Allogeneic hematopoietic stem cell (HSC) transplantation is an effective therapy for a number of ... more Allogeneic hematopoietic stem cell (HSC) transplantation is an effective therapy for a number of diseases. However, graft-versus-host disease (GVHD) remains a significant obstacle to the successful outcome of this procedure. We have demonstrated in Phase I clinical trials that co-transplantation of culture expanded human MSCs during allogeneic HSC transplantation can facilitate engraftment without increasing the risk of GVHD. MSC have been shown to have immunomodulatory activity, and decrease T-cell interferon (IFN)-γ production. More recently, clinical studies have suggested that MSC infusion can also reduce the severity of GVHD. Based on these data, we have initiated a Phase I clinical trial (CWRU 3Y03) using allogeneic same-sibling donor MSC infusion of 1–6 × 106 culture expanded MSCs per kg for the treatment of acute or chronic GVHD of clinical grade II – IV after sibling donor HSC transplant. One of the main hurdles to overcome in MSC infusion protocols is the expansion of single donor MSCs to achieve the prescribed cell dose in a specified time frame. Here we report that the addition of recombinant human FGF-2 to the culture medium expedites and enhances MSC expansion capacity in normal adult donors cultured under clinically relevant conditions. In pre-clinical studies, MSCs from 13 normal donors (median age 28.5; range 21 – 40) were cultured in standard growth medium with or without FGF-2. The cells were expanded for up to 8 passages. MSCs expanded in the presence of FGF-2 exhibited shorter population doubling times and achieved a greater number of population doublings (31 ± 3) than those expanded in standard conditions (23 ± 2). These FGF-stimulated MSCs exhibited the same phenotype and immunomodulatory potential as MSCs grown in conventional medium. For each patient enrolled on CWRU 3Y03, donor MSCs were harvested, culture expanded and cryopreserved until indicated. Twenty-three culture expansions were attempted from 21 different donors (18 without FGF; 3 with FGF; median donor age 52, range 38 – 67). From the cultures grown in the absence of FGF, 11 donors were expanded to an infusion dose of 0.5 – 2.4 × 106 cells/kg (based on patient weight) with a mean of 161 ± 54 × 106 MSCs at harvest and a median cell expansion time of 41 days (range 23 – 66). Nine cultures failed to reach a minimum cell dose of 0.5 × 106 cells/kg during an 8-week culture period. The three MSC cultures grown in the presence of FGF -2 (R&D systems) were successful and reached infusion doses of 1.65 – 2.4 × 106 cells/kg. In these 3 cultures, the mean number of MSCs was 135 × 106 and the median day to harvest was 28 (range 27 – 41). The immunomodulatory potential of these three MSC preparations was tested in vitro in an IFN-γ EliSpot assay in which they inhibited IFN-γ production by 87.6 ± 5.4%. Cells from one donor that failed to expand in media without FGF-2 reached 171 × 106 (2.8 × 106/kg) MSCs in 27 days when MSCs from a second marrow harvest from the same donor were cultured in medium containing FGF-2, suggesting that FGF-2 supplementation may rescue a culture that may be otherwise unable to expand. Thus, FGF-2 can facilitate MSC culture expansion for clinical use while retaining their immunomodulatory function.
Abstract 1907 Defining in vivo effects and biodistribution of human bone marrow-derived mesenchym... more Abstract 1907 Defining in vivo effects and biodistribution of human bone marrow-derived mesenchymal stem cell (hMSCs) following allogeneic bone marrow transplantation (alloBMT) could impact the clinical utility of MSC therapy for the prevention and treatment of graft-versus-host disease (GvHD). Using an established model of murine alloBMT, we defined hMSC effects on GvHD and graft-versus-leukemia (GvL) activity. We first studied whether hMSC could modulate in vitro murine T-cell (TC) alloreactivity in mixed leukocyte cultures (MLCs). Specifically, hMSCs added to MLCs significantly reduced TC proliferation in a concentration-dependent manner distinct from human fibroblasts. In contrast to MLC cultures alone, MLCs containing hMSCs had significant reduction in TNFα, IFNγ, and IL-10 levels and higher levels of PGE2 and TGFβ1. Modulation in the inflammatory milieu was associated with changes in TC phenotypes, including more naïve and less activated TC surface marker expression (CD62L+CD69−) and the induction of CD4+CD25+FoxP3+ T-regulatory cells. To determine whether hMSCs could modulate in vivo mTC alloreactivity, irradiated recipient B6D2F1 (H-2bxd) mice were transplanted with allogeneic C57BL/6 (H-2b) BM and purified splenic TCs (B6→B6D2F1) and then were tail-vein injected with hMSC infusions (1 million per injection) on days one and four post-transplant. Syngeneic transplant recipients (B6D2F1→B6D2F1) were used as controls. hMSC-treated alloBMT mice had significantly prolonged survival and improved clinical GvHD scores, reduced splenic TC expansion and TNFα and IFNγ-producing TCs, and lower circulating TNFα and IFNγ levels versus untreated alloBMT mice. Bioluminescence imaging showed redistribution of labeled hMSCs from the lungs to abdominal organs within 72 hours following infusion. Importantly, GvHD target tissues (small and large bowel and liver) harvested from hMSC-treated alloBMT mice had significantly lower GvHD pathology scores than untreated alloBMT mice. We next determined the effects of hMSCs on GvL activity using the murine mastocytoma cell line, P815 (H-2d). TCs co-cultured with hMSCs maintained potent in vitro cytotoxic T-lymphocyte (CTL) activity comparable to untreated control CTLs. After challenge with P815 tumor cells, hMSCs-treated alloBMT mice had less severe GvHD, eradication of tumor burden, and improved leukemia-free survival compared to alloBMT control mice. Lastly, indomethacin (IM) added to MLC-hMSC co-cultures significantly reversed attenuation in both murine TC alloreactivity and surface activation expression. In addition, IM administered to hMSC-treated alloBMT mice reversed hMSC-associated survival advantage, suggesting that PGE2 in part mediates hMSC immunomodulatory effects. Together, our results show that hMSC infusions effectively attenuate GvHD and maintain GvL potency in alloBMT mice and reveal potential biomarkers and mechanisms of action underlying hMSC effects. Disclosures: Solchaga: Bimemetic Therapeutics: Employment. Cooke:Amgen: Provides experimental drug and central pharmacy support for 2 trials for which I am Co-PI.
