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.

Effect of ectopic OCT4 expression on canine adipose tissue-derived mesenchymal stem cell proliferation.

Enhancing the proliferative capacity of mesenchymal stem cells (MSCs) is critical for increasing their therapeutic potential in a variety of diseases. We hypothesized that lentivirus-mediated overexpression of canine octamer-binding transcription factor 4 (OCT4) might influence the proliferation of canine adipose tissue-derived MSCs (cATMSCs). cOCT4-cATMSCs were generated by transducing cATMSCs with a cOCT4-lentiviral vector. Increased expression of cOCT4 was confirmed using RT-PCR and immunoblotting. Immunophenotypic characterization using flow cytometry indicated that the CD29, CD44, CD73, CD90, and CD105 surface markers were highly expressed by both cOCT4- and mock-transduced cATMSCs (mock-cATMSCs), whereas the CD31 and CD45 markers were absent. We performed the osteogenic differentiation assay to evaluate the effects of cOCT4 overexpression on the osteogenic differentiation potential of cATMSCs. The results showed that cOCT4-cATMSCs had a much higher potential for osteogenic differentiation than mock-cATMSCs. Next, the proliferative capacities of cOCT4- and mock-cATMSCs were evaluated using a WST-1 cell proliferation assay and trypan blue exclusion. cOCT4-cATMSCs showed a higher proliferative capacity than mock-cATMSCs. Cell cycle analysis indicated that overexpression of cOCT4 in cATMSCs induced an increase in the proportion of cells in S and G2/M phases. Consistent with this, immunoblot analysis showed that cyclin D1 expression was increased in cOCT4-cATMSCs. In conclusion, our results indicate that lentivirus-mediated overexpression of cOCT4 increased the proliferative capacity of cATMSCs. OCT4-mediated enhancement of cell proliferation may be a useful method for expanding MSC population rapidly without loss of stemness.

Amniotic membrane extract-loaded double-layered wound dressing: evaluation of gel properties and wound healing.

The conservative single-layered wound dressing system is decomposed when mixed in polyvinyl alcohol (PVA) solution, which means it cannot be used with a temperature-sensitive drug. The goal of this investigation was to make an amniotic membrane extract (AME)-loaded double-layered wound dressing with an improved healing result compared to the conservative single-layered wound dressing systems. The double-layered wound dressing was developed with PVA/sodium alginate using a freeze-melting technique; one layer was PVA layer and the other was the drug-loaded sodium alginate layer. Its gel properties were assessed compared to single-layered wound dressings. Moreover, in vivo wound-healing effects and histopathology were calculated compared to commercial products. The double-layered wound dressing gave a similar gel fraction and Young's module as single-layered wound bandages developed with only PVA, and a similar inflammation ability and WVTR as single-layered wound dressings developed with PVA and sodium alginate. Our data indicate that these double-layered wound bandages were just as swellable, but more elastic and stronger than single-layered wound dressings comprised of the same polymers and quantities, possibly giving an acceptable level of moisture and accumulation of exudates in the wound zone. Compared to the commercial product, the double-layered wound dressing comprising 6.7% PVA, 0.5% sodium alginate and 0.01% AME significantly enhanced the wound-healing effect in the wound-healing test. Histological investigations showed that superior full-thickness wound-healing effects compared to the commercial product. Therefore, the double-layered wound dressing would be an outstanding wound-dressing system with improved wound healing and good gel property.

Human adipose tissue-derived mesenchymal stem cells improve cognitive function and physical activity in ageing mice.

Brain ageing leads to atrophy and degeneration of the cholinergic nervous system, resulting in profound neurobehavioral and cognitive dysfunction from decreased acetylcholine biosynthesis and reduced secretion of growth and neurotrophic factors. Human adipose tissue-derived mesenchymal stem cells (ADMSCs) were intravenously (1 × 10(6) cells) or intracerebroventricularly (4 × 10(5) cells) transplanted into the brains of 18-month-old mice once or four times at 2-week intervals. Transplantation of ADMSCs improved both locomotor activity and cognitive function in the aged animals, in parallel with recovery of acetylcholine levels in brain tissues. Transplanted cells differentiated into neurons and, in part, into astrocytes and produced choline acetyltransferase proteins. Transplantation of ADMSCs restored microtubule-associated protein 2 in brain tissue and enhanced Trk B expression and the concentrations of brain-derived neurotrophic factor and nerve growth factor. These results indicate that human ADMSCs differentiate into neural cells in the brain microenvironment and can restore physical and cognitive functions of aged mice not only by increasing acetylcholine synthesis but also by restoring neuronal integrity that may be mediated by growth/neurotrophic factors. © 2013 Wiley Periodicals, Inc.

