Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis of the knee: a proof-of-concept clinical trial.

Mesenchymal stem cells (MSCs) are known to have a potential for articular cartilage regeneration. However, most studies focused on focal cartilage defect through surgical implantation. For the treatment of generalized cartilage loss in osteoarthritis, an alternative delivery strategy would be more appropriate. The purpose of this study was to assess the safety and efficacy of intra-articular injection of autologous adipose tissue derived MSCs (AD-MSCs) for knee osteoarthritis. We enrolled 18 patients with osteoarthritis of the knee and injected AD MSCs into the knee. The phase I study consists of three dose-escalation cohorts; the low-dose (1.0 × 10(7) cells), mid-dose (5.0 × 10(7)), and high-dose (1.0 × 10(8)) group with three patients each. The phase II included nine patients receiving the high-dose. The primary outcomes were the safety and the Western Ontario and McMaster Universities Osteoarthritis index (WOMAC) at 6 months. Secondary outcomes included clinical, radiological, arthroscopic, and histological evaluations. There was no treatment-related adverse event. The WOMAC score improved at 6 months after injection in the high-dose group. The size of cartilage defect decreased while the volume of cartilage increased in the medial femoral and tibial condyles of the high-dose group. Arthroscopy showed that the size of cartilage defect decreased in the medial femoral and medial tibial condyles of the high-dose group. Histology demonstrated thick, hyaline-like cartilage regeneration. These results showed that intra-articular injection of 1.0 × 10(8) AD MSCs into the osteoarthritic knee improved function and pain of the knee joint without causing adverse events, and reduced cartilage defects by regeneration of hyaline-like articular cartilage.

In vivo differentiation of undifferentiated human adipose tissue-derived mesenchymal stem cells in critical-sized calvarial bone defects.

Adult stem cells have recently drawn considerable attention for potential cell therapy applications. However, critical details about their specific in vivo environments and cellular activities are unclear. Adipose tissue-derived mesenchymal stem cells (ASCs) are attractive candidates for treating bone defects, but most studies focus on delivery of in vitro-differentiated cells. We assessed various scaffolding materials for the ability to support osteogenic differentiation of undifferentiated human ASCs in vivo, in athymic nude rat calvaria. Twenty-four 9- to 10-week-old athymic nude Sprague-Dawley rats (250 g) were used in these experiments. Fat tissue from 3 patients was harvested from abdominal tissue discarded during reconstructive breast surgery by transverse rectus abdominis myocutaneous flap, performed at the Asan Medical Center after resection of breast cancer. Human ASCs were extracted from discarded adipose tissue and isolated based on standard International Society for Cellular Therapy protocols. Adipose tissue-derived mesenchymal stem cells were seeded on polylactic glycolic acid, atelocollagen, and hydroxyapatite scaffolds, and osteogenesis was evaluated using bone mineral densitometry, histology, immunohistochemistry, and reverse transcription polymerase chain reaction. The gross appearance of scaffolds seeded with ASCs was strikingly different from that of scaffolds alone. Bone mineral densitometry analysis revealed a 2- to 3-fold increase in mineral density in ASC-seeded scaffolds. In addition, undifferentiated ASCs seeded onto hydroxyapatite scaffolds, but not onto collagen or polylactic glycolic acid scaffolds, expressed human messenger RNA for osteogenic markers such as alkaline phosphatase, osteopontin, osteocalcin, and osteonectin. These results indicate that undifferentiated human ASCs can differentiate into osteocytes or osteoblasts in athymic nude rat calvaria, and the importance of appropriate scaffolding for in vivo ASC differentiation.

Reversal of serologic, immunologic, and histologic dysfunction in mice with systemic lupus erythematosus by long-term serial adipose tissue-derived mesenchymal stem cell transplantation.