Human mesenchymal stem cells (MSCs) produce soluble factors that inhibit T-cell proliferation and... more Human mesenchymal stem cells (MSCs) produce soluble factors that inhibit T-cell proliferation and alloreactivity. We have previously shown that MSCs require activation by CD14+ monocytes in cell culture. Toll-like receptors (TLRs) critically modulate antigen-presenting cell (APC) activation, maturation and function. Therefore, we aimed to determine whether TLR agonists could enhance CD14-mediated MSC activation and subsequent MSC-mediated T-cell inhibition. TLR agonists and human IL-1β were used to stimulate CD14+ cells isolated from peripheral blood of normal donors. TLR agonists included formalin-fixed Staphylococcus aureus Cowan A strain (SAC, TLR-2), Pam3CysSerLys4 (Pam3Cys, TLR-2), Salmonella enteriditis lipopolysaccharide (LPS, TLR-4), and R848 (Resiquimod, TLR7/8) in complete RPMI media (heat-inactivated FBS, glutamine, and antibiotics). TLR-stimulated CD14+ cells and supernatants from TLR-stimulated CD14+ cells were then used to stimulate third- and fourth-passage human MSCs (CD45−CD105+CD90+CD80−CD73+HLA-I+) expanded from normal volunteer bone marrow aspirate specimens. After a 24-hour culture with stimulant cells or supernatants, stimuli were removed, MSCs were washed twice with sterile PBS, and then were cultured for an additional 24 h in FBS-free RPMI media. Supernatants from TLR-stimulated CD14+ cell cultures and from washed MSC cultures were used to measure cytokine and chemokine production and to inhibit T-cell alloreactivity using an established mixed lymphocyte reaction (MLR) IFN-γ ELISPOT. MLR was also performed in the presence of TLR agonists and media alone. TLR stimulation resulted in high-level soluble factor induction (IL-1β, IL-6, TNF-α, and RANTES) from CD14+ cells. For example, levels of inducible IL-6 measured in CD14+ cell culture supernatants following LPS, SAC and R848 stimulation were 5.2 (70.3 ± 26.2 ng/ml), 4.7 (64.1 ± 16.3 ng/ml), and 4.6 (63.4 ± 13.1 ng/ml)-fold higher than after IL-1β stimulation (13.7 ± 4.0 ng/ml) (Mean ± SEM, 4 independent experiments). Supernatants of TLR-stimulated CD14+ cells induced higher levels of soluble factors from MSCs than stimulation with CD14+ cells themselves, suggesting that soluble factors from CD14+ cells activate MSCs. Not all supernatants of TLR-stimulated CD14+ cells were similar in their capacity to activate MSC-mediated inhibition of T-cell alloreactivity. For example, supernatants of Pam3Cys-, R848- and SAC-stimulated CD14+ cells all induced high levels of TNF-α in washed MSC cultures; but only supernatant from Pam3Cys-stimulated CD14+ cells resulted in MSC-mediated inhibition of T-cell alloreactivity (63.7 ± 12.9 % inhibition, Mean ± SEM, 4 independent ELISPOTs). Ongoing studies are being performed to define these immunomodulatory factors. Together, these results suggest that ex vivo TLR stimulation enhances soluble factor production from CD14+ cells, which, in turn, increases MSC production of immunomodulatory factors that mediate inhibition of T-cell alloreactivity.
S265 ance of collagen in those cartilage tissue complex. Homologously, compared with none herbal ... more S265 ance of collagen in those cartilage tissue complex. Homologously, compared with none herbal intervention group, the expression of collagen II in cartilage-liked tissue in HBP-A intervention group was also improved in PCR assay with 0.80 [95%CI = 0.67,0.93] vs 0.49 [95%CI = 0.45,0.53] (P<0.05). However, the mRNA expression of aggrecan was failed to demonstrate difference with 0.59 [95%CI = 0.40,0.78] vs 0.50 [95%CI = 0.28,0.71] (P>0.05). Conclusions: In a summary,cartilage like tissue could be found after the injection of alginate hydrogel embedded with chondrocytes and HBP-A. The study documented that the potential pharmacological target of Chinese herbal extract HBP-A in the application of cartilage tissue engineering may be concerned with the inhibition of catabolic enzymes MMP-3 and ADAMTS-5, and increasing of collagen II expression,and further study should be explored.
The purpose of this study was to assess the joint cartilage's capacity for repair and the potenti... more The purpose of this study was to assess the joint cartilage's capacity for repair and the potential of various biological tissues as replacements for damaged cartilage. Methods: We operated 30.3 months old, lambs, creating a chondral lesion which was left untreated in group I and treated with a fresh chondral implant in group III, a frozen chondral implant in group IV, and a frozen periostal implant in group V; in group II the lesion extended as far as the subchondral bone. The lesions were performed in the loading area of the medial condyle of the knee. Follow-up time was 6 months, and the results were assessed histologically. Results: In the chondral lesions which remained untreated (group I), degeneration of the exposed layers occurred, and loss of both cartilage thickness and homogeneity of the matrix was noted. Where the lesion extended as far as the subchondral bone (group II), repair was found to have taken place with a fibrous tissue indistinguishable from cartilage. When cartilage was implanted (group III and IV), the integration of the implant depended on wether there was any contact between the implant and the surrounding tissue. Discussion: The integrity of the fresh implants was maintained better than that of the frozen ones, which were found to contain cells with a proliferative capacity. When periosteum was placed over the chondral lesion, we observed the formation of a very loose fibrous tissue in which the initial stages of differentiation could be appreciated in the deepest layers.
