Regulation of Immune Checkpoint Antigen CD276 (B7-H3) on Human Placenta-Derived Mesenchymal Stromal Cells in GMP-Compliant Cell Culture Media.

Therapies utilizing autologous mesenchymal cell delivery are being investigated as anti-inflammatory and regenerative treatments for a broad spectrum of age-related diseases, as well as various chronic and acute pathological conditions. Easily available allogeneic full-term human placenta mesenchymal stromal cells (pMSCs) were used as a potential pro-regenerative, cell-based therapy in degenerative diseases, which could be applied also to elderly individuals. To explore the potential of allogeneic pMSCs transplantation for pro-regenerative applications, such cells were isolated from five different term-placentas, obtained from the dissected maternal, endometrial (mpMSCs), and fetal chorion tissues (fpMSCs), respectively. The proliferation rate of the cells in the culture, as well as their shape, in vitro differentiation potential, and the expression of mesenchymal lineage and stem cell markers, were investigated. Moreover, we studied the expression of immune checkpoint antigen CD276 as a possible modulation of the rejection of transplanted non-HLA-matched homologous or even xeno-transplanted pMSCs. The expression of the cell surface markers was also explored in parallel in the cryosections of the relevant intact placenta tissue samples. The expansion of pMSCs in a clinical-grade medium complemented with 5% human platelet lysate and 5% human serum induced a significant expression of CD276 when compared to mpMSCs expanded in a commercial medium. We suggest that the expansion of mpMSCs, especially in a medium containing platelet lysate, elevated the expression of the immune-regulatory cell surface marker CD276. This may contribute to the immune tolerance towards allogeneic pMSC transplantations in clinical situations and even in xenogenic animal models of human diseases. The endurance of the injected comparably young human-term pMSCs may promote prolonged effects in clinical applications employing non-HLA-matched allogeneic cell therapy for various degenerative disorders, especially in aged adults.

Alleviation of Severe Skin Insults Following High-Dose Irradiation with Isolated Human Fetal Placental Stromal Cells.

Skin exposure to high-dose irradiation, as commonly practiced in radiotherapy, affects the different skin layers, causing dry and wet desquamation, hyperkeratosis fibrosis, hard to heal wounds and alopecia and damaged hair follicles. Fetal tissue mesenchymal stromal cells (f-hPSC) were isolated from excised human fetal placental tissue, based on their direct migration from the tissue samples to the tissue dish. The current study follows earlier reports on for the mitigation of acute radiation syndrome following whole body high-dose exposure with remotely injected f-hPSC. Both the head only and a back skin flap of mice were irradiated with 16 &18 Gy, respectively, by 6MeV clinical linear accelerator electron beam. In both locations, the irradiated skin areas developed early and late radiation induced skin damages, including cutaneous fibrosis, lesions, scaring and severe hair follicle loss and reduced hair pigmentation. Injection of 2 × 106 f-hPSC, 3 and 8 weeks following 16 Gy head irradiation, and 1 and 4 weeks following the 18 Gy back skin only irradiation, resulted in significantly faster healing of radiation induced damages, with reduction of wet desquamation as measured by surface moisture level and minor recovery of the skin viscoelasticity. Detailed histological morphometry showed a clear alleviation of radiation induced hyperkeratosis in f-hPSC treated mice, with significant regain of hair follicles density. Following 16 Gy head irradiation, the hair follicles density in the scalp skin was reduced significantly by almost a half relative to the controls. A nearly full recovery of hair density was found in the f-hPSC treated mice. In the 18 Gy irradiated back skin, the hair follicles density dropped in a late stage by ~70% relative to naïve controls. In irradiated f-hPSC treated mice, it was reduced by only ~30% and was significantly higher than the non-treated group. Our results suggest that local injections of xenogeneic f-hPSC could serve as a simple, safe and highly effective non-autologous pro-regenerative treatment for high-dose radiation induced skin insults. We expect that such treatment could also be applied for other irradiated organs.

Allogenic Use of Human Placenta-Derived Stromal Cells as a Highly Active Subtype of Mesenchymal Stromal Cells for Cell-Based Therapies.

