[Coronary malperfusion due to acute type A aortic dissection; surgical strategy and results].

Coronary malperfusion due to acute type A aortic dissection (DAA) is a lethal complication. It is especially difficult to rescue the patients with left coronary malperfusion because of acute global myocardial infarction (AMI), even with successful surgical treatments, including the replacement of the ascending aorta and coronary artery bypass grafting (CABG). We review our experience and illustrate our approach to these critically ill patients. In addition, we classify the mechanism of malperfusion into 4 types based upon perioperative findings and discuss surgical management indivisually. From January 1990 to April 2005, a total of 260 patients were operated for DAA in our institution. Twenty (7.7%) patients, 11 men and 9 women were suffering from coronary malperfusion due to DAA. The mean age was 55 (range 28-72) years. The right coronary artery was involved in 9 patients, and the left in 11. All procedures such as graft replacement and CABG were done on an emergent or urgent basis. Hospital mortality rate of right coronary malperfusion was 22% (2/9 patients), and that related to left coronary malperfusion was 5/11 (45%). Assisting device was required in 9 cases, veno-arterial bypass (VAB) in 6 cases, left ventricular assist system (LVAS) in 1, left heart bypass (LHB) in 1, LHB+right heart bypass (RHB) in 1. We lost all patients using VAB. Only 3 patients supported with strong assist device survived. Aggressive myocardial resuscitation and early operation are the key factors in the management of these critically ill patients. But once severe myocardial infarction occurs, V-A bypass (percutaneous cardiopulmonary support) is useless in treating patients with DAA who develop severe heart failure. We recommend to implant stronger assist device including LVAS immediately before exacerbation of multiple organ failure. In conclusion, surgical management is not easy for emergency patients with DAA in association with myocardial ischemia. However, reasonable surgical results can be obtained with supplemental CABG and strong mechanical support of the left ventricle.

Cardiac mesenchymal progenitors differentiate into adipocytes via Klf4 and c-Myc.

Direct reprogramming of differentiated cells to pluripotent stem cells has great potential to improve our understanding of developmental biology and disorders such as cancers, and has implications for regenerative medicine. In general, the effects of transcription factors (TFs) that are transduced into cells can be influenced by pre-existing transcriptional networks and epigenetic modifications. However, previous work has identified four key TFs, Oct4, Sox2, Klf4 and c-Myc, which can reprogram various differentiated cells to generate induced pluripotent stem cells. Here, we show that in the heart, the transduction of cardiac mesenchymal progenitors (CMPs) with Klf4 and c-Myc (KM) was sufficient to drive the differentiation of these cells into adipocytes without the use of adipogenic stimulation cocktail, that is, insulin, 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone. KM-transduced CMPs exhibited a gradually increased expression of adipogenic-related genes, such as C/Ebpα, Pparγ and Fabp4, activation of the peroxisome proliferator-activated receptor (PPAR) signaling pathway, inactivation of the cell cycle-related pathway and formation of cytoplasmic lipid droplets within 10 days. In contrast, NIH3T3 fibroblasts, 3T3-L1 preadipocytes, and bone marrow-derived mesenchymal stem cells transduced with KM did not differentiate into adipocytes. Both in vitro and in vivo cardiac ischemia reperfusion injury models demonstrated that the expression of KM genes sharply increased following a reperfusion insult. These results suggest that ectopic adipose tissue formation in the heart following myocardial infarction results from CMPs that express KM following a stress response.

A novel polymorphism in the promoter region for the human osteocalcin gene: the possibility of a correlation with bone mineral density in postmenopausal Japanese women.