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.

Therapeutic effect of adipose-derived stem cells and BDNF-immobilized PLGA membrane in a rat model of cavernous nerve injury.

INTRODUCTION: Cavernous nerve injury is the main reason for post-prostatectomy erectile dysfunction (ED). Stem cell and neuroprotection therapy are promising therapeutic strategy for ED. AIM: To evaluate the therapeutic efficacy of adipose-derived stem cells (ADSCs) and brain-derived neurotrophic factor (BDNF) immobilized Poly-Lactic-Co-Glycolic (PLGA) membrane on the cavernous nerve in a rat model of post-prostatectomy ED. Methods. Rats were randomly divided into five groups: normal group, bilateral cavernous nerve crush injury (BCNI) group, ADSC (BCNI group with ADSCs on cavernous nerve) group, BDNF-membrane (BCNI group with BDNF/PLGA membrane on cavernous nerve) group, and ADSC/BDNF-membrane (BCNI group with ADSCs covered with BDNF/PLGA membrane on cavernous nerve) group. BDNF was controlled-released for a period of 4 weeks in a BDNF/PLGA porous membrane system. MAIN OUTCOME MEASURES: Four weeks after the operation, erectile function was assessed by detecting the ratio of intra-cavernous pressure (ICP)/mean arterial pressure (MAP). Smooth muscle and collagen content were determined by Masson's trichrome staining. Neuronal nitric oxide synthase (nNOS) expression in the dorsal penile nerve was detected by immunostaining. Phospho-endothelial nitric oxide synthase (eNOS) protein expression and cyclic guanosine monophosphate (cGMP) level of the corpus cavernosum were quantified by Western blotting and cGMP assay, respectively. RESULTS: In the ADSC/BDNF-membrane group, erectile function was significantly elevated, compared with the BCNI and other treated groups. ADSC/BDNF-membrane treatment significantly increased smooth muscle/collagen ratio, nNOS content, phospho-eNOS protein expression, and cGMP level, compared with the BCNI and other treated groups. CONCLUSIONS: ADSCs with BDNF-membrane on the cavernous nerve can improve erectile function in a rat model of post-prostatectomy ED, which may be used as a novel therapy for post-prostatectomy ED.

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.

Generation of transgenic dogs that conditionally express green fluorescent protein.

We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.

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.

Establishment of efficacy and safety assessment of human adipose tissue-derived mesenchymal stem cells (hATMSCs) in a nude rat femoral segmental defect model.

Human adipose tissue-derived mesenchymal stem cell (hATMSC) have emerged as a potentially powerful tool for bone repair, but an appropriate evaluation system has not been established. The purpose of this study was to establish a preclinical assessment system to evaluate the efficacy and safety of cell therapies in a nude rat bone defect model. Segmental defects (5 mm) were created in the femoral diaphyses and transplanted with cell media (control), hydroxyapatite/tricalcium phosphate scaffolds (HA/TCP, Group I), hATMSCs (Group II), or three cell-loading density of hATMSC-loaded HA/TCP (Group III-V). Healing response was evaluated by serial radiography, micro-computed tomography and histology at 16 weeks. To address safety-concerns, we conducted a GLP-compliant toxicity study. Scanning electron microscopy studies showed that hATMSCs filled the pores/surfaces of scaffolds in a cell-loading density-dependent manner. We detected significant increases in bone formation in the hATMSC-loaded HA/TCP groups compared with other groups. The amount of new bone formation increased with increases in loaded cell number. In a toxicity study, no significant hATMSC-related changes were found in body weights, clinical signs, hematological/biochemical values, organ weights, or histopathological findings. In conclusion, hATMSCs loaded on HA/TCP enhance the repair of bone defects and was found to be safe under our preclinical efficacy/safety hybrid assessment system.