OBJECTIVE: To investigate the efficacy of human adipose tissue-derived mesenchymal stem cell (AD-MSC) transplantation in systemic lupus erythematosus (SLE) and to determine the optimal transplantation window for stem cells either before or after disease onset. METHODS: (NZB×NZW)F1 mice with SLE were administered human AD-MSCs (5×10(5)) intravenously every 2 weeks from age 6 weeks until age 60 weeks, while the control group received saline vehicle on the same schedule. Another experiment was carried out with a different initiation time point for serial transplantation (age 6 weeks or age 32 weeks). RESULTS: Long-term serial administration (total of 28 times) of human AD-MSCs ameliorated SLE without any adverse effects. Compared with the control group, the human AD-MSC-treated group had a significantly higher survival rate with improvement of histologic and serologic abnormalities and immunologic function, and also had a decreased incidence of proteinuria. Anti-double-stranded DNA antibodies and blood urea nitrogen levels decreased significantly with transplantation of human AD-MSCs, and serum levels of granulocyte-macrophage colony-stimulating factor, interleukin-4 (IL-4), and IL-10 increased significantly. A significant increase in the proportion of CD4+FoxP3+ cells and a marked restoration of capacity for cytokine production were observed in spleens from the human AD-MSC-treated group. In the second experiment, an early stage treatment group showed better results (higher survival rates and lower incidence of proteinuria) than an advanced stage treatment group. CONCLUSION: Serial human AD-MSC transplantation had beneficial effects in the treatment of SLE, without adverse effects. Transplantation of human AD-MSCs before disease onset was preferable for amelioration of SLE and restoration of immune homeostasis.

Clinical application of human adipose tissue-derived mesenchymal stem cells in progressive hemifacial atrophy (Parry-Romberg disease) with microfat grafting techniques using 3-dimensional computed tomography and 3-dimensional camera.

BACKGROUND: Parry-Romberg disease is a rare condition that results in progressive hemifacial atrophy, involving the skin, dermis, subcutaneous fat, muscle, and, finally, cartilage and bone. Patients have been treated with dermofat or fat grafts or by microvascular free flap transfer. We hypothesized that adipose-derived stem cells (ASCs) may improve the results of microfat grafting through enhancing angiogenesis. We evaluated the utility of ASC in microfat grafting of patients with Parry-Romberg disease by measuring the change in the hemifacial volumes after injection of ASCs with microfat grafts or microfat grafts alone. METHODS: In April 2008, this investigation was approved by the Korean Food and Drug Administration and the institutional review board of the Asan Medical Center (Seoul, Korea) that monitor investigator-initiated trials. Between May 2008 and January 2009, 10 volunteers with Parry-Romberg disease (5 men and 5 women; mean age, 28 y) were recruited; 5 received ASC and microfat grafts and 5 received microfat grafts only. The mean follow-up period was 15 months. Adipose-derived stem cells were obtained from abdominal fat by liposuction and were cultured for 2 weeks. On day 14, patients were injected with fat grafts alone or plus (in the test group) 1 × 10 ASCs. Patients were evaluated postoperatively using a 3-dimensional camera and 3-dimensional CT scans, and grafted fat volumes were objectively calculated. RESULTS: Successful outcomes were evident in all 5 patients receiving microfat grafts and ASCs, and the survival of grafted fat was better than in patients receiving microfat grafts alone. Before surgery, the mean difference between ipsilateral and contralateral hemiface volume in patients receiving microfat grafts and ASCs was 21.71 mL decreasing to 4.47 mL after surgery. Overall resorption in this ASC group was 20.59%. The mean preoperative difference in hemiface volume in those receiving microfat grafts alone was 8.32 mL decreasing to 3.89 mL after surgery. Overall resorption in this group was 46.81%. The preoperative and postoperative volume differences between the groups was statistically significant (P = 0.002; random-effects model [SAS 9.1]). CONCLUSIONS: Adipose-derived stem cells enhance the survival of fat grafted into the face. A microfat graft with simultaneous ASC injection may be used to treat Parry-Romberg disease without the need for microvascular free flap transfer.

Zika Virus Infection During Research Vaccine Development: Investigation of the Laboratory-Acquired Infection via Nanopore Whole-Genome Sequencing.