The application of mesenchymal stromal cells (MSCs) from different sources, including bone marrow (BM, bmMSCs), adipose tissue (atMSCs), and human term placenta (hPSCs) has been proposed for various clinical purposes. Accumulated evidence suggests that the activity of the different MSCs is indirect and associated with paracrine release of pro-regenerative and anti-inflammatory factors. A major limitation of bmMSCs-based treatment for autologous application is the limited yield of cells harvested from BM and the invasiveness of the procedure. Similar effects of autologous and allogeneic MSCs isolated from various other tissues were reported. The easily available fresh human placenta seems to represent a preferred source for harvesting abundant numbers of human hPSCs for allogenic use. Cells derived from the neonate tissues of the placenta (f-hPSC) can undergo extended expansion with a low risk of senescence. The low expression of HLA class I and II on f-hPSCs reduces the risk of rejection in allogeneic or xenogeneic applications in normal immunocompetent hosts. The main advantage of hPSCs-based therapies seems to lie in the secretion of a wide range of pro-regenerative and anti-inflammatory factors. This renders hPSCs as a very competent cell for therapy in humans or animal models. This review summarizes the therapeutic potential of allogeneic applications of f-hPSCs, with reference to their indirect pro-regenerative and anti-inflammatory effects and discusses clinical feasibility studies.

Isolation and expansion of high yield of pure mesenchymal stromal cells from fresh and cryopreserved placental tissues.

The injection of placental stromal cells isolated from fetal human tissues (f-hPSC) was reported to indirectly induce tissue regeneration in different animal models. A procedure of f-hPSC isolation from fragments of both selected fresh or cryopreserved bulk placental neonate tissues is proposed, based on their high migratory potential,. The fragments of the desired fetal placental tissues are adhered to a culture dish by traces of diluted fibrin and covered with culture medium. Spontaneous migration of pure f-hPSC from the tissue fragments to the cell culture dishes is followed by their rapid expansion by numerous passages. The isolated f-hPSC express typical mesenchymal surface antigens, including CD29, CD105, CD166 and CD146, with negative expression of white blood cell lineage and endothelial cells markers. Optimal yields of f-hPSC cultures can also be obtained from tissue samples cryopreserved in medium composed of 10% dimethyl sulfoxide (M2SO) and 50% fetal calf serum. Slightly better yields are obtained with media supplemented with 1% human albumin. Medium with 5% M2SO and/or 0.25 mg/ml PEG yielded inferior results. The f-hPSC from fresh or cryopreserved tissues express similar cell markers and growth kinetics. The proposed isolation protocol may also be applied for high yield isolation of stromal cells from fresh and cryopreserved tissue of other organs.

Modulating the innate immune activity in murine tumor microenvironment by a combination of inducer molecules attached to microparticles.

Targeted cancer immunotherapy is challenging due to the cellular diversity and imposed immune tolerance in the tumor microenvironment (TME). A promising route to overcome those drawbacks may be by activating innate immune cells (IIC) in the TME, toward tumor destruction. Studies have shown the ability to "re-educate" pro-tumor-activated IIC toward antitumor responses. The current research aims to stimulate such activation using a combination of innate activators loaded onto microparticles (MP). Four inducers of Toll-like receptors 4 and 7, complement C5a receptor (C5aR) and gamma Fc receptor and their combinations were loaded on MP, and their influence on immune cell activation evaluated. MP stimulation of immune cell activation was tested in vitro and in vivo using a subcutaneous B16-F10 melanoma model induced in C57BL6 mice. Exposure to the TLR4 ligand lipopolysaccharide (LPS) bound to MP-induced acute inflammatory cytokine and chemokine activity in vitro and in vivo, with the elevation of CD45(+) leukocytes in particular GR-1(+) neutrophils and F4/80 macrophages in the TME. Nevertheless, LPS alone on MP was insufficient to significantly delay tumor progression. LPS combined with the C5aR ligand C5a-pep on the same MP resulted in a similar inflammation activation pattern. However, interleukin-10 levels were lower, and tumor growth was significantly delayed. Mixtures of these two ligands on separate MP did not yield the same cytokine activation pattern, demonstrating the importance of the cells' dual activation. The results suggest that combining inducers of distinct innate immune activation pathways holds promise for successful redirection of TME-residing IIC toward anti-tumoral activation.

Mast cell activation by fibrinogen-related homologous c-terminal peptides (haptides) modulates systemic blood pressure.

