Long-Term Results of Intracardiac Mesenchymal Stem Cell Transplantation in Patients With Cardiomyopathy.

BACKGROUND: Mesenchymal stem cells (MSCs), which have the potential to differentiate into cardiomyocytes or vascular endothelial cells, have been used clinically as therapy for cardiomyopathy. In this study, we aimed to evaluate the long-term follow-up results.Methods and Results:We studied 8 patients with symptomatic heart failure (HF) on guideline-directed therapy (ischemic cardiomyopathy, n=3; nonischemic cardiomyopathy, n=5) who underwent intracardiac MSC transplantation using a catheter-based injection method between May 2004 and April 2006. Major adverse events and hospitalizations were investigated up to 10 years afterward. Compared with baseline, there were no significant differences in B-type natriuretic peptide (BNP) (from 211 to 173 pg/mL), left ventricular ejection fraction (LVEF) (from 24% to 26%), and peak oxygen uptake (from 16.5 to 19.2 mL/min/kg) at 2 months. During the follow-up period, no patients experienced serious adverse events such as arrhythmias. Three patients died of pneumonia in the 1st year, liver cancer in the 6th year, and HF in the 7th year. Of the remaining 5 patients, 3 patients were hospitalized for exacerbated HF, 1 of whom required heart transplantation in the 2nd year; 2 patients survived for 10 years without worsening HF. CONCLUSIONS: The results of this exploratory study of intracardiac MSCs administration suggest further research regarding the feasibility and efficacy is warranted.

Reg Gene Expression in Periosteum after Fracture and Its In Vitro Induction Triggered by IL-6.

The periosteum is a thin membrane that surrounds the outer surface of bones and participates in fracture healing. However, the molecular signals that trigger/initiate the periosteal reaction are not well established. We fractured the rat femoral bone at the diaphysis and fixed it with an intramedullary inserted wire, and the expression of regenerating gene (Reg) I, which encodes a tissue regeneration/growth factor, was analyzed. Neither bone/marrow nor muscle showed RegI gene expression before or after the fracture. By contrast, the periosteum showed an elevated expression after the fracture, thereby confirming the localization of Reg I expression exclusively in the periosteum around the fractured areas. Expression of the Reg family increased after the fracture, followed by a decrease to basal levels by six weeks, when the fracture had almost healed. In vitro cultures of periosteal cells showed no Reg I expression, but the addition of IL-6 significantly induced Reg I gene expression. The addition of IL-6 also increased the cell number and reduced pro-apoptotic gene expression of Bim. The increased cell proliferation and reduction in Bim gene expression were abolished by transfection with Reg I siRNA, indicating that these IL-6-dependent effects require the Reg I gene expression. These results indicate the involvement of the IL-6/Reg pathway in the osteogenic response of the periosteum, which leads to fracture repair.

Biomineral/Agarose Composite Gels Enhance Proliferation of Mesenchymal Stem Cells with Osteogenic Capability.

Hydroxyapatite (HA) or calcium carbonate (CaCO3) formed on an organic polymer of agarose gel is a biomaterial that can be used for bone tissue regeneration. However, in critical bone defects, the regeneration capability of these materials is limited. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming osteoblasts. In this study, we loaded MSCs on HA- or CaCO3-formed agarose gel and cultured them with dexamethasone, which triggers the osteogenic differentiation of MSCs. High alkaline phosphatase activity was detected on both the HA- and CaCO3-formed agarose gels; however, basal activity was only detected on bare agarose gel. Bone-specific osteocalcin content was detected on CaCO3-formed agarose gel on Day 14 of culture, and levels subsequently increased over time. Similar osteocalcin content was detected on HA-formed agarose on Day 21 and levels increased on Day 28. In contrast, only small amounts of osteocalcin were found on bare agarose gel. Consequently, osteogenic capability of MSCs was enhanced on CaCO3-formed agarose at an early stage, and both HA- and CaCO3-formed agarose gels well supported the capability at a later stage. Therefore, MSCs loaded on either HA- or CaCO3-formed agarose could potentially be employed for the repair of critical bone defects.

[Current status of bone/cartilage tissue engineering towards clinical applications].