Zika virus (ZIKV) emerged as a serious public health problem since the first major outbreak in 2007. Current ZIKV diagnostic methods can successfully identify known ZIKV but are impossible to track the origin of viruses and pathogens other than known ZIKV strains. We planned to determine the ability of Whole Genome Sequencing (WGS) in clinical epidemiology by evaluating whether it can successfully detect the origin of ZIKV in a suspected case of laboratory-acquired infection (LAI). ZIKV found in the patient sample was sequenced with nanopore sequencing technology, followed by the production of the phylogenetic tree, based on the alignment of 38 known ZIKV strains with the consensus sequence. The closest viral strain with the consensus sequence was the strain used in the laboratory, with a percent identity of 99.27%. We think WGS showed its time-effectiveness and ability to detect the difference between strains to the level of a single base. Additionally, to determine the global number of LAIs, a literature review of articles published in the last 10 years was performed, and 53 reports of 338 LAIs were found. The lack of a universal reporting system was worrisome, as in the majority of cases (81.1%), the exposure route was unknown.

Transplantation of CTLA4Ig gene-transduced adipose tissue-derived mesenchymal stem cells reduces inflammatory immune response and improves Th1/Th2 balance in experimental autoimmune thyroiditis.

BACKGROUND: Autoimmune thyroiditis is one of common organ-specific autoimmune disease. The aim of this study was to observe the effect of adipose tissue derived mesenchymal stem cells (ATMSC) and CTLA4Ig gene-transduced ATMSC on autoimmune thyroiditis. METHODS: Experimental autoimmune thyroiditis was induced by immunization with thyroglobulin. Animals were divided into three groups: (i) a half million of human ATMSC, (ii) a half million of murine CLTA4Ig gene-transduced human ATMSC (CTLA4Ig-MSC), or (iii) normal saline (as control), which were administered intravenously four times within a 3-week period. Blood and tissue samples were collected 1 week after the last cell transplantation. RESULTS: The absorbance of serum thyroglobulin autoantibody (TgAA) in the CTLA4Ig-MSC group was considerably lower than those in other groups. In culture supernatant of LPS-stimulated spleen cells, both of the MSC-treated groups showed significantly lower absorbances of TgAA than the control. Flow cytometric analysis of spleen cells revealed a significant decrease in the proportion of CD3+ and CD11b in the CTLA4Ig-MSC group compared to the other groups. Lymphocyte infiltration in the thyroid glands was also dramatically decreased in both of MSC-treated groups. Cytokine analysis showed that ATMSC decreased the production of proinflammatory cytokines and improved the Th1/Th2 balance by down-regulating Th1 cytokines. CONCLUSION: Although CTLA4Ig-MSC transplantation had better result in reduction of serum TgAA, both of ATMSC and CTLA4Ig-MSC transplantations are promising treatments for autoimmune thyroiditis judging from the results of histopathology and cytokine analysis. They may be attractive candidates for treating organ-specific autoimmune disease.

Stem cell treatment for patients with autoimmune disease by systemic infusion of culture-expanded autologous adipose tissue derived mesenchymal stem cells.

Prolonged life expectancy, life style and environmental changes have caused a changing disease pattern in developed countries towards an increase of degenerative and autoimmune diseases. Stem cells have become a promising tool for their treatment by promoting tissue repair and protection from immune-attack associated damage. Patient-derived autologous stem cells present a safe option for this treatment since these will not induce immune rejection and thus multiple treatments are possible without any risk for allogenic sensitization, which may arise from allogenic stem cell transplantations. Here we report the outcome of treatments with culture expanded human adipose-derived mesenchymal stem cells (hAdMSCs) of 10 patients with autoimmune associated tissue damage and exhausted therapeutic options, including autoimmune hearing loss, multiple sclerosis, polymyotitis, atopic dermatitis and rheumatoid arthritis. For treatment, we developed a standardized culture-expansion protocol for hAdMSCs from minimal amounts of fat tissue, providing sufficient number of cells for repetitive injections. High expansion efficiencies were routinely achieved from autoimmune patients and from elderly donors without measurable loss in safety profile, genetic stability, vitality and differentiation potency, migration and homing characteristics. Although the conclusions that can be drawn from the compassionate use treatments in terms of therapeutic efficacy are only preliminary, the data provide convincing evidence for safety and therapeutic properties of systemically administered AdMSC in human patients with no other treatment options. The authors believe that ex-vivo-expanded autologous AdMSCs provide a promising alternative for treating autoimmune diseases. Further clinical studies are needed that take into account the results obtained from case studies as those presented here.