It has been proposed that high energy shockwaves could be used to creare microfractures in cortic... more It has been proposed that high energy shockwaves could be used to creare microfractures in cortical bone. This quality might be exploited clinically to perforen closed osteotomies and promote healing in nonunion (15). However; no study has previously documented the effect of shockwaves on cortical bone "in vivo". We report an investigation designed to demonstrate the effect of shockwaves on mature cortical and healing bone. An osteotomy was performed on the tibiae of 37 lambs; two weeks later the operation site was exposed to shockwaves. Three weeks later the lambs were killed and specimens of the bone examined histologically and radiographically. Shockwaves had no effect on the periosteal surface of mature cortical bone, but on the endosteal surface some new trabecular bone was seen. Healing of bone was delayed by the shockwave therapy. We conclude that there is currently little place for shockwave treatment in clinical orthopaedics. RÉSUMÉ Le but de cette étude est d'évaluer les effets directs des ondes de choc de haute énergie sur l'os cortical du mouton, afin de préciser leur utilisation éventuelle en chirurgie orthopédique. Les animaux ont été divisés en plusieurs groupes selon le type d'intervention pratiqué, fenétres ou perforations, et selon le nombre et l'intensité des ondes de choc appliquées. Dans aucun des cas il n'y a eu de fragmentation ou d'érosion osseuse. Les ondes de choc ont entraîné une réaction intramédullaire au niveau de la corticale opposée aux orifices bombardés, sous forme de tissu mésenchymateux, siège de nombreuses cellules et d'une augmentation de la vascularisation. Les ondes de choc peuvent également perturber la reconstruction normale de l'os. Dans le groupe de contrôle les travées osseuses passent d'un bord l'autre de la fenêtre, contrairement à ce qui se produit chez les animaux bombardés où les travées proviennent de la corticale opposée. Nous avons noté que les ondes de choc, aussi bien à haute qu'à basse énergie, provoquent d'importants hématomes musculaires et sous-cutanés.
The purpose of this study was to assess the joint cartilage's capacity for repair and the potenti... more The purpose of this study was to assess the joint cartilage's capacity for repair and the potential of various biological tissues as replacements for damaged cartilage. METHODS We operated 30.3 months old, lambs, creating a chondral lesion which was left untreated in group I and treated with a fresh chondral implant in group III, a frozen chondral implant in group IV, and a frozen periostal implant in group V ; in group II the lesion extended as far as the subchondral bone. The lesions were performed in the loading area of the medial condyle of the knee. Follow-up time was 6 months, and the results were assessed histologically. RESULTS In the chondral lesions which remained untreated (group I), degeneration of the exposed layers occurred, and loss of both cartilage thickness and homogeneity of the matrix was noted. Where the lesion extended as far as the subchondral bone (group II), repair was found to have taken place with a fibrous tissue indistinguishable from cartilage. When cartilage was implanted (groups III and IV), the integration of the implant depended on wether there was any contact between the implant and the surrounding tissue. DISCUSSION The integrity of the fresh implants was maintained better than that of the frozen ones, which were found to contain cells with a proliferative capacity. When periosteum was placed over the chondral lesion, we observed the formation of a very loose fibrous tissue in which the initial stages of differentiation could be appreciated in the deepest layers.
Correlative laboratory studies were developed in a phase I trial to evaluate the safety of intrac... more Correlative laboratory studies were developed in a phase I trial to evaluate the safety of intracoronary injection of escalating doses of bone marrow (BM) CD133+ cells in patients with chronic coronary ischemia. Concurrent with patient cellular therapy, CD133+ cells were phenotyped and tested functionally with endothelial cell colony formation and in vitro and in vivo transmigration. BM (194 ± 11 ml) was isolated from patients meeting study inclusion criteria. CD133+ cells (20 ± 13 x 106, 84 ± 7% purity and 76 ± 7% viability (7AAD)) were isolated using the CliniMACS device (Miltenyi). Contaminating cells following the CliniMACS selection were: < 5% of CD3, CD3neg/CD56, CD19 (immature/mature), CD14, and CD71 cells with 5% CD61, 8% CD13+ SSChigh. BM, PB (peripheral blood), cord blood (CB)-derived endothelial progenitor cells (EPC) were assessed by a culture assay (StemCell Technologies) scoring early outgrowth CFU-EC. SEACOAST patients yielded significantly less colonies compared t...
Mesenchymal Stem Cells (MSCs) are non-hematopoietic multipotent cells residing in the stroma of t... more Mesenchymal Stem Cells (MSCs) are non-hematopoietic multipotent cells residing in the stroma of the bone marrow that are capable of differentiating into both mesenchymal and non-mesenchymal lineages [1]. In fact, in addition to bone, cartilage, fat, and myoblasts, it has been demonstrated that MSCs are capable of differentiating into neurons and astrocytes in vitro and in vivo [2, 3]. Multipotent
Natural killer (NK) cells are innate immune cells that play a key role in tumor immune surveillan... more Natural killer (NK) cells are innate immune cells that play a key role in tumor immune surveillance. NK cells have self-tolerance to healthy cells and can kill allogeneic tumor cells- both hematological and solid malignancies. Recent advances to derive immune cells from human induced pluripotent stem cells (iPSCs) allow the production of NK cells that can be used for immunotherapy. Recent clinical trials suggest that high doses of NK cells (approx. 0.5-1 x 109 cells per dose) and multiple doses are both safe and likely necessary for clinical efficacy. Manufacturing large number of NK cells from a clonal master iPSC line provides a promising strategy to enable off-the-shelf, next generation allogenic cell therapies. To do this, we have developed a novel method to produce clinical scale iPSC-NK cells and improved the process to expand NK cells efficiently and consistently. Briefly, hematopoietic progenitor cells were induced using an improved spin embryoid body (EB) method and the hem...
ABSTRACT Culture-expanded bone marrow-derived mesenchymal progenitor cells (MPCs) can differentia... more ABSTRACT Culture-expanded bone marrow-derived mesenchymal progenitor cells (MPCs) can differentiate into chondrocytes and/or osteoblasts when combined with a porous calcium phosphate ceramic delivery vehicle and implanted subcutaneously in vivo 1 . We use this assay to test new materials as putative delivery vehicles in skeletal tissue engineering models. A new family of hyaluronan-based biomaterials has been developed. These polymers can be manufactured in many different formats and its chemistry can be tailored to specific requirements. HYAFF®-11p75HE is one of these materials, consisting of polymers of partially derivatized hyaluronic acid. HYAFF®-11p75HE sponges were tested as carriers for bone marrow-derived MFC preparations in the subcutaneous implantation model. Three weeks after implantation the HYAFF®-11p75HE sponge had dissolved and the tissue collected had the appearance of trabecular bone with undifferentiated mesenchyme filling the space between the trabeculae. Six weeks after implantation, the bone had matured and remodeled into an ossicle containing bone marrow-like tissue in its core. Hyaluronan is a multifunctional molecule that affects different cells or the same cells in a different manner depending on its molecular size and concentration. These complex, multifunctional effects can be used to develop hyaluronan-based scaffolds as delivery vehicles for tissue engineering of various skeletal tissues including bone.