Osteo/chondrogenic differentiation capabilities are seen after in vivo implantation of mesenchymal stem cells (MSCs), which are currently used for the patients having bone/cartilage defects. Importantly, the differentiation capabilities are induced by culturing technology, resulting in in vitro bone/cartilage formation. Especially, the in vitro bone tissue is useful for bone tissue regeneration. For cartilage regeneration, culture expanded chondrocytes derived from patient's normal cartilage are also used for the patients having cartilage damages. Recently, the cultured chondrocytes embedded in atelocollagen gel are obtainable as tissue engineered products distributed by Japan Tissue Engineering Co. Ltd. The products are available in the well-regulated hospitals by qualified orthopedic surgeons. The criteria for these hospitals/surgeons have been established. This review paper focuses on current status of bone/cartilage tissue engineering towards clinical applications in Japan.

N-Cadherin is a prospective cell surface marker of human mesenchymal stem cells that have high ability for cardiomyocyte differentiation.

Mesenchymal stem cells (MSCs) are among the most promising sources of stem cells for regenerative medicine. However, the range of their differentiation ability is very limited. In this study, we explored prospective cell surface markers of human MSCs that readily differentiate into cardiomyocytes. When the cardiomyogenic differentiation potential and the expression of cell surface markers involved in heart development were analyzed using various immortalized human MSC lines, the MSCs with high expression of N-cadherin showed a higher probability of differentiation into beating cardiomyocytes. The differentiated cardiomyocytes expressed terminally differentiated cardiomyocyte-specific markers such as α-actinin, cardiac troponin T, and connexin-43. A similar correlation was observed with primary human MSCs derived from bone marrow and adipose tissue. Moreover, N-cadherin-positive MSCs isolated with N-cadherin antibody-conjugated magnetic beads showed an apparently higher ability to differentiate into cardiomyocytes than the N-cadherin-negative population. Quantitative polymerase chain reaction analyses demonstrated that the N-cadherin-positive population expressed significantly elevated levels of cardiomyogenic progenitor-specific transcription factors, including Nkx2.5, Hand1, and GATA4 mRNAs. Our results suggest that N-cadherin is a novel prospective cell surface marker of human MSCs that show a better ability for cardiomyocyte differentiation.

Early fixation of cobalt-chromium based alloy surgical implants to bone using a tissue-engineering approach.

To establish the methods of demonstrating early fixation of metal implants to bone, one side of a Cobalt-Chromium (CoCr) based alloy implant surface was seeded with rabbit marrow mesenchymal cells and the other side was left unseeded. The mesenchymal cells were further cultured in the presence of ascorbic acid, ß-glycerophosphate and dexamethasone, resulting in the appearance of osteoblasts and bone matrix on the implant surface. Thus, we succeeded in generating tissue-engineered bone on one side of the CoCr implant. The CoCr implants were then implanted in rabbit bone defects. Three weeks after the implantation, evaluations of mechanical test, undecalcified histological section and electron microscope analysis were performed. Histological and electron microscope images of the tissue engineered surface exhibited abundant new bone formation. However, newly formed bone tissue was difficult to detect on the side without cell seeding. In the mechanical test, the mean values of pull-out forces were 77.15 N and 44.94 N for the tissue-engineered and non-cell-seeded surfaces, respectively. These findings indicate early bone fixation of the tissue-engineered CoCr surface just three weeks after implantation.

Induction of pluripotent stem cells from human third molar mesenchymal stromal cells.

The expression of four transcription factors (OCT3/4, SOX2, KLF4, and MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. We generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without MYC, which is considered as oncogene. Interestingly, some of the clonally expanded MSCs could be used for iPS cell generation with 30-100-fold higher efficiency when compared with that of other clonally expanded MSCs and human dermal fibroblasts. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient clones, suggesting that the processes of chromatin remodeling have important roles in the cascade of iPS cells generation. The generated iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES marker expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that clonally expanded MSCs derived from human third molars are a valuable cell source for the generation of iPS cells.

In vivo study of dendronlike nanoparticles for stem cells "tune-up": from nano to tissues.

