Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells.

Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×10(6) cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective "off the shelf" therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia.

Marrow-derived stromal cell delivery on fibrin microbeads can correct radiation-induced wound-healing deficits.

Skin that is exposed to radiation has an impaired ability to heal wounds. This is especially true for whole-body irradiation, where even moderate nonlethal doses can result in wound-healing deficits. Our previous attempts to administer dermal cells locally to wounds to correct radiation-induced deficits were hampered by poor cell retention. Here we improve the outcome by using biodegradable fibrin microbeads (FMBs) to isolate a population of mesenchymal marrow-derived stromal cells (MSCs) from murine bone marrow by their specific binding to the fibrin matrix, culture them to high density in vitro, and deliver them as MSCs on FMBs at the wound site. MSCs are retained locally, proliferate in site, and assist wounds in gaining tensile strength in whole-body irradiated mice with or without additional skin-only exposure. MSC-FMBs were effective in two different mouse strains but were ineffective across a major histocompatability barrier. Remarkably, irradiated mice whose wounds were treated with MSC-FMBs showed enhanced hair regrowth, suggesting indirect effect on the correction of radiation-induced follicular damage. Further studies showed that additional wound-healing benefit could be gained by administration of granulocyte colony-stimulating factor and AMD3100. Collagen strips coated with haptides and MSCs were also highly effective in correcting radiation-induced wound-healing deficits.

Fibrin microbeads loaded with mesenchymal cells support their long-term survival while sealed at room temperature.

Efficient transfer of progenitor cells without affecting their survival is a key factor in any practical cell therapy. Fibrin microbeads (FMB) were developed as hard biodegradable cell carriers. The FMB could efficiently isolate mesenchymal stem cells (MSCs) from different sources and support the expansion of matrix-dependent cell types in a three-dimensional culture in slow rotation. The cells on FMB could also undergo induced differentiation for their eventual implantation to enhance tissue regeneration. FMB loaded with isolated human MSC (hMSC) were sealed in tubes topped up with medium. Almost full cell survival was recorded when the sealed cells were maintained in room temperature for up to 10 days, followed by a recovery period of 24 hrs at optimal conditions. Assay of cells recovery after such long room temperature storage showed ∼80%-100% survival of the cells on FMB, with only a marginal survival of cells that were kept in suspension without FMB in the same conditions. The hMSC that survived storage at room temperature preserved their profile of mesenchymal cell surface markers, their rate of proliferation, and their differentiation potential. The cell protective effect was not dependent on the presence of serum in the storage medium. It was clearly shown that over-expression of hypoxia induced factor-1α in hMSC with time, which may have protected the sealed cells on FMB at room temperature storage, was not necessarily related to extreme hypoxic stress. Foreskin normal fibroblasts on FMB sealed at room temperature were similarly protected, but with no elevation of their hypoxia-induced factor-1α expression. The results also show that FMB, unlike other commercially available cell carriers, could be used for delivery and shipping of progenitor cells at room temperature for extended time intervals. This could be highly useful for cell transfer for therapeutic application and for simplified cell transfer between different research centers.

A family of cell-adhering peptides homologous to fibrinogen C-termini.

A family of cell-adhesive peptides homologous to sequences on different chains of fibrinogen was investigated. These homologous peptides, termed Haptides, include the peptides Cß, preCγ, and CαE, corresponding to sequences on the C-termini of fibrinogen chains ß, γ, and αE, respectively. Haptides do not affect cell survival and rate of proliferation of the normal cell types tested. The use of new sensitive assays of cell adhesion clearly demonstrated the ability of Haptides, bound to inert matrices, to mediate attachment of different matrix-dependent cell types including normal fibroblasts, endothelial, and smooth muscle cells. Here we present new active Haptides bearing homologous sequences derived from the C-termini of other proteins, such as angiopoietin 1&2, tenascins C&X, and microfibril-associated glycoprotein-4. The cell adhesion properties of all the Haptides were found to be associated mainly with their 11 N-terminal residues. Mutated preCγ peptides revealed that positively charged residues account for their attachment effect. These results suggest a mechanism of direct electrostatic interaction of Haptides with the cell membrane. The extended Haptides family may be applied in modulating adhesion of cells to scaffolds for tissue regeneration and for enhancement of nanoparticulate transfection into cells.