Effects of expanded human adipose tissue-derived mesenchymal stem cells on the viability of cryopreserved fat grafts in the nude mouse.

Adipose-derived mesenchymal stem cells (AdMSCs) augment the ability to contribute to microvascular remodeling in vivo and to modulate vascular stability in fresh fat grafts. Although cryopreserved adipose tissue is frequently used for soft tissue augmentation, the viability of the fat graft is poor. The effects of culture-expanded human adipose tissue-derived mesenchymal stem cells (hAdMSCs) on the survival and quality of the cryopreserved fat graft were determined. hAdMSCs from the same donor were mixed with fat tissues cryopreserved at -70 °C for 8 weeks and injected subcutaneously into 6-week-old BALB/c-nu nude mice. Graft volume and weight were measured, and histology was evaluated 4 and 15 weeks post-transplantation. The hAdMSC-treated group showed significantly enhanced graft volume and weight. The histological evaluation demonstrated significantly better fat cell integrity compared with the vehicle-treated control 4 weeks post-transplantation. No significant difference in graft weight, volume, or histological parameters was found among the groups 15 weeks post-transplantation. The hAdMSCs enhanced the survival and quality of transplanted cryopreserved fat tissues. Cultured and expanded hAdMSCs have reconstructive capacity in cryopreserved fat grafting by increasing the number of stem cells.

Effects of GM-CSF gene transfer using silica-nanoparticles as a vehicle on white blood cell production in dogs.

OBJECTIVE: We sought to test two concepts: that nanoparticles can be used for in vivo gene delivery and that canine granulocyte-macrophage colony-stimulating factor (GM-CSF)/nanoparticles can have possibility to be used to treat transient (acute) canine leukopenia. MATERIALS AND METHODS: We have generated a novel fluorescent-silica nanoparticle binding of canine GM-CSF gene; canine GM-CSF gene was inserted between the cytomegalovirus promoter and poly-adenylation sequences of simian virus 40, and the gene construct was ligated to fluorescent silica nanoparticles functionalized with tertiary amine. RESULTS: When the GM-CSF/nanoparticles were injected into normal dogs, the GM-CSF was expressed in peripheral blood mononuclear cells for at least 9 days and there were significant increases in white blood cell counts, as confirmed by complete blood count, differential count, and flow cytometry. Significant increases in expression of major histocompatibility complex class II on granulocytes and in serum GM-CSF were also observed. Readministration of the nanoparticles was also effective and expression in various tissues was confirmed by reverse transcriptase polymerase chain reaction. CONCLUSIONS: These GM-CSF/nanoparticles may be useful for correction of acute leukopenia, such as chemotherapy-induced myelosuppression without developing neutralizing antibodies.

Preventive and therapeutic effects of gene therapy using silica nanoparticles-binding of GM-CSF gene on white blood cell production in dogs with leukopenia.

OBJECTIVE: Our previous study has shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) gene/silica nanoparticles have a leukocytosis effect in normal dogs. Therefore, this study was conducted to determine whether treatment of canine GM-CSF gene/silica nanoparticles has preventive or therapeutic effects in dogs with leukopenia. MATERIALS AND METHODS: To induce leukopenia, vinblastine was administered intravenously at a dose of 2 mg/m(2) of body surface area on day 0. Then 7.5 microg GM-CSF/nanoparticles (1:100, w/w) were administered intravenously to each of four dogs in the prevention group on day 2 and an equivalent amount of GM-CSF/nanoparticles was administered to the post-nadir group on day 4 (other groups were administered phosphate-buffered saline intravenously). RESULTS: Therapeutic GM-CSF gene was expressed in peripheral blood mononuclear cells for 10 days and both the prevention and post-nadir groups showed significant increases in white blood cell counts when compared with the control group, as confirmed by complete blood count, differential count, and flow cytometry. CONCLUSIONS: GM-CSF/nanoparticles can be useful for correction of acute leukopenia, such as chemotherapy-induced myelosuppression, without developing neutralizing antibodies.