BACKGROUND: Haptides are a family of short peptides homologous to C-termini sequences of fibrinogen chains ß and γ (haptides Cß and preCγ, respectively) which were previously shown to penetrate and bind cells. OBJECTIVES: This work investigates the systemic effect of the haptides with possible clinical implications. METHODS: Intra-arterial monitoring in rats recorded the haptides' effects on systemic blood pressure. In parallel, their effect was also tested in vitro on isolated rat peritoneal mast cells and on human mast cells. RESULTS: Intra-arterial monitoring in rats showed that intravenous administration of low haptides concentrations (35-560 µg/kg rat) caused a shocklike behavior with transient decrease in the systolic and diastolic blood pressure by up to 55% (P < .05) in a dose-dependent manner and a minor increase in their heart rate. Randomly scrambled sequences of the haptides had no such effect, suggesting a specific interaction with receptors. Intravenous administration of blockers to histamine receptors H1 and H2 before haptides administration attenuated this effect. Furthermore, in vitro incubation of human LAD2 mast cell line or isolated rat peritoneal mast cells with the haptides caused degranulation of the mast cells. We found that the haptides Cß and preCγ activated mast cells causing histamine release, resulting in a steep decrease in blood pressure, comparable to anaphylactic shock. CONCLUSION: In treating vascular occlusive diseases, massive fibrinolysis is induced, and haptide-containing sequences are released. We suggest that treatment with histamine receptor blockers or with mast cell stabilizing agents in such pathological conditions may overcome this effect.

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.

Short homologous peptides based on C-terminal sequences of fibrinogen ß- and γ-chains (Haptides) affect cardiovascular function by eNOS inhibition.

Haptides are a family of 19-21-mer cell-binding and permeating peptides homologous to sequences in the C termini on both fibrinogen ß- and γ-chain (Cß and preCγ, respectively). The effect of the Haptides on the cardiovascular system was studied by different assays, including the activity of isolated perfused rat heart and blood vessels in the organ bath. Haptides (50-80 µg/ml) decreased the hemodynamic functions of perfused rat hearts by up to 60% (p < 0.05) in a dose-dependent manner. Whole fibrinogen or a control nonrelated peptide (Cα) did not show such an effect. The NO donor, sodium nitroprusside, reversed the inhibitory effects of Haptides. L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, did not further augment the effect of the Haptides. Perfused (FITC)Haptides were attached to the coronary endothelium. In myocardial homogenates and HUVEC, Haptides significantly decreased eNOS activity, but had no effect on the contraction of isolated cultured adult cardiomyocytes. Haptides also significantly enhanced the contraction of rings of rat aorta and human mammary artery vessels ex vivo only when the endothelium was intact. Haptides seem to affect the coronary endothelium, but not the cardiomyocytes, by inhibiting eNOS activity, causing vasoconstriction, temporary ischemia and impaired myocardial function that seem to be related to the amino acid composition of the Haptides.

An in-vitro tumour microenvironment model using adhesion to type I collagen reveals Akt-dependent radiation resistance in renal cancer cells.

BACKGROUND: Renal cell carcinoma (RCC) is considered resistant to ionizing radiation. Recently, the extracellular matrix (ECM) has been shown to play a role in both drug resistance and radiation resistance (RR). While fibronectin has been extensively investigated in the context of RR, the role of type I collagen [col(I)], a principal constituent of the ECM in tumour metastases, in RR of RCC is unknown. METHODS: RCC cell adhesion to matrix was studied via pre-coating a variety of ECM glycoproteins onto plates. Cancer cell apoptosis and cell cycle were evaluated with flow cytometry using annexin V and propidium iodide stains, respectively. Activation of cellular survival signalling was analysed with western blots, and specific molecular inhibitors were correspondingly employed to block signalling. Hypoxia (<1%) was induced via N(2)/CO(2) gas flow in a specialized chamber. RESULTS: While adherence to col(I) enhanced RCC cell proliferation in general, col(I) and fibronectin, but not fibrinogen, could confer specific anti-apoptotic RR to RCC cells. The radioprotective effect of col(I) was maintained during both hypoxia/reoxygenation and normoxia conditions. In contrast to intact col(I), micronized col(I), lacking the natural fibrillar structure, was not radioprotective. The effect of col(I) in RCC cells is mediated via attenuation of apoptosis rather than cell cycle redistribution, involving the PI3 kinase/Akt pathway but not the MAP kinase pathway. CONCLUSIONS: Adherence to col(I) appears to be a relevant environmental cue enhancing RR in RCC cells, Akt dependently. Our results support inhibition of the PI3-kinase/Akt pathway as a radiosensitizing approach.

Alleviation of acute radiation-induced bone marrow failure in mice with human fetal placental stromal cell therapy.