Coated cross-species antibodies by mannosamine-biotin adduct confer protection against snake venom without eliciting humoral immune response.

Passive immunization with cross-species antibodies triggers the patient's immune response, thereby preventing repeated treatment. Mannosamine-biotin adduct (MBA) has been described as a masking agent for immunogenic reduction and here, the immunogenicity and biological activity of MBA-coated horse anti-viper venom (hsIgG) were compared to those of uncoated or PEGylated hsIgG. In in vitro tests, hsIgG binding was not affected by MBA conjugation. The immune response to hsIgG-MBA was about 8-fold and 32-fold lower than to PEG-coated and uncoated hsIgG, respectively. In vivo, hsIgG-MBA showed efficient venom-neutralization activity. We thus demonstrate the feasibility of using MBA as a masking agent for passive immunization with cross-species antibodies.

Targeted microbeads for attraction and induction of specific innate immune response in the tumor microenvironment.

Antitumor activity of molecules and cells of the innate immune system has been reported. Here we propose a method for targeting preferred innate immune cells and magnifying their tumoricidal effect at the tumor microenvironment, by modular multiple-component complexes (termed TILTAN). As a model, micro-scale complexes were assembled carrying monoclonal anti-HER2 antibodies, lipopolysaccharide and/or mannose. The complexes showed high binding capacity to HER2-positive cancer cells in vitro, high induction of interleukin-1 RNA transcription by the activated monocytes and ability to mediate monocytes' attachment to HER2-positive cells. TILTAN treatment was found safe in in vivo testing and induced change in interleukin-1 RNA transcription in tumors xenografts. We thus present a new vision of targeting a desired innate immune response to the tumor microenvironment.

Efficient isolation and chondrogenic differentiation of adult mesenchymal stem cells with fibrin microbeads and micronized collagen sponges.

BACKGROUND: Mesenchymal stem cells (MSCs) have been demonstrated to potentially undergo chondrogenic differentiation. We propose a new matrix for stem cell-based chondrogenesis using dense fibrin microbeads (FMBs) combined with grounded dehydrothermally crosslinked collagen sponges (micronized collagen). METHODS: In this study, MSCs were isolated from bone marrow of transgenic green fluorescent protein C57/Bl mice by FMBs in high yield. After 48 h in slowly rotating suspension culture, micronized collagen was added. RESULTS: The cells on the FMBs migrated to the collagen pieces and formed aggregates that developed into cartilage-like structures. Following chondrogenic differentiation, alcian blue staining and collagen type II immunohistochemistry demonstrated the presence of chondrocytes in the 3D structures. PCR for the expression of aggrecan and collagen type II genes supported these findings. The in vitro structures that formed were used for ectopic subdermal implantation in wild-type C57/Bl mice. However, the chondrogenic markers faded relative to the pre-implant in vitro structures. CONCLUSION: We propose that FMBs with micronized collagen could serve as a simple technology for MSC isolation and chondrogenesis as a basis for implantation.

Isolation and implantation of bone marrow-derived mesenchymal stem cells with fibrin micro beads to repair a critical-size bone defect in mice.

Fibrin microbeads (FMBs) made using thermal treatment of fibrin drops in oil can efficiently isolate mesenchymal stem cells (MSCs) from bone marrow (BM) and other similar sources and culture them continuously in suspension culture. The pure mesenchymal profile of MSCs isolated using FMBs and their differentiation potency to different mesenchymal lineages were previously described in detail. In the current study, MSCs were isolated from the BM of (GFP+) C57/bl mice using FMBs. Addition of pro-osteogenic medium with 10 mM of ss-glycerolphosphate, 50 microg/mL of ascorbic acid, and 10(-8) M of dexamethasone for 1 month resulted in ossified bone-like solid cellular structures, as seen using fluorescence and scanning electron microscopy (SEM). Such spontaneously formed structures were implanted in full-depth approximately 5-mm-diameter drilled defects in the skulls of wild-type c57/bl mice. Two months later, the excised upper parts of the skulls with the defects were viewed using fluorescence microscopy for green fluorescence protein of the cells in the defect and using SEM. They were also scanned using micro-computed tomography to visualize the formation of new hard tissue. Then the samples were processed and sectioned for hematoxylin and eosin staining and immunohistochemistry. Implanted FMBs loaded with (GFP+) MSCs formed partially mature, dense bone-like tissue using a residual moderate inflammatory process containing remnants of FMBs and neo-angiogenesis. The filled defect with bone-like tissue had a Ca/P ratio similar to that of native bone. Limited merging of the implant with the skull indicated that the induced bone regeneration derived from the MSCs that were delivered with the implant. No repair was seen in the control animals without implants or where the defect was filled with FMBs only. Repair scoring (on a 0-5 scale) was found to be 3.38+/-0.35 in the experimental arm, relative to 0 in the controls (p < 0.001).