Introduction: Human multipotent mesenchymal stem cell (MSC) therapies are being tested clinically... more Introduction: Human multipotent mesenchymal stem cell (MSC) therapies are being tested clinically for a variety of disorders, including Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, and cartilage defects. However, despite the remarkable clinical advancements in this eld, most applications still use traditional culture media containing fetal bovine serum. The ill-de ned and highly variable nature of traditional culture media remains a challenge, hampering both the basic and clinical human MSC research elds. To date, no reliable serum-free medium for human MSCs has been available. Methods: In this study, we developed and tested a serum-free growth medium on human bone marrow-derived MSCs through the investigation of multiple parameters including primary cell isolation, multipassage expansion, mesoderm di erentiation, cellular phenotype, and gene-expression analysis. Results: Similar to that achieved with traditional culture medium, human MSCs expanded in serum-free medium supplemented with recombinant human platelet-derived growth factor-BB (PDGF-BB), basic broblast growth factor (bFGF), and transforming growth factor (TGF)-β1 showed extensive propagation with retained phenotypic, di erentiation, and colony-forming unit potential. To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded under serum-free and serum-containing conditions were compared, revealing similar expression pro les. In addition, the described serum-free culture medium supported the isolation of human MSCs from primary human marrow aspirate with continual propagation. Conclusions: Although the described serum-free MSC culture medium is not free of xenogeneic components, this medium provides a substitute for serum-containing medium for research applications, setting the stage for future clinical applications.
Mesenchymal stem cells (MSCs) are non-hematopoietic, pluripotent cells that give rise to stromal ... more Mesenchymal stem cells (MSCs) are non-hematopoietic, pluripotent cells that give rise to stromal cells in the marrow. MSCs have been shown to be immunosuppressive and have become an attractive therapeutic option for the modulation of undesired immune responses. Currently, ex vivo expanded human (h)MSCs are being utilized in clinical trials both in the USA and in Europe to treat a variety of immune disorders. hMSCs need to be harvested, iso- lated and expanded in culture. This necessary expansion may also result in decrease or loss of the immunomodula- tory potential of hMSCs. Ideally, the intrinsic immunomodulatory activity (potency) of an hMSC preparation should be assessed prior to its administration. The goal of the experiments described here was to develop a simple potency assay for the immunomodulatory properties of hMSCs. The immunosuppressive activity of hMSCs conditioned media was tested in enzyme-linked immunosorbent spot assays (ELISpot) and the immunosuppressive activity ...
Introduction Spinal fusion is a procedure used to correct spinal deformities and treat fractured ... more Introduction Spinal fusion is a procedure used to correct spinal deformities and treat fractured vertebrae, spinal instabilities or chronic back pain. In interbody fusion, all or part of the intervertebral disc is removed and a supporting spacer inserted between the vertebral bodies for support and to facilitate bone growth between them. In most cases, bone growth is enhanced with graft materials placed within the spacer. Autologous bone graft (autograft) is one of the materials commonly used to facilitate fusion. Although autograft is considered the “gold standard” due to its osteoconductive and osteoinductive properties, it has limitations including availability, graft quality, and donor site morbidity. Synthetic bone graft substitutes are alternatives to autograft that eliminate donor-site morbidity and graft material variability. Bone graft substitutes have other advantages including unlimited supply, off-theshelf availability and easy sterilization and storage. Disadvantages ca...
The specialty of craniofacial surgery is broad and includes trauma, esthetics, reconstruction of ... more The specialty of craniofacial surgery is broad and includes trauma, esthetics, reconstruction of congenital deformities, and regeneration of tissues. Moreover, craniofacial surgery deals with a diverse range of tissues including both „soft‟ and „hard‟ tissues. Technological advances in materials and biological sciences and improved surgical techniques have remarkably improved clinical outcomes. The quest to raise the bar for patient care continues to inspire advances for predictable biological regeneration of „soft‟ and „hard‟ tissues. As a consequence of this quest for advancement, a wide spectrum of biologicals are becoming available to surgeons. Is the use of recombinant DNA engineered biologicals daring? Sensible? Logical? Timely? Safe? It is crucial for the practicing craniofacial surgeon to take a step back periodically and carefully review the biological factors that have the potential for dramatically altering the discipline of craniofacial surgery. With this emphasis, the c...
FT538 is an investigational, off-the-shelf, multiplexed engineered natural killer (NK) cell cance... more FT538 is an investigational, off-the-shelf, multiplexed engineered natural killer (NK) cell cancer immunotherapy that is derived from a clonal master induced pluripotent stem cell (iPSC) line engineered with three functional components to enhance innate immunity: a novel high-affinity, non-cleavable CD16 (hnCD16) Fc receptor; an IL-15/IL-15 receptor fusion (IL-15RF); and the elimination of CD38 expression. The use of a clonal master engineered iPSC line as a starting cell source enables routine mass production of FT538 and supports off-the-shelf product availability to reach many patients. The clonal master iPSC line for the manufacture of FT538 was made by reprogramming and engineering donor-consented human fibroblasts to induce pluripotency using a proprietary non-integrating system and to integrate a bicistronic cassette containing hnCD16 and IL-15RF into the CD38 locus, which resulted in complete disruption of the CD38 gene. The engineered iPSC population was then sorted to isol...
Tissue engineering requires large numbers of cells with enhanced differentiation properties. Thus... more Tissue engineering requires large numbers of cells with enhanced differentiation properties. Thus, the effect of expansion conditions must be explored. Human and rat marrow-derived mesenchymal stem cells (hMSCs and rMSCs, respectively) were comparatively culture expanded through seven passages in the presence of either fibroblast growth factor-2 (FGF-2) or platelet-derived growth factor BB (PDGF-BB). Proliferation of both hMSCs and rMSCs was enhanced by FGF-2 and PDGF-BB. Population doubling times for hMSCs were 2.4 days for control and 1.75 and 2.0 days for FGF-2 and PDGF-BB, respectively, and 3.25, 3.06, and 2.95 days for rMSCs. Supplementation with FGF-2 during cell expansion resulted in significantly greater in vivo bone formation for hMSCs. Use of PDGF-BB resulted in greater bone formation than that observed for control conditions, but the differences were only significant for P1. For rMSCs, significant increases in bone formation were noted in either FGF-2 or PDGF-BB expanded ...