The effect of gene therapy using CTLA4Ig/silica-nanoparticles on canine experimental autoimmune thyroiditis.

BACKGROUND: The present study aimed to determine the effect of canine CTLA4Ig on canine autoimmune thyroiditis. In a previous study, we established a canine model of autoimmune thyroiditis by immunizing normal dogs with bovine thyroglobulin. An in vitro study using recombinant CTLA4Ig revealed that this protein can inhibit the expression of Th1-type cytokines and the pro-inflammatory cytokines tested. METHODS: As a result of the in vitro study, we constructed therapeutic CTLA4Ig/silica-nanoparticles and applied them to the treatment of experimentally induced canine autoimmune thyroiditis. RESULTS: Gene therapy resulted in significant reductions in anti-canine-thyroglobulin autoantibody titer, anti-T4 antibody titer and T-cell proliferation against thyroglobulin and in the mRNA expressions of interleukin-18 in fresh peripheral blood mononuclear cells (PBMC) from all dogs. There was also a significant reduction compared to day 0 in tumor necrosis factor-alpha and interferon-gamma levels in the supernatant from cultured PBMC. CONCLUSIONS: The CTLA4Ig-induced suppression of Th1 cytokines is relatively more significant than it appears because autoimmune thyroiditis is a Th1-polarized disease. Thus, CTLA4Ig can improve Th1/Th2 cytokine balance in autoimmune thyroiditis by downregulating Th1 cytokines.

In vitro and in vivo gene therapy with CMV vector-mediated presumed dog beta-nerve growth factor in pyridoxine-induced neuropathy dogs.

Due to the therapeutic potential of gene therapy for neuronal injury, many studies of neurotrophic factors, vectors, and animal models have been performed. The presumed dog beta-nerve growth factor (pdbeta-NGF) was generated and cloned and its expression was confirmed in CHO cells. The recombinant pdbeta-NGF protein reacted with a human beta-NGF antibody and showed bioactivity in PC12 cells. The pdbeta-NGF was shown to have similar bioactivity to the dog beta-NGF. The recombinant pdbeta-NGF plasmid was administrated into the intrathecal space in the gene therapy group. Twenty-four hours after the vector inoculation, the gene therapy group and the positive control group were intoxicated with excess pyridoxine for seven days. Each morning throughout the test period, the dogs' body weight was taken and postural reaction assessments were made. Electrophysiological recordings were performed twice, once before the experiment and once after the test period. After the experimental period, histological analysis was performed. Dogs in the gene therapy group had no weight change and were normal in postural reaction assessments. Electrophysiological recordings were also normal for the gene therapy group. Histological analysis showed that neither the axons nor the myelin of the dorsal funiculus of L4 were severely damaged in the gene therapy group. In addition, the dorsal root ganglia of L4 and the peripheral nerves (sciatic nerve) did not experience severe degenerative changes in the gene therapy group. This study is the first to show the protective effect of NGF gene therapy in a dog model.

Cloning, expression and bioassay of canine CTLA4Ig.