We present a polymorphism of the human osteocalcin gene (also known as BGP, for bone Gla protein) due to a 1 base pair (bp) substitution from cytosine to thymine at position 298 nucleotides (nt), which is at position 198 nt upstream from the BGP exon 1. This mutation was detected by single-strand conformation polymorphism analysis after polymerase chain reaction for the osteocalcin gene fragment (326 bp) and sequencing analysis. The cytosine/thymine polymorphism can be defined by restriction fragment length polymorphism analysis using a modified primer pair and the restriction endonuclease HindIII. The osteocalcin genotype was determined in 160 postmenopausal Japanese women (age 48-80 years). Osteocalcin alleles were designated according to the absence (H) or presence (h) of the HindIII restriction site. There were 12 HH, 49 Hh, and 99 hh individuals, and the allele frequencies were 22.8% for H and 77.2% for h. To determine if genetic variation influences bone mineral density (BMD) and thus can be a determinant of susceptibility to osteoporosis in older women, we examined the association of BMD with the osteocalcin genotypes found in the present study. The subjects with genotype HH had the smallest BMD and those with hh had the greatest BMD among subjects, but these differences did not reach statistical significance. The HindIII genotype showed a significant effect on the prevalence of osteopenia in the subjects, that is, women with genotype HH had a 5.74 times greater risk for osteopenia (p < 0.05) and those with genotype Hh had a 1.59 times greater risk than women with genotype hh. We identified the osteocalcin gene polymorphism, detected with the HindIII genotype, which was suggested to influence bone density and is a possible genetic marker for bone metabolism.

Gene therapy and transplantation.

Advances in molecular biology and in techniques of gene transfer have resulted in the development of practical approaches to human gene therapy. Many applications are of relevance to manipulation of the immune system and have potential in organ and cell transplantation. For example, gene therapy approaches may facilitate the induction of immunological tolerance to a donor organ or protect it locally against the host's immune response. Based on a comprehensive review of the world literature, examples of current research efforts in both allogeneic and xenogeneic transplantation are presented and discussed.

Anti-Galalpha1-3Gal antibody levels in organ transplant recipients receiving immunosuppressive therapy.

The effect of long-term pharmacologic immunosuppression (PI) on anti-Galalpha1-3Gal (alphaGal) antibody (Ab) levels has not been determined previously in humans. In this study, we measured alpha Gal Ab levels by ELISA in 14 healthy volunteers (controls) and in 70 patients with grafts (kidney, heart, liver) who had received different combinations of PI (including cyclosporine, tacrolimus, azathioprine, mycophenolate mofetil, and steroids) for >3 months. There was great variation in Gal IgM (<80-fold) and IgG (<160-fold). There was no difference in Gal IgM or Gal IgG between any one group and any other. In kidney patients with either high (mean 68%) or low (mean 6%) panel-reactive alloantibodies, there was no difference in alpha Gal Ab level or serum cytotoxicity to pig cells. In vitro immunoadsorption of alphaGal Ab from the serum did not change panel-reactive alloantibody positivity. Therapy with OKT3, a mouse product that might stimulate alphaGal Ab production, led to no significant change in patient Ab levels. We conclude that long-term (>3 months) PI does not reduce Gal Ab levels sufficiently to be of clinical value in xenotransplantation.

Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates.