Allogeneic hematopoietic stem cell (HSC) transplantation is an effective therapy for a number of ... more Allogeneic hematopoietic stem cell (HSC) transplantation is an effective therapy for a number of diseases. However, graft-versus-host disease (GVHD) remains a significant obstacle to the successful outcome of this procedure. We have demonstrated in Phase I clinical trials that co-transplantation of culture expanded human MSCs during allogeneic HSC transplantation can facilitate engraftment without increasing the risk of GVHD. MSC have been shown to have immunomodulatory activity, and decrease T-cell interferon (IFN)-γ production. More recently, clinical studies have suggested that MSC infusion can also reduce the severity of GVHD. Based on these data, we have initiated a Phase I clinical trial (CWRU 3Y03) using allogeneic same-sibling donor MSC infusion of 1–6 × 106 culture expanded MSCs per kg for the treatment of acute or chronic GVHD of clinical grade II – IV after sibling donor HSC transplant. One of the main hurdles to overcome in MSC infusion protocols is the expansion of single donor MSCs to achieve the prescribed cell dose in a specified time frame. Here we report that the addition of recombinant human FGF-2 to the culture medium expedites and enhances MSC expansion capacity in normal adult donors cultured under clinically relevant conditions. In pre-clinical studies, MSCs from 13 normal donors (median age 28.5; range 21 – 40) were cultured in standard growth medium with or without FGF-2. The cells were expanded for up to 8 passages. MSCs expanded in the presence of FGF-2 exhibited shorter population doubling times and achieved a greater number of population doublings (31 ± 3) than those expanded in standard conditions (23 ± 2). These FGF-stimulated MSCs exhibited the same phenotype and immunomodulatory potential as MSCs grown in conventional medium. For each patient enrolled on CWRU 3Y03, donor MSCs were harvested, culture expanded and cryopreserved until indicated. Twenty-three culture expansions were attempted from 21 different donors (18 without FGF; 3 with FGF; median donor age 52, range 38 – 67). From the cultures grown in the absence of FGF, 11 donors were expanded to an infusion dose of 0.5 – 2.4 × 106 cells/kg (based on patient weight) with a mean of 161 ± 54 × 106 MSCs at harvest and a median cell expansion time of 41 days (range 23 – 66). Nine cultures failed to reach a minimum cell dose of 0.5 × 106 cells/kg during an 8-week culture period. The three MSC cultures grown in the presence of FGF -2 (R&amp;D systems) were successful and reached infusion doses of 1.65 – 2.4 × 106 cells/kg. In these 3 cultures, the mean number of MSCs was 135 × 106 and the median day to harvest was 28 (range 27 – 41). The immunomodulatory potential of these three MSC preparations was tested in vitro in an IFN-γ EliSpot assay in which they inhibited IFN-γ production by 87.6 ± 5.4%. Cells from one donor that failed to expand in media without FGF-2 reached 171 × 106 (2.8 × 106/kg) MSCs in 27 days when MSCs from a second marrow harvest from the same donor were cultured in medium containing FGF-2, suggesting that FGF-2 supplementation may rescue a culture that may be otherwise unable to expand. Thus, FGF-2 can facilitate MSC culture expansion for clinical use while retaining their immunomodulatory function.
Abstract 1907 Defining in vivo effects and biodistribution of human bone marrow-derived mesenchym... more Abstract 1907 Defining in vivo effects and biodistribution of human bone marrow-derived mesenchymal stem cell (hMSCs) following allogeneic bone marrow transplantation (alloBMT) could impact the clinical utility of MSC therapy for the prevention and treatment of graft-versus-host disease (GvHD). Using an established model of murine alloBMT, we defined hMSC effects on GvHD and graft-versus-leukemia (GvL) activity. We first studied whether hMSC could modulate in vitro murine T-cell (TC) alloreactivity in mixed leukocyte cultures (MLCs). Specifically, hMSCs added to MLCs significantly reduced TC proliferation in a concentration-dependent manner distinct from human fibroblasts. In contrast to MLC cultures alone, MLCs containing hMSCs had significant reduction in TNFα, IFNγ, and IL-10 levels and higher levels of PGE2 and TGFβ1. Modulation in the inflammatory milieu was associated with changes in TC phenotypes, including more naïve and less activated TC surface marker expression (CD62L+CD69−) and the induction of CD4+CD25+FoxP3+ T-regulatory cells. To determine whether hMSCs could modulate in vivo mTC alloreactivity, irradiated recipient B6D2F1 (H-2bxd) mice were transplanted with allogeneic C57BL/6 (H-2b) BM and purified splenic TCs (B6→B6D2F1) and then were tail-vein injected with hMSC infusions (1 million per injection) on days one and four post-transplant. Syngeneic transplant recipients (B6D2F1→B6D2F1) were used as controls. hMSC-treated alloBMT mice had significantly prolonged survival and improved clinical GvHD scores, reduced splenic TC expansion and TNFα and IFNγ-producing TCs, and lower circulating TNFα and IFNγ levels versus untreated alloBMT mice. Bioluminescence imaging showed redistribution of labeled hMSCs from the lungs to abdominal organs within 72 hours following infusion. Importantly, GvHD target tissues (small and large bowel and liver) harvested from hMSC-treated alloBMT mice had significantly lower GvHD pathology scores than untreated alloBMT mice. We next determined the effects of hMSCs on GvL activity using the murine mastocytoma cell line, P815 (H-2d). TCs co-cultured with hMSCs maintained potent in vitro cytotoxic T-lymphocyte (CTL) activity comparable to untreated control CTLs. After challenge with P815 tumor cells, hMSCs-treated alloBMT mice had less severe GvHD, eradication of tumor burden, and improved leukemia-free survival compared to alloBMT control mice. Lastly, indomethacin (IM) added to MLC-hMSC co-cultures significantly reversed attenuation in both murine TC alloreactivity and surface activation expression. In addition, IM administered to hMSC-treated alloBMT mice reversed hMSC-associated survival advantage, suggesting that PGE2 in part mediates hMSC immunomodulatory effects. Together, our results show that hMSC infusions effectively attenuate GvHD and maintain GvL potency in alloBMT mice and reveal potential biomarkers and mechanisms of action underlying hMSC effects. Disclosures: Solchaga: Bimemetic Therapeutics: Employment. Cooke:Amgen: Provides experimental drug and central pharmacy support for 2 trials for which I am Co-PI.