Blockade of the B7:CD28 costimulatory pathway has been shown to inhibit humoral immunity, graft rejection, graft versus host disease and ameliorate autoimmune diseases. A soluble chimeric fusion protein, CTLA4Ig, binds to B7 with greater affinity than CD28 and blocks the binding of CD28 to B7. We describe the cloning and expression of canine CTLA4Ig, a recombinant chimeric fusion protein composed of the extracellular domain of canine CTLA-4 and the CH2-CH3 domains of canine immunoglobulin alpha constant region (IGHA) genes, linked via an immunologically inert flexible peptide. The recombinant CTLA4Ig protein of approximately 45kDa molecular weight was expressed mainly as insoluble inclusion bodies in Escherichia coli. The protein was solubilized in denaturing buffer and purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography followed by refolding. The yield was about 6mg of recombinant CTLA4Ig per liter of culture. The purified protein was biologically active in one-way mixed lymphocyte reactions, demonstrating immunosuppressive activities in a dose-dependent manner. The findings suggest that recombinant canine CTLA4Ig protein could be valuable in assessing the function of CTLA-4 in the canine immune system and may be effective in autoimmune disease therapy.

Mesenchymal stem cell transplantation can restore lupus disease-associated miRNA expression and Th1/Th2 ratios in a murine model of SLE.

C3.MRL-Faslpr/J mice spontaneously develop high titers of anti-dsDNA, mild glomerular nephritis, and severe lymphoproliferation symptoms. This study aimed to compare the effects of long-term serial administration of human adipose tissue-derived mesenchymal stem cells (ASCs), and cyclophosphamide treatment in C3.MRL-Faslpr/J mice using a murine SLE model. C3.MRL-Faslpr/J mice were divided into saline (C), cyclophosphamide (Y), and ASC (H) treatment groups. Background-matched control C3H mice treated with saline (N) were also compared. The Y group showed the greatest improvement in disease parameters, but with damaged trabecular integrity. ASC transplantation reduced anti-dsDNA levels, glomerular C3 deposition and CD138 proportion significantly, without trabecular damage. Furthermore, both cyclophosphamide and ASC treatment significantly decreased the ratio of Th1/Th2 compared with the saline-treatment. The expression levels of miR-31-5p, miR-96-5p, miR-182-5p, miR-183-5p, and miR-379-5p were significantly higher, while those of miR150-5p were significantly lower in the C group than in the N group. The expression levels of miR-96-5p, miR-182-5p in the Y and H groups were significantly lower than in the C group. Thus, treatment with cyclophosphamide or ASC can change miRNAs and decrease miR-96-5p and miR-182-5p expression, as well as decreasing the CD138 proportion and the Th1/Th2 ratio, which might be involved in the therapeutic mechanism.

Comparative Efficacies of Long-Term Serial Transplantation of Syngeneic, Allogeneic, Xenogeneic, or CTLA4Ig-Overproducing Xenogeneic Adipose Tissue-Derived Mesenchymal Stem Cells on Murine Systemic Lupus Erythematosus.

Allogeneic and xenogeneic transplantation are suitable alternatives for treating patients with stem cell defects and autoimmune diseases. The purpose of this study was to compare the effects of long-term serial transplantation of adipose tissue-derived mesenchymal stem cells (ASCs) from (NZB × NZW) F1 mice (syngeneic), BALB/c mice (allogeneic), or humans (xenogeneic) on systemic lupus erythematosus (SLE). The effects of transplanting human ASCs overproducing CTLA4Ig (CTLA4Ig-hASC) were also compared. Animals were divided into five experimental groups, according to the transplanted cell type. Approximately 500,000 ASCs were administered intravenously every 2 weeks from 6 to 60 weeks of age to all mice except for the control mice, which received saline. The human ASC groups (hASC and CTLA4Ig-hASC) showed a 13-week increase in average life spans and increased survival rates and decreased blood urea nitrogen, proteinuria, and glomerular IgG deposition. The allogeneic group also showed higher survival rates compared to those of the control, up to 40, 41, 42, 43, 44, 45, 52, and 53 weeks of age. Syngeneic ASC transplantation did not accelerate the mortality of the mice. The mean life span of both the syngeneic and allogeneic groups was prolonged for 6-7 weeks. Both human ASC groups displayed increased serum interleukin-10 and interleukin-4 levels, whereas both mouse ASC groups displayed significantly increased GM-CSF and interferon-γ levels in the serum. The strongest humoral immune response was induced by xenogeneic transplantation, followed by allogeneic, CTLA4Ig-xenogeneic, and syngeneic transplantations. Long-term serial transplantation of the ASCs from various sources displayed different patterns of cytokine expression and humoral responses, but all of them increased life spans in an SLE mouse model.