PURPOSE: Selected placental mesenchymal stromal cells isolated from the fetal mesenchymal placental tissues (f-hPSCs) were tested as cell therapy of lethal acute radiation syndrome (ARS) with bone marrow regeneration and induced extramedullary hematopoiesis. METHODS AND MATERIALS: f-hPSCs were isolated from the chorionic plate of human placentae and further expanded in regular culture conditions. 2 × 106 f-hPSCs were injected on days 1 and 4 to 8-Gy total body irradiated (TBI) C3H mice, both intramuscularly and subcutaneously. Pre-splenectomized TBI mice were used to test the involvement of extramedullary spleen hematopoiesis in the f-hPSC-induced hematopoiesis recovery in the TBI mice. Weight and survival of the mice were followed up within the morbid period of up to 23 days following irradiation. The role of hematopoietic progenitors in the recovery of treated mice was evaluated by flow cytometry, blood cell counts, and assay of possibly relevant growth factors. RESULTS AND CONCLUSIONS: The survival rate of all groups of TBI f-hPSC-treated mice at the end of the follow-up was dramatically elevated from < 10% in untreated to ~ 80%, with a parallel regain of body weight, bone marrow (BM) recovery, and elevated circulating progenitors of blood cell lineages. Blood erythropoietin levels were elevated in all f-hPSC-treated mice. Extramedullary splenic hematopoiesis was recorded in the f-hPSC-treated mice, though splenectomized mice still had similar survival rate. Our findings suggest that the indirect f-hPSC life-saving therapy of ARS may also be applied for treating other conditions with a failure of the hematopoietic system and severe pancytopenia.

In Vivo Formation of Stable Hyaline Cartilage by Naïve Human Bone Marrow Stromal Cells with Modified Fibrin Microbeads.

Osteoarthritic and other types of articular cartilage defects never heal on their own. Medicinal and surgical approaches are often ineffective, and the supply of autologous chondrocytes for tissue engineering is very limited. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested as an adequate cell source for cartilage reconstruction. However, the majority of studies employing BMSCs for cartilage tissue engineering have used BMSCs predifferentiated into cartilage prior to implantation. This strategy has failed to achieve formation of stable, hyaline-like cartilage, resistant to hypertrophy in vivo. We hypothesized that in vitro predifferentiation of BMSCs is not necessary when cells are combined with an adequate scaffold that supports the formation of stable cartilage in vivo. In this study, naïve (undifferentiated) human BMSCs were attached to dehydrothermally crosslinked stable fibrin microbeads (FMBs) without and with other scaffolds and implanted subcutaneously into immunocompromised mice. Optimal formation of abundant, hypertrophy-resistant, ectopic hyaline-like cartilage was achieved when BMSCs were attached to FMBs covalently coated with hyaluronic acid. The cartilage that was formed was of human origin and was stable for at least 28 weeks in vivo. Stem Cells Translational Medicine 2019;8:586-592.

A 3D rotary renal and mesenchymal stem cell culture model unveils cell death mechanisms induced by matrix deficiency and low shear stress.

BACKGROUND: In epithelial and endothelial cells, detachment from the matrix results in anoikis, a form of apoptosis, whereas stromal and cancer cells are often anchorage independent. The classical anoikis model is based on static 3D epithelial cell culture conditions (STCK). METHODS: We characterized a new model of renal, stromal and mesenchymal stem cell (MSC) matrix deprivation, based on slow rotation cell culture conditions (ROCK). This model induces anoikis using a low shear stress, laminar flow. The mechanism of cell death was determined via FACS (fluorescence-activated cell sorting) analysis for annexin V and propidium iodide uptake and via DNA laddering. RESULTS: While only renal epithelial cells progressively died in STCK, the ROCK model could induce apoptosis in stromal and transformed cells; cell survival decreased in ROCK versus STCK to 40%, 52%, 62% and 7% in human fibroblast, rat MSC, renal cell carcinoma (RCC) and human melanoma cell lines, respectively. Furthermore, while ROCK induced primarily apoptosis in renal epithelial cells, necrosis was more prevalent in transformed and cancer cells [necrosis/apoptosis ratio of 72.7% in CaKi-1 RCC cells versus 4.3% in MDCK (Madin-Darby canine kidney) cells]. The ROCK-mediated shift to necrosis in RCC cells was further accentuated 3.4-fold by H(2)O(2)-mediated oxidative stress while in adherent HK-2 renal epithelial cells, oxidative stress enhanced apoptosis. ROCK conditions could also unveil a similar pattern in the LZ100 rat MSC line where in ROCK 44% less apoptosis was observed versus STCK and 45% less apoptosis versus monolayer conditions. Apoptosis in response to oxidative stress was also attenuated in the rat MSC line in ROCK, thereby highlighting rat MSC transformation. CONCLUSIONS: The ROCK matrix-deficiency cell culture model may provide a valuable insight into the mechanism of renal and MSC cell death in response to matrix deprivation.

