signatures with the BMI. Comparative analysis of peripheral blood mononuclear cells (PBMC) from 12 nonobese (BMI <30 kg/m(2)) and 14 obese (BMI >30 kg/m(2)) disease-free donors by flow cytometry revealed that obesity is associated with a fivefold increased frequency of circulating progenitor cells (CPC), a population consisting of hematopoietic and endothelial precursors. Our data also indicate that obesity is associated with increased frequency of circulating mesenchymal stromal progenitor cells (MSC). In contrast, the frequencies of mature endothelial cells (EC) and CD34-bright leukocytes are unaffected by obesity. Combined, our results indicate that obesity promotes mobilization of progenitor cells, which may have clinical relevance.
[Show abstract][Hide abstract] ABSTRACT: A novel bioactive sponge was created with a composite of type I collagen sponges or porous poly(-caprolactone) (PCL) scaffolds, platelet-rich plasma (PRP), BMP2-loaded nanoporous silicon enclosure (NSE) microparticles, mineralizing peptide amphiphiles (PA), and mesenchymal stem cells (MSC). Primary MSC from cortical bone (CB) tissue proved to form more and larger colony units, as well as produce more mineral matrix under osteogenic differentiation, than MSC from bone marrow (BM). Coating OPEN ACCESS J. Funct. Biomater. 2011, 2 40 pre-treatments were optimized for maximum cell adhesion and mineralization, while a PRP-based gel carrier was created to efficiently deliver and retain MSC and microparticles within a porous scaffold while simultaneously promoting cell recruitment, proliferation, and angiogenesis. Components and composite sponges were evaluated for osteogenic differentiation in vitro. Osteogenic sponges were loaded with MSC, PRP, PA, and NSE and implanted subcutaneously in rats to evaluate the formation of bone tissue and angiogenesis in vivo. It was found that the combination of a collagen sponge with CB MSC, PRP, PA, and the BMP2-releasing NSE formed the most bone and was most vascularized by four weeks compared to analogous composites featuring BM MSC or PCL or lacking PRP, PA, and NSE. This study indicates that CB MSC should be considered as an alternative to marrow as a source of stem cells, while the PRP-PA cell and microparticle delivery system may be utilized for diverse tissue engineering applications.
[Show abstract][Hide abstract] ABSTRACT: Here, we have prospectively isolated and characterized, for the first time, clonogenic cells with self-renewal capacities from mantle cell lymphoma (MCL), a particularly deadly form of non-Hodgkin's lymphoma (NHL). Self-renewal and tumorigenic activities were enriched in MCL cell fractions that lacked expression of the prototypic B-cell surface marker, CD19. CD45+CD19- cells represented a relatively small fraction of the total MCL tumor cells; however, they recapitulated the heterogeneity of original patient tumors on transplantation into immunodeficient mice. As few as 100 of these cells displayed self-renewal capacities in secondary and tertiary recipient mice by in vivo limiting dilution assays. Similar to leukemic stem cells, CD45+CD19- MCL cells also displayed a quiescent status as determined by dye efflux assays. In summary, this study is the first to isolate subpopulations of MCL cells that have self-renewal and tumorigenic capacities. Identification and characterization of MCL-ICs are important first steps toward understanding how self-renewal and tumorigenicity are regulated in MCL and designing targeted therapies against MCL-ICs will ultimately lead to improved outcomes for MCL patients.
Stem Cell Research 11/2010; 5(3):212-25. · 4.47 Impact Factor