Effects of Transplantation of CTLA4Ig-Overexpressing Adipose Tissue-Derived Mesenchymal Stem Cells in Mice With Sustained Severe Rheumatoid Arthritis.

CTLA4Ig has therapeutic potential for rheumatoid arthritis patients unresponsive to methotrexate (MTX) or TNF-α blockers. However, recombinant CTLA4Ig proteins are short acting and expensive. Adipose tissue-derived mesenchymal stem cells (ASCs) present an ideal stem cell source for practical regenerative medicine due to their abundant availability and their beneficial properties including immunomodulation, homing activity, paracrine effects, and differentiation ability. Therefore, we aimed to determine whether CTLA4Ig and human ASCs show synergistic effects on immunomodulation and clinical improvement of sustained severe rheumatoid arthritis in a mouse model. hASCs overexpressing CTLA4Ig (CTLA4Ig-hASC) were serially transplanted into mice with collagen-induced arthritis. Arthritic mice were subjected to four treatments based on their arthritis score on day 62 postimmunization: control (C group), hASC (H group), CTLA4Ig-hASC (CT group), and MTX (MTX group). A group of healthy mice was used as a normal control (N). Mice in the N and C groups were infused with 150 µl saline, and 2 × 10(6) hASCs or CTLA4Ig-hASCs in 150 µl of saline were intravenously administered to those in the H and CT groups, respectively, on days 63, 70, 77, and 84 after CII immunization. About 1 mg/kg of methotrexate was intraperitoneally administered to the MTX group three times a week for 4 weeks. Serial hASC and CTLA4Ig-hASC transplantation modulated various cytokines and chemokines related to the development of rheumatoid arthritis. Both treatments protected against destruction of cartilage, with CTLA4Ig-hASCs being most effective. Serum levels of CII autoantibodies and C-telopeptide of type II collagen were significantly low in the group transplanted with CTLA4Ig-hASCs. In vitro, ASC and CTLA4Ig-hASC treatment significantly decreased T-bet and GATA-3 expression in splenocytes from arthritic mice, and CTLA4Ig-hASC treatment significantly increased the ratio of Treg/Th17 (CD4(+)CD25(+)FoxP3(+)/CD4(+)CD25(+)RORγt) cells. Serial hASC and CTLA4Ig-hASC transplantation offers promising treatment for rheumatoid arthritis, and CTLA4Ig-hASCs showed stronger therapeutic effects than nontransduced hASCs.

Gene therapy using non-viral peptide vector in a canine systemic lupus erythematosus model.

Although viral vectors are commonly used for therapeutic gene delivery, their applications are limited due to their specific cell membrane receptor-mediated infection and host immune response. In the present study, we constructed a non-viral peptide vector and applied it in the treatment of experimentally induced systemic lupus erythematosus-like disease in dogs. For therapeutic gene construction, the extracellular domain of canine CTLA-4, and the CH2-CH3 domains of canine immunoglobulin alpha constant region were inserted between the cytomegalovirus promoter and poly-adenylation sequence of bovine growth hormone. The constructed therapeutic gene was ligated to the non-viral synthetic peptide vector and was applied to systemic lupus erythematosus-like disease induced dogs. After gene therapy, clinical signs of systemic lupus erythematosus were reduced dramatically: the anti-nuclear antibody titers and urine protein/creatinine ratios were recovered to normal values, and the skin regained its normal histological features. The peptide vector did not show either tissue specific tropism or host induced immune response.

Molecular cloning of the cDNA of canine homeodomain-interacting protein kinase 2.