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.

Rescue from lethal acute radiation syndrome (ARS) with severe weight loss by secretome of intramuscularly injected human placental stromal cells.

BACKGROUND: Most current cell-based regenerative therapies are based on the indirect induction of the affected tissues repair. Xenogeneic cell-based treatment with expanded human placenta stromal cells, predominantly from fetal origin (PLX-RAD cells), were shown to mitigate significantly acute radiation syndrome (ARS) following high dose irradiation in mice, with expedited regain of weight loss and haematopoietic function. The current mechanistic study explores the indirect effect of the secretome of PLX-RAD cells in the rescue of the irradiated mice. METHODS: The mitigation of the ARS was investigated following two intramuscularly (IM) injected 2 × 106 PLX-RAD cells, 1 and 5 days following 7.7 Gy irradiation. The mice survival rate and their blood or bone marrow (BM) cell counts were followed up and correlated with multiplex immunoassay of a panel of related human proteins of PLX-RAD derived secretome, as well as endogenous secretion of related mouse proteins. PLX-RAD secretome was also tested in vitro for its effect on the induction of the migration of BM progenitors. RESULTS: A 7.7 Gy whole body mice irradiation resulted in ~25% survival by 21 days. Treatment with two IM injections of 2 × 106 PLX-RAD cells on days 1 and 5 after irradiation mitigated highly significantly the subsequent lethal ARS, with survival rate increase to nearly 100% and fast regain of the initial weight loss (P < 0,0001). This was associated with a significant faster haematopoiesis recovery from day 9 onwards (P < 0.01). Nine out of the 65 human proteins tested were highly significantly elevated in the mouse circulation, peaking on days 6-9 after irradiation, relative to negligible levels in non-irradiated PLX-RAD injected mice (P < 0.01). The highly elevated proteins included human G-CSF, GRO, MCP-1, IL-6 and lL-8, reaching >500 pg/mL, while MCP-3, ENA, Eotaxin and fractalkine levels ranged between ~60-160pg/mL. The detected radiation-induced PLX-RAD secretome correlated well with the timing of the fast haematopoiesis regeneration. The radiation-induced PLX-RAD secretome seemed to reinforce the delayed high levels secretion of related mouse endogenous cytokines, including GCSF, KC, MCP-1 and IL-6. Additional supportive in vitro studies also confirmed the ability of cultured PLX-RAD secretome to induce accelerated migration of BM progenitors. CONCLUSIONS: A well-regulated and orchestrated secretion of major pro-regenerative BM supporting secretome in high dose irradiated mice, treated with xenogeneic IM injected PLX-RAD cells, can explain the observed mitigation of ARS. This seemed to coincide with faster haematopoiesis regeneration, regain of severe weight loss and the increased survival rate. The ARS-related stress signals activating the IM injected PLX-RAD cells for the remote secretion of the relevant human proteins deserve further investigation.

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.

Effect of halofuginone, a collagen alpha1(I) inhibitor, on wound healing in normal and irradiated skin: implication for hematopoietic stem cell transplantation.

It is a major interest in the field of hematopoietic stem cell transplantation to reduce scarring of healing wounds with overdeposition of collagen due to radiation injury or graft-versus-host disease. Halofuginone (HF) inhibits collagen alpha1(I) gene expression and overdeposition of collagen. We examined the effect of HF on the healing of full-depth incision wounds inflicted in normal skin or skin areas compromised by local preirradiation with 18 Gy. Preirradiation significantly decreased the tensile strength of the healing wounds at day 14 (by approximately 60%, p < 0.0001). In contrast, HF treatment did not significantly decrease the strength of wounds inflicted in both normal and preirradiated skin. Histological evaluation revealed that HF induced moderate thinning of the dermis accompanied by elevated thickness of the epidermis and enhanced rejoining of subdermal muscles in the wound area. HF only minimally reduced total collagen deposition in both groups, with minor changes in the level of more matured fibrillar collagen network. Our study demonstrates that HF does not significantly affect wound strength. This encourages the possible use of HF as an antifibrotic agent with minimal complications for post-hematopoietic stem cell transplantation complications including radiation toxicity and graft-versus-host disease.