Human mesenchymal stem cells (MSCs) produce soluble factors that inhibit T-cell proliferation and... more Human mesenchymal stem cells (MSCs) produce soluble factors that inhibit T-cell proliferation and alloreactivity. We have previously shown that MSCs require activation by CD14+ monocytes in cell culture. Toll-like receptors (TLRs) critically modulate antigen-presenting cell (APC) activation, maturation and function. Therefore, we aimed to determine whether TLR agonists could enhance CD14-mediated MSC activation and subsequent MSC-mediated T-cell inhibition. TLR agonists and human IL-1β were used to stimulate CD14+ cells isolated from peripheral blood of normal donors. TLR agonists included formalin-fixed Staphylococcus aureus Cowan A strain (SAC, TLR-2), Pam3CysSerLys4 (Pam3Cys, TLR-2), Salmonella enteriditis lipopolysaccharide (LPS, TLR-4), and R848 (Resiquimod, TLR7/8) in complete RPMI media (heat-inactivated FBS, glutamine, and antibiotics). TLR-stimulated CD14+ cells and supernatants from TLR-stimulated CD14+ cells were then used to stimulate third- and fourth-passage human MSCs (CD45−CD105+CD90+CD80−CD73+HLA-I+) expanded from normal volunteer bone marrow aspirate specimens. After a 24-hour culture with stimulant cells or supernatants, stimuli were removed, MSCs were washed twice with sterile PBS, and then were cultured for an additional 24 h in FBS-free RPMI media. Supernatants from TLR-stimulated CD14+ cell cultures and from washed MSC cultures were used to measure cytokine and chemokine production and to inhibit T-cell alloreactivity using an established mixed lymphocyte reaction (MLR) IFN-γ ELISPOT. MLR was also performed in the presence of TLR agonists and media alone. TLR stimulation resulted in high-level soluble factor induction (IL-1β, IL-6, TNF-α, and RANTES) from CD14+ cells. For example, levels of inducible IL-6 measured in CD14+ cell culture supernatants following LPS, SAC and R848 stimulation were 5.2 (70.3 ± 26.2 ng/ml), 4.7 (64.1 ± 16.3 ng/ml), and 4.6 (63.4 ± 13.1 ng/ml)-fold higher than after IL-1β stimulation (13.7 ± 4.0 ng/ml) (Mean ± SEM, 4 independent experiments). Supernatants of TLR-stimulated CD14+ cells induced higher levels of soluble factors from MSCs than stimulation with CD14+ cells themselves, suggesting that soluble factors from CD14+ cells activate MSCs. Not all supernatants of TLR-stimulated CD14+ cells were similar in their capacity to activate MSC-mediated inhibition of T-cell alloreactivity. For example, supernatants of Pam3Cys-, R848- and SAC-stimulated CD14+ cells all induced high levels of TNF-α in washed MSC cultures; but only supernatant from Pam3Cys-stimulated CD14+ cells resulted in MSC-mediated inhibition of T-cell alloreactivity (63.7 ± 12.9 % inhibition, Mean ± SEM, 4 independent ELISPOTs). Ongoing studies are being performed to define these immunomodulatory factors. Together, these results suggest that ex vivo TLR stimulation enhances soluble factor production from CD14+ cells, which, in turn, increases MSC production of immunomodulatory factors that mediate inhibition of T-cell alloreactivity.
S265 ance of collagen in those cartilage tissue complex. Homologously, compared with none herbal ... more S265 ance of collagen in those cartilage tissue complex. Homologously, compared with none herbal intervention group, the expression of collagen II in cartilage-liked tissue in HBP-A intervention group was also improved in PCR assay with 0.80 [95%CI = 0.67,0.93] vs 0.49 [95%CI = 0.45,0.53] (P<0.05). However, the mRNA expression of aggrecan was failed to demonstrate difference with 0.59 [95%CI = 0.40,0.78] vs 0.50 [95%CI = 0.28,0.71] (P>0.05). Conclusions: In a summary,cartilage like tissue could be found after the injection of alginate hydrogel embedded with chondrocytes and HBP-A. The study documented that the potential pharmacological target of Chinese herbal extract HBP-A in the application of cartilage tissue engineering may be concerned with the inhibition of catabolic enzymes MMP-3 and ADAMTS-5, and increasing of collagen II expression,and further study should be explored.
The purpose of this study was to assess the joint cartilage's capacity for repair and the potenti... more The purpose of this study was to assess the joint cartilage's capacity for repair and the potential of various biological tissues as replacements for damaged cartilage. Methods: We operated 30.3 months old, lambs, creating a chondral lesion which was left untreated in group I and treated with a fresh chondral implant in group III, a frozen chondral implant in group IV, and a frozen periostal implant in group V; in group II the lesion extended as far as the subchondral bone. The lesions were performed in the loading area of the medial condyle of the knee. Follow-up time was 6 months, and the results were assessed histologically. Results: In the chondral lesions which remained untreated (group I), degeneration of the exposed layers occurred, and loss of both cartilage thickness and homogeneity of the matrix was noted. Where the lesion extended as far as the subchondral bone (group II), repair was found to have taken place with a fibrous tissue indistinguishable from cartilage. When cartilage was implanted (group III and IV), the integration of the implant depended on wether there was any contact between the implant and the surrounding tissue. Discussion: The integrity of the fresh implants was maintained better than that of the frozen ones, which were found to contain cells with a proliferative capacity. When periosteum was placed over the chondral lesion, we observed the formation of a very loose fibrous tissue in which the initial stages of differentiation could be appreciated in the deepest layers.