The control of stem cell differentiation to obtain osteoblasts in vivo is still regarded as a challenge in stem-cell-based and bone-tissue engineering strategies. Biodegradable dexamethasone-loaded dendron-like nanoparticles (NPs) of carboxymethylchitosan/poly(amidoamine) dendrimer have been proposed as intracellular drug-delivery systems of bioactive molecules. In this study, combination of nanotechnology, stem-cell engineering and tissue engineering is proposed in pre-programming the fate of rat bone marrow stromal cells (RBMSCs) towards osteoblasts cells and development of new bone tissue, in vivo. This work demonstrated that the developed NPs were able to be taken up by RBMSCs, and exhibited a noncytotoxic behavior in vitro. The performance of the developed dendronlike NP system for the intracellular delivery of dexamethasone was investigated by seeding the engineered RBMSCs onto starch-polycaprolactone scaffolds ex vivo, and implanting subcutaneously in the back of Fischer 344/N rats (Syngeneic), in the absence of the typical osteogenic supplements. Favorable results were observed in vivo, thus suggesting that stem cell "tune-up" strategy can open up a new regenerative strategy for bone-tissue engineering. FROM THE CLINICAL EDITOR: In this study, a combination of nanotechnology, stem-cell engineering and tissue engineering is proposed in pre-programming the fate of rat bone marrow stromal cells (RBMSCs) towards osteoblasts cells and development of new bone tissue in vivo.

[Regenerative therapy using allogeneic mesenchymal stem cells].

The use of mesenchymal stem cells (MSC) for tissue and organ regeneration offers advantages because the MSC contain multipotent progenitor cells and reported to be immunoprivileged as well as immunosuppressive. Therefore, cell therapy with allogeneic MSC has been reported as a promising treatment for severe acute graft versus host disease (GVHD). We reported a pilot study for GVHD treatments using a small number of allogeneic MSC. We also reported that MSC can show osteogenic differentiation capability when implanted in vivo as well as cultured in vitro. Based on these findings, we attempted to use allogeneic MSC for the treatment of genetic disorder of hypophosphatasia patient. Present paper summarizes our clinical experiences of allogeneic MSC for the purpose of regenerative medicine.

Safety of Intravenous Autologous Bone Marrow-Derived Mesenchymal Cell Transplantation in 5 Patients With Reduced Left Ventricular Ejection Fraction.

Background: Although intracardiac injection or intracoronary delivery of mesenchymal stem cells (MSCs) has been reported, there have been few studies on the intravenous injection of MSCs, particularly in Japan. Methods and Results: Five patients with left ventricular ejection fraction (LVEF) ≤45% received 1.0×108 MSCs intravenously. The procedure did not induce significant changes in vital signs. One patient had an elevated body temperature after 1 day, but recovered spontaneously. Laboratory tests remained normal for 1 month after cell delivery. Computed tomography was performed after 1-2 years, and there was no evidence of malignancy. Conclusions: In this pilot study of patients with reduced LVEF, intravenous MSC delivery had no adverse effects.

Single intra-arterial injection of mesenchymal stromal cells for treatment of steroid-refractory acute graft-versus-host disease: a pilot study.

BACKGROUND AIMS: Cell therapy with mesenchymal stromal cells (MSC) has been reported recently as a promising treatment for severe acute graft-versus-host disease (GvHD). METHODS: We designed a pilot study to treat severe hepatic or gut GvHD using MSC derived from only the donor and cultured without bovine serum. Because the number of cultured MSC is smaller using this method, we planned to treat patients by intra-arterial regional administration directly to the target organs. RESULTS: Three patients were enrolled, and the MSC could be expanded using donor serum. There were no obvious side-effects immediately after arterial injection. The maximum response was partial in one of three patients and did not continue for more than 2 months. Idiopathic pneumonia syndrome developed in two of the three patients. CONCLUSIONS: A single local arterial MSC injection was unable to save these patients' lives and so might not be more effective than multiple systemic intravenous MSC injection. Further clinical research and additional strategies are required to develop appropriate methods for using MSC to achieve extended remission of GvHD.

Development of a high-specificity enzyme-linked immunosorbent assay (ELISA) system for the quantification and validation of intact rat osteocalcin.

Osteocalcin (OC) exhibits hard tissue-specific expression and binding activity to hydroxyapatite. Therefore, measurement of secreted OC is a very useful index for evaluating osteoblastic differentiation in regenerative bone. In the present study, we established a high-specificity sandwich enzyme-linked immunosorbent assay (ELISA) system for the quantification of intact rat OC, which could be useful for validating tissue-engineered bone samples nondestructively and continuously. The range of detection with the sandwich ELISA system was 0.1-100 ng OC/mL of cell culture media or rat sera. No cross-reactivities were detected with OCs from other species, including human, bovine and mouse OCs, and other mammalian sera, which would contain the corresponding endogenous OCs. The intra- and inter-assay coefficients of variation were < or =4.9% and

Stem cell technology using bioceramics: hard tissue regeneration towards clinical application.