Haptide-coated collagen sponge as a bioactive matrix for tissue regeneration.

We previously described a new class of conserved, cell adhesive (haptotactic) peptides, termed Haptides, based on sequences first identified in fibrinogen. Here, we describe a new biomaterial, Haptide-coated Collagen, in which the carbodiimide reagent, EDC, was used to covalently couple a Haptide (preC gamma), equivalent to the carboxy terminus of the fibrinogen gamma chain, to a cross-linked sponge composed of bovine collagen type I. The dose response of Haptide bound to collagen on cell attachment response reached a plateau at a concentration of 5-10 mg Haptide/g collagen. The Haptized-collagen was more stable to 1N NaOH, with a degradation half-time (T(1/2)) of 1.7 h, compared to 0.9 h for untreated control. Haptized collagen discs could be loaded with approximately 30% more human dermal fibroblasts or bovine aortic endothelial cells than unmodified collagen discs (p < 0.001). After a proliferation phase, Haptized collagen discs contained approximately 45% more fibroblasts than non-Haptized discs (p < 0.01). Histological analysis following sub-dermal implantation in rats indicated that at day 8, Haptized collagen sponge was less degraded than unmodified collagen sponge, attracted more endogenous fibroblasts with newly deposited collagen, and provoked less inflammatory or other adverse reactions. These results suggest potential clinical applications for Haptized collagen sponge for tissue regeneration, soft tissue augmentation, skin repair, and wound healing.

Heat denaturation of fibrinogen to develop a biomedical matrix.

Native and heat denatured fibrinogen are the basis for various matrices used to establish hemostasis as well as for constructing biomedical devices. For example, fibrin microbeads (FMB) prepared by a heated ( approximately 70 degrees C) oil emulsion process were reported to be attractive to mesenchymal-type cells, such as fibroblasts, endothelial and smooth muscle cells, and useful for isolating mesenchymal stem cells from bone marrow. Here, we examined the solution properties of fibrinogen subjected to heat (47-60 degrees C). Fibrinogen exhibited maximal stability of pH(max stab) = 6.8. At physiologically relevant concentrations, Ca(II) stabilized and Zn(II) destabilized fibrinogen against heat denaturation. Scanning electron micrographs (SEM) of precipitated, heat denatured, fibrinogen showed globular structures ( approximately 400 nm diameter), composed of aggregates of >3000 fibrinogen monomers. Monoclonal antibodies (MAb) to various regions of fibrinogen, as well as two polyclonal antibody (Ab) to haptotactic peptides (Haptides) equivalent to or near the C-termini of beta and gamma-chains (beta(463-483) and gamma(372-391/411)), were used to monitor epitopic changes of fibrinogen bound to and heated on plastic ELISA plates. The pattern of altered Ab binding indicated that fibrinogen heat denaturation on plastic exposed the C-terminal epitope gamma(397-411) as well as Haptide epitopes (beta(463-483) and gamma(372-391)). Immuno-staining of FMB prepared by a heated (below 75 degrees C) oil emulsion process, also presented many exposed Haptide epitopes, which probably helped to attract cells. Our results indicated that moderately heat-denatured fibrinogen, in the form of FMB, could be used for cell culturing and biomedical applications.

High-yield isolation, expansion, and differentiation of murine bone marrow-derived mesenchymal stem cells using fibrin microbeads (FMB).