It has been proposed that high energy shockwaves could be used to creare microfractures in cortic... more It has been proposed that high energy shockwaves could be used to creare microfractures in cortical bone. This quality might be exploited clinically to perforen closed osteotomies and promote healing in nonunion (15). However; no study has previously documented the effect of shockwaves on cortical bone "in vivo". We report an investigation designed to demonstrate the effect of shockwaves on mature cortical and healing bone. An osteotomy was performed on the tibiae of 37 lambs; two weeks later the operation site was exposed to shockwaves. Three weeks later the lambs were killed and specimens of the bone examined histologically and radiographically. Shockwaves had no effect on the periosteal surface of mature cortical bone, but on the endosteal surface some new trabecular bone was seen. Healing of bone was delayed by the shockwave therapy. We conclude that there is currently little place for shockwave treatment in clinical orthopaedics. RÉSUMÉ Le but de cette étude est d'évaluer les effets directs des ondes de choc de haute énergie sur l'os cortical du mouton, afin de préciser leur utilisation éventuelle en chirurgie orthopédique. Les animaux ont été divisés en plusieurs groupes selon le type d'intervention pratiqué, fenétres ou perforations, et selon le nombre et l'intensité des ondes de choc appliquées. Dans aucun des cas il n'y a eu de fragmentation ou d'érosion osseuse. Les ondes de choc ont entraîné une réaction intramédullaire au niveau de la corticale opposée aux orifices bombardés, sous forme de tissu mésenchymateux, siège de nombreuses cellules et d'une augmentation de la vascularisation. Les ondes de choc peuvent également perturber la reconstruction normale de l'os. Dans le groupe de contrôle les travées osseuses passent d'un bord l'autre de la fenêtre, contrairement à ce qui se produit chez les animaux bombardés où les travées proviennent de la corticale opposée. Nous avons noté que les ondes de choc, aussi bien à haute qu'à basse énergie, provoquent d'importants hématomes musculaires et sous-cutanés.
The purpose of this study was to assess the joint cartilage's capacity for repair and the potenti... more The purpose of this study was to assess the joint cartilage's capacity for repair and the potential of various biological tissues as replacements for damaged cartilage. METHODS We operated 30.3 months old, lambs, creating a chondral lesion which was left untreated in group I and treated with a fresh chondral implant in group III, a frozen chondral implant in group IV, and a frozen periostal implant in group V ; in group II the lesion extended as far as the subchondral bone. The lesions were performed in the loading area of the medial condyle of the knee. Follow-up time was 6 months, and the results were assessed histologically. RESULTS In the chondral lesions which remained untreated (group I), degeneration of the exposed layers occurred, and loss of both cartilage thickness and homogeneity of the matrix was noted. Where the lesion extended as far as the subchondral bone (group II), repair was found to have taken place with a fibrous tissue indistinguishable from cartilage. When cartilage was implanted (groups III and IV), the integration of the implant depended on wether there was any contact between the implant and the surrounding tissue. DISCUSSION The integrity of the fresh implants was maintained better than that of the frozen ones, which were found to contain cells with a proliferative capacity. When periosteum was placed over the chondral lesion, we observed the formation of a very loose fibrous tissue in which the initial stages of differentiation could be appreciated in the deepest layers.
Correlative laboratory studies were developed in a phase I trial to evaluate the safety of intrac... more Correlative laboratory studies were developed in a phase I trial to evaluate the safety of intracoronary injection of escalating doses of bone marrow (BM) CD133+ cells in patients with chronic coronary ischemia. Concurrent with patient cellular therapy, CD133+ cells were phenotyped and tested functionally with endothelial cell colony formation and in vitro and in vivo transmigration. BM (194 ± 11 ml) was isolated from patients meeting study inclusion criteria. CD133+ cells (20 ± 13 x 106, 84 ± 7% purity and 76 ± 7% viability (7AAD)) were isolated using the CliniMACS device (Miltenyi). Contaminating cells following the CliniMACS selection were: < 5% of CD3, CD3neg/CD56, CD19 (immature/mature), CD14, and CD71 cells with 5% CD61, 8% CD13+ SSChigh. BM, PB (peripheral blood), cord blood (CB)-derived endothelial progenitor cells (EPC) were assessed by a culture assay (StemCell Technologies) scoring early outgrowth CFU-EC. SEACOAST patients yielded significantly less colonies compared t...
Mesenchymal Stem Cells (MSCs) are non-hematopoietic multipotent cells residing in the stroma of t... more Mesenchymal Stem Cells (MSCs) are non-hematopoietic multipotent cells residing in the stroma of the bone marrow that are capable of differentiating into both mesenchymal and non-mesenchymal lineages [1]. In fact, in addition to bone, cartilage, fat, and myoblasts, it has been demonstrated that MSCs are capable of differentiating into neurons and astrocytes in vitro and in vivo [2, 3]. Multipotent
Natural killer (NK) cells are innate immune cells that play a key role in tumor immune surveillan... more Natural killer (NK) cells are innate immune cells that play a key role in tumor immune surveillance. NK cells have self-tolerance to healthy cells and can kill allogeneic tumor cells- both hematological and solid malignancies. Recent advances to derive immune cells from human induced pluripotent stem cells (iPSCs) allow the production of NK cells that can be used for immunotherapy. Recent clinical trials suggest that high doses of NK cells (approx. 0.5-1 x 109 cells per dose) and multiple doses are both safe and likely necessary for clinical efficacy. Manufacturing large number of NK cells from a clonal master iPSC line provides a promising strategy to enable off-the-shelf, next generation allogenic cell therapies. To do this, we have developed a novel method to produce clinical scale iPSC-NK cells and improved the process to expand NK cells efficiently and consistently. Briefly, hematopoietic progenitor cells were induced using an improved spin embryoid body (EB) method and the hem...
ABSTRACT Culture-expanded bone marrow-derived mesenchymal progenitor cells (MPCs) can differentia... more ABSTRACT Culture-expanded bone marrow-derived mesenchymal progenitor cells (MPCs) can differentiate into chondrocytes and/or osteoblasts when combined with a porous calcium phosphate ceramic delivery vehicle and implanted subcutaneously in vivo 1 . We use this assay to test new materials as putative delivery vehicles in skeletal tissue engineering models. A new family of hyaluronan-based biomaterials has been developed. These polymers can be manufactured in many different formats and its chemistry can be tailored to specific requirements. HYAFF®-11p75HE is one of these materials, consisting of polymers of partially derivatized hyaluronic acid. HYAFF®-11p75HE sponges were tested as carriers for bone marrow-derived MFC preparations in the subcutaneous implantation model. Three weeks after implantation the HYAFF®-11p75HE sponge had dissolved and the tissue collected had the appearance of trabecular bone with undifferentiated mesenchyme filling the space between the trabeculae. Six weeks after implantation, the bone had matured and remodeled into an ossicle containing bone marrow-like tissue in its core. Hyaluronan is a multifunctional molecule that affects different cells or the same cells in a different manner depending on its molecular size and concentration. These complex, multifunctional effects can be used to develop hyaluronan-based scaffolds as delivery vehicles for tissue engineering of various skeletal tissues including bone.