The research of p53 is being conducted to find the mechanisms of tumorigenesis and to treat various cancers. Homeodomain-interacting protein kinase2 (HIPK2) is an important factor to regulate p53 and to increase the stability of p53. Activation of HIPK2 leads to the selective phosphorylation of p53, resulting in growth arrest and the enhancement of apoptosis. In this study, the canine HIPK2 cDNA fragments were obtained, and their overlapping regions were aligned to give a total sequence of 3489 bp. The canine HIPK2 cDNA (GenBank accession number; AY800385) shares 93% and 90% sequence identity with those of human and mouse HIPK2, respectively. The canine HIPK2 cDNA contains an open reading frame encoding 1163 amino acid residues and the predicted amino acid sequence has 98% and 96% identity with those of human and mouse, respectively. The deduced amino acid sequence of canine HIPK2 has also all domains' sites compared with human and mouse HIPK2. Therefore, these structural similarities suggested that the canine HIPK2 shares the basic biological functions that HIPK2 exhibit in other species.

Transplanted Human Amniotic Epithelial Cells Secrete Paracrine Proangiogenic Cytokines in Rat Model of Myocardial Infarction.

Human amniotic epithelial cells (h-AECs) have been shown to differentiate into cardiomyocyte-like cells in vivo that can regenerate myocardial tissue and improve cardiac function in a rat model of myocardial infarction (MI). In this study, we investigated the paracrine factors released from h-AECs under hypoxic conditions to elucidate the possible mechanisms underlying this previously reported phenomenon of h-AEC-mediated cardiac repair. We used hypoxic cell culture conditions to simulate myocardial infarction in vitro. In comparison to normal conditions, we found that h-AECs secreted higher levels of several cytokines, including angiogenin (ANG), epidermal growth factor (EGF), interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1. To determine whether transplanted h-AECs express these proangiogenic cytokines in vivo, we ligated the coronary artery of rats to cause MI and injected either h-AECs or saline into the infarcted area. We found that the infarct and border zones of rat myocardium treated with h-AECs had higher expression levels of the human-origin cytokines ANG, EGF, IL-6, and MCP-1 compared to the tissues of saline-treated rats. In conclusion, h-AECs secreted proangiogenic cytokines in a rat model of MI, which may suggest that the paracrine effect by h-AECs could regenerate myocardial tissue and improve cardiac function.

Transplantation of Adipose Tissue-Derived Mesenchymal Stem Cells Prevents the Development of Lupus Dermatitis.

MRL/lpr mice spontaneously develop high titers of anti-dsDNA antibodies and symptoms such as glomerular nephritis and organ weight gain. They also develop spontaneous skin inflammation similar to the cutaneous lesions common in human lupus erythematosus. This study aimed to compare the effects of long-term serial administration of human adipose tissue-derived mesenchymal stem cells (ASCs), CTLA4Ig-overexpressing ASCs, and cyclophosphamide treatment in MRL/lpr mice. MRL/lpr mice were divided into saline (C), cyclophosphamide (Y), ASC early (E), ASC late (L), and CTLA4Ig-overexpressing ASC (CT) treatment groups. Background-matched control MRL/MPJ mice treated with saline (N) were also compared. The treatment period was 5-23 weeks, except for the L group (15-23 weeks). Blood and tissue samples were collected when the mice were 24 weeks old. Organ weight, anti-dsDNA antibodies, urine protein, skin and kidney histologic abnormalities, and trabecular bone volume were evaluated. The Y group showed the greatest decrease in anti-dsDNA antibodies, organ weight, degree of kidney inflammation and glomerular infiltration of C3, and incidence rate of severe proteinuria; the E, L, and CT treatment groups showed better results than the C group. ASC transplantation reduced anti-dsDNA antibody levels significantly. Mice treated with ASCs or CTLA4Ig-ASCs starting from the early disease stage did not show dermatitis upon gross examination; they demonstrated significant improvement in hyperkeratosis, acanthosis, and inflammatory cell infiltration scores in histopathology. Micro-CT analysis revealed that cyclophosphamide treatment significantly decreased bone volume and increased bone spacing in the trabecular bone. Thus, we found that ASC and CTLA4-ASC treatments prevent lupus dermatitis development in MRL/lpr mice without adverse effects.