Mesenchymal stem cells (MSCs) are adult stem cells which show differentiation capabilities toward various cell lineages. We have already used MSCs for treatments of osteoarthritis, bone necrosis and bone tumor. For this purpose, culture expanded MSCs were combined with various ceramics and then implanted. Because of rejection response to allogeneic MSC implantation, we have utilized patients' own MSCs for the treatment. Bone marrow is a good cell source of MSCs, although the MSCs also exist in adipose tissue. When comparing osteogenic differentiation of these MSCs, bone marrow MSCs show more extensive bone forming capability than adipose MSCs. Thus, the bone marrow MSCs are useful for bone tissue regeneration. However, the MSCs show limited proliferation and differentiation capabilities that hindered clinical applications in some cases. Recent advances reveal that transduction of plural transcription factors into human adult cells results in generation of new type of stem cells called induced pluripotent stem cells (iPS cells). A drawback of the iPS cells for clinical applications is tumor formation after their in vivo implantation; therefore it is difficult to use iPS cells for the treatment. To circumvent the problem, we transduced a single factor of either SOX2 or NANOG into the MSCs and found high proliferation as well as osteogenic differentiation capabilities of the MSCs. The stem cells could be combined with bioceramics for clinical applications. Here, we summarize our recent technologies using adult stem cells in viewpoints of bone tissue regeneration.

[Genetic basis for skeletal disease. Stem cell therapy for genetic bone disorders].

Mesenchymal stem cells (MSCs) can show osteogenic differentiation capability when implanted in vivo , as well as cultured in vitro; therefore we attempted to use allogeneic MSCs for a patient with hypophosphatasia, which is caused by mutations in tissue non-specific alkaline phosphatase (TNSALP) gene. Donor MSCs were obtained by culture expansion of fresh marrow from the patient's father. Some of the MSCs were further cultured under osteogenic conditions on a culture dish or porous hydroxyapatite ceramics, resulting in cultured osteoblasts and osteogenic constructs, respectively. After traditional bone marrow transplantation, The donor MSCs and osteoblasts were injected into the patient and the constructs were implanted subcutaneously or intraosseous lesions. The patient's respiratory condition improved and donor cells were detected in newly formed bone tissue. These findings showed the importance of allogeneic MSC transplantation for the hypophosphatasia patient.

Molecular cloning and gene expression of the prox1a and prox1b genes in the medaka, Oryzias latipes.

Prox1 is a prospero-related homeobox gene. Prox1 is expressed in various internal organs and is related to those differentiations. Small fishes such as the zebrafish and the medaka are useful model animals in the clarification of the mechanism of development. The zebrafish prox1 is also identified, and it contributes to clarifying the function of prox1. However, it is necessary to note that many genes are duplicated in teleost fishes. In this study, we identified the orthologs of the mammalian prox1 gene in the medaka. The gene was also duplicated in the medaka, and we named it prox1a and prox1b. In silico analysis from the perspective of synteny indicated that medaka prox1a was similar to the prox1 gene of other vertebrates. Medaka prox1a was expressed in all internal organs that we have examined by RT-PCR. In contrast, medaka prox1b expression was limited to the brain, heart, liver, kidney, thymus, gill, testis, and ovary. This suggests that the two prox1 genes do not have a complementary relationship. In addition, we examined their expression patterns during embryonic development using whole-mount in situ hybridization. The expression pattern of prox1a showed a pattern similar to that of zebrafish prox1. In contrast, medaka prox1b was expressed asymmetrically in part of the central nervous system, especially strongly in the right side of the habenula.

New bone formation by allogeneic mesenchymal stem cell transplantation in a patient with perinatal hypophosphatasia.