Introduction: Human multipotent mesenchymal stem cell (MSC) therapies are being tested clinically... more Introduction: Human multipotent mesenchymal stem cell (MSC) therapies are being tested clinically for a variety of disorders, including Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, and cartilage defects. However, despite the remarkable clinical advancements in this eld, most applications still use traditional culture media containing fetal bovine serum. The ill-de ned and highly variable nature of traditional culture media remains a challenge, hampering both the basic and clinical human MSC research elds. To date, no reliable serum-free medium for human MSCs has been available. Methods: In this study, we developed and tested a serum-free growth medium on human bone marrow-derived MSCs through the investigation of multiple parameters including primary cell isolation, multipassage expansion, mesoderm di erentiation, cellular phenotype, and gene-expression analysis. Results: Similar to that achieved with traditional culture medium, human MSCs expanded in serum-free medium supplemented with recombinant human platelet-derived growth factor-BB (PDGF-BB), basic broblast growth factor (bFGF), and transforming growth factor (TGF)-β1 showed extensive propagation with retained phenotypic, di erentiation, and colony-forming unit potential. To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded under serum-free and serum-containing conditions were compared, revealing similar expression pro les. In addition, the described serum-free culture medium supported the isolation of human MSCs from primary human marrow aspirate with continual propagation. Conclusions: Although the described serum-free MSC culture medium is not free of xenogeneic components, this medium provides a substitute for serum-containing medium for research applications, setting the stage for future clinical applications.
Mesenchymal stem cells (MSCs) are non-hematopoietic, pluripotent cells that give rise to stromal ... more Mesenchymal stem cells (MSCs) are non-hematopoietic, pluripotent cells that give rise to stromal cells in the marrow. MSCs have been shown to be immunosuppressive and have become an attractive therapeutic option for the modulation of undesired immune responses. Currently, ex vivo expanded human (h)MSCs are being utilized in clinical trials both in the USA and in Europe to treat a variety of immune disorders. hMSCs need to be harvested, iso- lated and expanded in culture. This necessary expansion may also result in decrease or loss of the immunomodula- tory potential of hMSCs. Ideally, the intrinsic immunomodulatory activity (potency) of an hMSC preparation should be assessed prior to its administration. The goal of the experiments described here was to develop a simple potency assay for the immunomodulatory properties of hMSCs. The immunosuppressive activity of hMSCs conditioned media was tested in enzyme-linked immunosorbent spot assays (ELISpot) and the immunosuppressive activity ...
Introduction Spinal fusion is a procedure used to correct spinal deformities and treat fractured ... more Introduction Spinal fusion is a procedure used to correct spinal deformities and treat fractured vertebrae, spinal instabilities or chronic back pain. In interbody fusion, all or part of the intervertebral disc is removed and a supporting spacer inserted between the vertebral bodies for support and to facilitate bone growth between them. In most cases, bone growth is enhanced with graft materials placed within the spacer. Autologous bone graft (autograft) is one of the materials commonly used to facilitate fusion. Although autograft is considered the “gold standard” due to its osteoconductive and osteoinductive properties, it has limitations including availability, graft quality, and donor site morbidity. Synthetic bone graft substitutes are alternatives to autograft that eliminate donor-site morbidity and graft material variability. Bone graft substitutes have other advantages including unlimited supply, off-theshelf availability and easy sterilization and storage. Disadvantages ca...
The specialty of craniofacial surgery is broad and includes trauma, esthetics, reconstruction of ... more The specialty of craniofacial surgery is broad and includes trauma, esthetics, reconstruction of congenital deformities, and regeneration of tissues. Moreover, craniofacial surgery deals with a diverse range of tissues including both „soft‟ and „hard‟ tissues. Technological advances in materials and biological sciences and improved surgical techniques have remarkably improved clinical outcomes. The quest to raise the bar for patient care continues to inspire advances for predictable biological regeneration of „soft‟ and „hard‟ tissues. As a consequence of this quest for advancement, a wide spectrum of biologicals are becoming available to surgeons. Is the use of recombinant DNA engineered biologicals daring? Sensible? Logical? Timely? Safe? It is crucial for the practicing craniofacial surgeon to take a step back periodically and carefully review the biological factors that have the potential for dramatically altering the discipline of craniofacial surgery. With this emphasis, the c...
FT538 is an investigational, off-the-shelf, multiplexed engineered natural killer (NK) cell cance... more FT538 is an investigational, off-the-shelf, multiplexed engineered natural killer (NK) cell cancer immunotherapy that is derived from a clonal master induced pluripotent stem cell (iPSC) line engineered with three functional components to enhance innate immunity: a novel high-affinity, non-cleavable CD16 (hnCD16) Fc receptor; an IL-15/IL-15 receptor fusion (IL-15RF); and the elimination of CD38 expression. The use of a clonal master engineered iPSC line as a starting cell source enables routine mass production of FT538 and supports off-the-shelf product availability to reach many patients. The clonal master iPSC line for the manufacture of FT538 was made by reprogramming and engineering donor-consented human fibroblasts to induce pluripotency using a proprietary non-integrating system and to integrate a bicistronic cassette containing hnCD16 and IL-15RF into the CD38 locus, which resulted in complete disruption of the CD38 gene. The engineered iPSC population was then sorted to isol...
Tissue engineering requires large numbers of cells with enhanced differentiation properties. Thus... more Tissue engineering requires large numbers of cells with enhanced differentiation properties. Thus, the effect of expansion conditions must be explored. Human and rat marrow-derived mesenchymal stem cells (hMSCs and rMSCs, respectively) were comparatively culture expanded through seven passages in the presence of either fibroblast growth factor-2 (FGF-2) or platelet-derived growth factor BB (PDGF-BB). Proliferation of both hMSCs and rMSCs was enhanced by FGF-2 and PDGF-BB. Population doubling times for hMSCs were 2.4 days for control and 1.75 and 2.0 days for FGF-2 and PDGF-BB, respectively, and 3.25, 3.06, and 2.95 days for rMSCs. Supplementation with FGF-2 during cell expansion resulted in significantly greater in vivo bone formation for hMSCs. Use of PDGF-BB resulted in greater bone formation than that observed for control conditions, but the differences were only significant for P1. For rMSCs, significant increases in bone formation were noted in either FGF-2 or PDGF-BB expanded ...
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Papers by Luis Solchaga