Comparison of Anti-Oxidative Effect of Human Adipose- and Amniotic Membrane-Derived Mesenchymal Stem Cell Conditioned Medium on Mouse Preimplantation Embryo Development.

Oxidative stress is a major cause of damage to the quantity and quality of embryos produced in vitro. Antioxidants are usually supplemented to protect embryos from the suboptimal in vitro culture (IVC) environment. Amniotic membrane-derived mesenchymal stem cells (AMSC) have emerged as a promising regenerative therapy, and their paracrine factors with anti-oxidative effects are present in AMSC conditioned medium (CM). We examined the anti-oxidative potential of human AMSC-CM treatment during IVC on mouse preimplantation embryo development and antioxidant gene expression in the forkhead box O (FoxO) pathway. AMSC-CM (10%) was optimal for overall preimplantation embryo developmental processes and upregulated the expression of FoxOs and their downstream antioxidants in blastocysts (BL). Subsequently, compared to adipose-derived mesenchymal stem cell (ASC)-CM, AMSC-CM enhanced antioxidant gene expression and intracellular GSH levels in the BL. Total antioxidant capacity and SOD activity were greater in AMSC-CM than in ASC-CM. Furthermore, SOD and catalase were more active in culture medium supplemented with AMSC-CM than in ASC-CM. Lastly, the anti-apoptotic effect of AMSC-CM was observed with the regulation of apoptosis-related genes and mitochondrial membrane potential in BL. In conclusion, the present study established AMSC-CM treatment at an optimal concentration as a novel antioxidant intervention for assisted reproduction.

Anti-influenza diarylheptanoids from the bark of Alnus japonica.

This study to investigate anti-influenza components from the bark of Alnus japonica resulted in the isolation of two rare acylated diarylheptanoids, named oregonoyl A (5) and oregonoyl B (6), along with nine known compounds (1-4 and 7-11). Their structures were elucidated on the basis of extensive spectroscopic and chemical methods. Antiviral testing of compounds 1-11 against KBNP-0028 (H9N2) avian influenza virus showed that platyphyllone (10) was strongly active, and platyphyllonol-5-xylopyranoside (9) was moderately active against KBNP-0028 as compared with the positive control, zanamivir, respectively.

Phenolic compounds from Artemisia iwayomogi and their effects on osteoblastic MC3T3-E1 cells.

One new (4) and twelve known phenolic compounds (1-3, 5-13) were isolated from a 70% MeOH extract of the aerial parts of Artemisia iwayomogi. The new compound was identified as 7,8-dimethoxy-coumarin-9-O-(6'-O-(E)-coumaroyl)-beta-D-glucopyranoside (4) and named iwayomin. The effects of compounds 1-13 on the function of osteoblastic MC3T3-E1 cells were examined by evaluating cell viability, alkaline phosphatase (ALP) activity, collagen synthesis, and mineralization in the presence of each compound. Compounds 3, 4, 7, and 9 showed potential in stimulating osteoblastic bone formation and may be useful for the prevention and/or treatment of osteoporosis.

Antioxidative and hepatoprotective diarylheptanoids from the bark of Alnus japonica.

The present study to evaluate the potential of constituents of the bark of Alnus japonica as a functional food with medicinal properties led to the identification of one new diarylheptanoid, named alusenone (1A), and 11 known ones (1B and 2-11). Their antioxidative and hepatoprotective activities were accessed by, respectively, a TOSC assay and a TBH-induced hepatotoxicity rat model. Mixtures 1, 2-6, 10, and 11 showed good antioxidative and hepatoprotective effects as compared with the positive controls.

A new diarylheptanoid from the bark of Alnus japonica.

A new diarylheptanoid, epihirsutanonol (1), was isolated from the bark of Alnus japonica, along with two known ones (2 and 3). Their structures were elucidated on the basis of extensive spectroscopic evidence. The new compound 1 showed significant hepatoprotective activity on the basis of t-butylhydroperoxide-induced hepatocyte injury in vitro assay.