Mesenchymal stem cells (MSCs) can show osteogenic differentiation capability when implanted in vivo, as well as cultured in vitro; therefore we attempted to use allogeneic MSCs for an 8-month-old patient with hypophosphatasia. MSCs were obtained by culture expansion of fresh marrow from the patient's father. Some of the MSCs were further cultured under osteogenic conditions on a culture dish or porous hydroxyapatite ceramics, resulting in cultured osteoblasts and osteogenic constructs, respectively. The MSCs and osteoblasts were injected into the patient, and the constructs were implanted locally. After traditional bone marrow transplantation, the MSCs, osteoblasts, and osteogenic constructs were used for treatment and to improve the patient's respiratory condition and skeletal abnormality. The condition worsened again, and an MSC booster shot was administered. At the same time, the construct was retrieved. The respiratory condition improved, and the retrieved construct showed de novo bone derived from both donor and patient cells. We demonstrated the importance of allogeneic MSC transplantation for hypophosphatasia and the constructs as an alternative to bone fragments that provided further osteogenic capability in the patient.

In vivo osteogenic capability of human mesenchymal cells cultured on hydroxyapatite and on beta-tricalcium phosphate.

The aim of the current study was to examine in vitro osteogenic capability and in vivo bone formation of mesenchymal stromal cells (MSCs) on two kinds of calcium phosphate ceramics. MSCs derived from human bone marrow were seeded on either hydroxyapatite (HA) ceramic or beta-tricalcium phosphate (beta-TCP) ceramic and then cultured in a medium supplemented with a donor's serum, vitamin C, beta-glycerophosphate, and dexamethasone. The culture revealed the expression of alkaline phosphatase activity, indicating the osteogenic differentiation of the MSCs on the ceramics (fabrication of tissue-engineered construct). The constructs were then implanted subcutaneously into nude rats for 8 weeks. New bone formation was observed in both types of ceramics, and human-specific Alu sequence was detected by in situ hybridization analysis. Quantitative microcomputed tomography showed that the volume of the new bone in the HA ceramic was greater than that in the beta-TCP ceramic in six of seven cases. These results suggest that human MSCs cultured on ceramics could retain their osteogenic capability even after ectopic implantation and provide a rationale for the use of tissue-engineered constructs derived from a patient's MSCs and calcium phosphate ceramics in bone tissue regeneration.

Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease.

Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl4)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl4-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.

Raloxifene: its ossification-promoting effect on female mesenchymal stem cells.

BACKGROUND: Raloxifene acts like estrogen in preventing bone loss in postmenopausal women, but it selectively activates biological responses in bone tissue. It has a direct effect on osteoblasts' differentiation and bone formation in bone marrow culture. However, the point at which raloxifene has an effect on bone marrow-derived mesenchymal stem cells (MSCs), regardless of sex difference, is not known. The purpose of this study was to examine the osteogenic effect of raloxifene on MSCs derived from female and male rats and to assess the sex difference of raloxifene with or without osteogenic supplements (OSs) in the regulation of bone formation. METHODS: Female and male rat bone marrow cells were cultured with or without OSs. In each experimental group, 10-6 M or 10-8 M raloxifene was added. As a control, cells were cultured without raloxifene. Histologically, mineralization was assessed by alizarin red S staining. Biochemically, alkaline phosphatase (ALP) activity, calcium content, and osteocalcin content were assessed. RESULTS: On histological analysis, mineralized nodules were seen on alizarin red S staining in the groups treated with OS. On the biochemical analysis, OS increased ALP activity, calcium content, and osteocalcin content. Among female groups with OSs, 10-6 M raloxifene significantly increased ALP activity, calcium content, and osteocalcin content compared with the controls. Among male groups, raloxifene had negligible effects. CONCLUSIONS: 10-6 M Raloxifene had no ossification-inducing effect on female MSCs, but it had an ossification-promoting effect; it had no osteogenic effect on male MSCs. Therefore, raloxifene has a sex difference with regard to its osteogenic effect on MSCs. Moreover, combined treatment with raloxifene plus OS has an effect on female MSCs. These results provide a useful insight into the possible influence of raloxifene after MSC transplantation in clinical practice.

[Progress of research in osteoarthritis. Tissue engineering therapy for osteoarthritis].

To repair articular cartilage defects, transplantation of various tissue or cells have been investigated. In 1994, autologous chondrocyte implantation was introduced by Brittberg et al. and the technology has been widely applied to repair cartilage defects caused by trauma. However, it is hard to be applied for articular cartilage defects in osteoarthritic patients. Recently, several researchers are trying to treat osteoarthritis using tissue-engineering approaches on the basis of stem cells and scaffold. In this paper, we introduce the trend of the approaches for cartilage defects and refer to the possibility for the treatments of osteoarthritic patients.