Transplantation of adult mesenchymal stem cells (MSCs) could provide a basis for tissue regeneration. MSCs are typically isolated from bone marrow (BM) based on their preferential adherence to plastic, although with low efficiency in terms of yield and purity. Extensive expansion is needed to reach a significant number of MSCs for any application. Fibrin microbeads (FMB) were designed to attach mesenchymal cells and to provide a matrix for their expansion. The current study was aimed at isolating a high yield of purified BM-derived mouse MSCs based on their preferential adherence and proliferation on FMB in suspension cultures. MSCs could be downloaded to plastics or further expanded on FMB. The yield of MSCs obtained by the FMB isolation technique was about one order of magnitude higher than that achieved by plastic adherence, suggesting that these cells are more abundant than previously reported. FMB-isolated cells were classified as MSCs by their fibroblastic morphology, self-renewal ability, and expression profile of their surface antigens, as examined by flow cytometry and immunostaining. In cell culture, the isolated MSCs could be induced to differentiate into three different mesodermal lineages, as demonstrated by histochemical stains and by RT-PCR analyses of tissue-specific genes. MSCs were also able to differentiate into osteocytes while still cultured on FMB. Our results suggest that FMB might serve as an efficient platform for the isolation, expansion, and differentiation of mouse BM-derived MSCs to be subsequently implanted for tissue regeneration.

Interactions of carboplatin with fibrin(ogen), implications for local slow release chemotherapy.

The effect of carboplatin (CPt) on fibrin(ogen) clot formation and the possible use of this combination for local slow release chemotherapy were examined. CPt significantly reduced thrombin-induced fibrin clotting time (CT) and increased clot turbidity in a concentration-dependent manner. When CPt was mixed with physiological levels of fibrinogen (>1 mg/ml), electron-dense nanoparticles (3 nm) were formed, as demonstrated by both optical particle counter and transmission electron microscopy (TEM). Upon thrombin-induced coagulation, the CPt nanoparticles were trapped within the fibrin mesh. At higher fibrinogen levels (>5 mg/ml), the 3-nm CPt nanoparticles aggregated, so that approximately 2% and approximately 0.5% of the CPt on the fibrinogen appeared as larger particles of 10 and 50 nm, respectively. Dialysis experiments showed that 60-70% of the CPt was released from the fibrin clot within one hour as a non-particulate soluble form, while approximately 30% of particulate CPt were retained. Up to 5 mg/ml this portion of firmly attached CPt was dependent of the initial drug level. CPt released from the fibrin by either diffusion or by fibrinolysis exhibited cytotoxic activity towards retinoblastoma (RB) cell lines (Y-79 and Weri RB1) equivalent to free drug. Our study indicates that CPt enhances fibrin clot formation and suggests the use of fibrin with high dose CPt for slow release chemotherapy against localized tumors such as retinoblastoma.

The protein phosphatase calcineurin determines basal parathyroid hormone gene expression.

Calcium and phosphate regulate PTH mRNA stability through differences in binding of parathyroid (PT) proteins to a minimal 63-nucleotide (nt) cis-acting instability element in its 3'-untranslated region. One of these proteins is adenosine-uridine-rich binding factor (AUF1), whose levels are not regulated in PT extracts from rats fed the different diets. However, two-dimensional gels showed posttranslational modification of AUF1 that included phosphorylation. There is no PT cell line, but in HEK 293 cells the 63-nt element is recognized as an instability element, and RNA interference for AUF1 decreased human PTH secretion in cotransfection experiments. Stably transfected cells with a chimeric GH gene containing the PTH 63-nt cis-acting element were used to study the signal transduction pathway that regulates AUF1 modification and chimeric gene mRNA stability. Cyclosporine A, the calcineurin inhibitor, regulated AUF1 posttranslationally, and this correlated with an increase in the stability of GH-PTH 63-nt mRNA but not of the control GH mRNA. Mice with genetic deletion of the calcineurin Abeta gene had markedly increased PTH mRNA levels that were still regulated by low calcium and phosphorus diets. Therefore, calcineurin regulates AUF1 posttranslationally in vitro and PTH gene expression in vivo but still allows its physiological regulation by calcium and phosphate.