Melanoma antigens and related immunological markers.

Melanoma is a highly lethal cancer deriving from transformed dermal melanocytes. Early diagnosed primary melanoma may be curable, but the cure-rate of more advanced stages is limited, with high mortality rate. With the progression of the tumor, the melanocytes overexpress intracellular or cell-surface molecules, including ectopic normal and tumor-specific proteins. Some of these induce a specific immune response by T and B lymphocytes. Antibodies raised against melanoma antigens were proposed for differential disease diagnosis, staging, prognosis and evaluation of treatment efficiency. Nevertheless, treatments based on stimulation of specific anti-melanoma immune responses have had only limited success. It seems that efficient immunotherapy should become more feasible pending on finding new adequate antigens to target. New insights into immune regulation of the tumor microenvironment and its progression may help the development of more successful treatments. We present here up-to-date information on known major melanoma-associated antigens, which could serve as tools for diagnosis as well as for clinical immunotherapy. This approach with promising results for treating some other selected malignancies is still experimental with a very limited success in melanoma. The development of new immune modulators of the tumor microenvironment and neo-antigens may be additional promising directions and may open new opportunities for the immunotherapy of melanoma.

Placental Stromal Cell Therapy for Experimental Autoimmune Encephalomyelitis: The Role of Route of Cell Delivery.

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS) with no effective treatment available for the chronic-progressive stage. Cell therapy is a promising therapeutic approach for attenuating the immune-mediated CNS process. Isolated and expanded human placental stromal cells (hPSCs) possess potent immunomodulatory and trophic properties, making them a good candidate for MS therapy. We examined the potential of hPSC therapy in preventing the onset or attenuating the course of established disease in a murine MS model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. We examined the feasibility of hPSC systemic delivery by intramuscular (i.m.) implantation rather than the commonly used intravenous injection, which is dose-limiting and carries the risk of pulmonary obstruction. Our findings showed significant attenuation of the disease only when hPSCs were injected directly to the central nervous system. Intramuscular implanted hPSCs survived at the site of injection for at least 2 months and elicited extensive local immune responses. Intramuscular hPSC implantation before disease onset caused a delay in the appearance of clinical signs and reduced the severity of a relapse induced by repeated challenge with the autoantigen. Intramuscular implantation after disease onset did not affect its course. Thus, pathological analysis of CNS tissue did not show inhibition of neuroinflammation in i.m. hPSC-implanted mice. Moreover, no apparent effect was seen on the proliferative response of peripheral lymph node cells in these animals. We conclude that to maximize their therapeutic potential in MS, hPSCs should be delivered directly to the affected CNS. Stem Cells Translational Medicine 2017;6:1286-1294.

High-yield isolation, expansion, and differentiation of rat bone marrow-derived mesenchymal stem cells with fibrin microbeads.

Fibrin microbeads (FMB), made of extensively cross-linked dense and partially denatured fibrin, were used as a matrix for efficient isolation of mesenchymal stem cells (MSC) from rat bone marrow (BM). After 2 days of incubation of FMB with whole BM in suspension, a high number of cells of mesenchymal origin attached to the FMB. On the 14th day after their transfer to plastic, the yield of the cells isolated via FMB was approximately 3-4 times higher than that obtained by currently used protocols based solely on plastic adhesion. This implies that the number of MSC in BM may be higher than previously reported. FACS analyses and immunostaining showed the mesenchymal characteristics of these cells by positive staining for fibronectin, vimentin, CD49E, and CD29. Immediately after isolation, less than 20% of the cells still expressed the hematopoietic markers CD11b and CD45. Most of these cells were eventually eliminated after further expansion of the isolated cells on plastic. Cells isolated via FMB were expanded in culture for more than 4 months and could be defined as MSC along this time period based on their ability to differentiate into precursors of mesenchymal tissues, such as osteogenic, adipogenic, and chondrogenic cells. Similar differentiation plasticity was observed in clones derived from single cells from whole MSC populations isolated via FMB. Based on our results we propose that FMB can serve as a 3-dimensional biodegradable matrix for isolation, differentiation, and possibly implantation of MSC 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.