Anti-Oxidative Effects of Human Adipose Stem Cell Conditioned Medium with Different Basal Medium during Mouse Embryo In Vitro Culture.

The quality of embryos produced by assisted reproductive techniques should be advanced by the improvement of in vitro culture conditions for successful implantation and pregnancy maintenance. We investigated the anti-oxidative effect of human adipose stem cell (ASC) conditioned medium with its optimal basal medium, Dulbecco's modified Eagle's medium (DMEM-CM), or keratinocyte serum-free medium (KSFM-CM) as supplements during in vitro culture (IVC) of in vitro fertilized mouse embryo. At first, preimplantation embryo development was evaluated in KSFM-CM and DMEM-CM supplemented cultures at various concentrations. The blastocyst (BL) and hatched BL formation rates were significantly increased in 5% DMEM-CM, while no difference was observed from KSFM-CM. Next, comparing the efficacy of KSFM-CM and DMEM-CM at the same concentration, DMEM-CM enhanced the developmental rate of 16 cells, morula, BL, and hatched BL. The expression level of reactive oxygen species decreased and that of glutathione increased in BL cultured with DMEM-CM, which confirms its anti-oxidative effect. Furthermore, apoptosis in BL cultured with DMEM-CM was reduced compared with that in KSFM-CM. This study demonstrated that the comparative effect of human ASC-CM made of two different basal media during mouse embryo IVC and anti-oxidative effect of 5% DMEM-CM was optimal to improve preimplantation embryo development.

Conditioned Medium from Canine Amniotic Membrane-Derived Mesenchymal Stem Cells Improved Dog Sperm Post-Thaw Quality-Related Parameters.

This study investigated the effects of conditioned medium (CM) from canine amniotic membrane-derived MSCs (cAMSCs) on dog sperm cryopreservation. For this purpose, flow cytometry analysis was performed to characterize cAMSCs. The CM prepared from cAMSCs was subjected to proteomic analysis for the identification of proteins present in the medium. Sperm samples were treated with freezing medium supplemented with 0%, 5%, 10%, and 15% of the CM, and kinetic parameters were evaluated after 4-6 h of chilling at 4 °C to select the best concentration before proceeding to cryopreservation. Quality-related parameters of frozen-thawed sperm were investigated, including motility; kinetic parameters; viability; integrity of the plasma membrane, chromatin, and acrosome; and mitochondrial activity. The results showed that 10% of the CM significantly enhanced motility, viability, mitochondrial activity, and membrane integrity (p < 0.05); however, the analysis of chromatin and acrosome integrity showed no significant differences between the treatment and control groups. Therefore, we concluded that the addition of 10% CM derived from cAMSC in the freezing medium protected dog sperm during the cryopreservation process.

High Frequency of Intravenous Injection of Human Adipose Stem Cell Conditioned Medium Improved Embryo Development of Mice in Advanced Maternal Age through Antioxidant Effects.

Advanced maternal age (AMA) has become prevalent globally. With aging, weakened antioxidant defense causes loss of normal function in the ovary and uterus due to oxidative stress. Here, we aimed to improve embryo development in AMA mice by intravenous injection (IV) of human adipose stem cell conditioned medium (ASC-CM) at various frequencies and intervals as an antioxidant intervention. Four- and six-month-old female ICR (Institute of Cancer Research) mice were randomly divided into groups IV treated with human ASC-CM under different conditions, and in vitro and in vivo embryo development were evaluated. Consequently, compared to the control group, blastocyst formation rate of parthenotes was significantly promoted in 4-month-old mice and the mean number of implanted fetuses after natural mating was significantly increased by approximately two-fold in 6-month-old mice. Through gene analysis, the anti-apoptotic and anti-oxidative effects of human ASC-CMs were confirmed in the ovaries and uterus of pregnant mice at both ages. In particular, ovarian expression of gpx1 and catalase drastically increased in 6-month-old mice. Furthermore, the levels of gpx1 and catalase were further increased, with a high frequency of injection regardless of age. Thus, we demonstrated for the first time the anti-oxidative effect of human ASC-CM administration against ovarian aging and the optimal injection condition.