BACKGROUND: Efforts to achieve tolerance to transplanted pig organs in nonhuman primates by the induction of a state of mixed hematopoietic chimerism have been associated with disorders of coagulation and thrombosis. Activation of recipient vascular endothelium and platelets by porcine hematopoietic cells and/or activation of donor organ vascular endothelium and/or molecular differences between the species may play roles. Irradiation or drug therapy could possibly potentiate endothelial cell activation and/or injury. METHODS: We have investigated parameters of coagulation and platelet activation in nonhuman primates after (1) a regimen aimed at inducing mixed hematopoietic chimerism and tolerance (TIR that included total body irradiation, T cell depletion, and splenectomy; (2) pig bone marrow or pig peripheral blood mobilized progenitor cell transplantation (PCTx); and/or (3) pig organ transplantation (POTx). Five experimental groups were studied. Baboons were the recipient subjects in all groups except Group 1. Gp 1 Cynomolgus monkeys (n=6) underwent TIR + allotransplantation of hematopoietic cells and a kidney or heart or TIR + concordant xenotransplantation (using baboons as donors) of cells and a kidney; Gp 2 Baboons (n=4) underwent TIR with or without (+/-) autologous hematopoietic cell infusion; Gp 3 (n=12) PCTx+/-TIR; Gp 4 (n=5) POTx+/-TIR; Gp 5 (n=4) TIR + PCTx + POTx. Platelet counts, with plasma prothrombin time, partial thromboplastin time, fibrinogen levels, fibrin split products and/or D-dimer were measured. RESULTS: In the absence of a discordant (porcine) cellular or organ transplant (Groups 1 and 2), TIR resulted in transient thrombocytopenia only, in keeping with bone marrow depression from irradiation. PCTx alone (Group 3) was associated with the rapid development of a thrombotic thrombocytopenic (TTP)-like microangiopathic state, that persisted longer when PCTx was combined with TIR. POTx (+/-TIR) (Group 4) was associated with a gradual fall (over several days) in platelet counts and fibrinogen with disseminated intravascular coagulation (DIC); after graft excision, the DIC generally resolved. When TIR, PCTx and POTx were combined (Group 5), an initial TTP-like state was superseded by a consumptive picture of DIC within the first week, necessitating graft removal. CONCLUSIONS: Both PCTx and POTx lead to profound alterations in hemostasis and coagulation parameters that must be overcome if discordant xenotransplantation of hematopoietic cells and organs is to be fully successful. Disordered thromboregulation could exacerbate vascular damage and potentiate activation of coagulation pathways after exposure to xenogeneic cells or a vascularized xenograft.

High-dose porcine hematopoietic cell transplantation combined with CD40 ligand blockade in baboons prevents an induced anti-pig humoral response.

BACKGROUND: In pig-to-primate organ transplantation, hyperacute rejection can be prevented, but the organ is rejected within days by acute vascular rejection, in which induced high-affinity anti-Gal alpha1-3Gal (alphaGal) IgG and possibly antibodies directed against new porcine (non-alphaGal) antigenic determinants are considered to play a major role. We have explored the role of an anti-CD40L monoclonal antibody in modifying the humoral response to porcine hematopoietic cells in baboons pretreated with a nonmyeloablative regimen. METHODS: Porcine peripheral blood mobilized progenitor cells obtained by leukapheresis from both major histocompatibility complex-inbred miniature swine (n=7) and human decay-accelerating factor pigs (n=3) were transplanted into baboons. Group 1 baboons (n=3) underwent whole body (300 cGy) and thymic (700 cGy) irradiation, T cell depletion with ATG, complement depletion with cobra venom factor, short courses of cyclosporine, mycophenolate mofetil, porcine hematopoietic growth factors, and anti-alphaGal antibody depletion by immunoadsorption before transplantation of high doses (2-4 x 10(10)/cells/kg) of peripheral blood mobilized progenitor cells. In group 2 (n=5), cyclosporine was replaced by eight doses of anti-CD40L monoclonal antibodies over 14 days. The group 3 baboons (n=2) received the group 1 regimen plus 2 doses of anti-CD40L monoclonal antibodies (on days 0 and 2). RESULTS: In group 1, sensitization to alphaGal (with increases in IgM and IgG of 3- to 6-fold and 100-fold, respectively) and the development of antibodies to new non-alphaGal porcine antigens occurred within 20 days. In group 2, no sensitization to alphaGal or non-alphaGal determinants was seen, but alphaGal-reactive antibodies did return to their pre- peripheral blood mobilized progenitor cells transplant levels. In group 3, attenuated sensitization to alphaGal antigens was seen after cessation of cyclosporine and mycophenolate mofetil therapy at 30 days (IgM 4-fold, IgG 8-30-fold), but no antibodies developed against new porcine determinants. In no baboon did anti-CD40L monoclonal antibodies prevent sensitization to its own murine antigens. CONCLUSIONS: We believe these studies are the first to consistently demonstrate prevention of a secondary humoral response after cell or organ transplantation in a pig-to-primate model. The development of sensitization to the murine elements of the anti-CD40L monoclonal antibodies suggests that nonresponsiveness to cell membrane-bound antigen (e.g., alphaGal) is a specific phenomenon and not a general manifestation of immunological unresponsiveness. T cell costimulatory blockade may facilitate induction of mixed hematopoietic chimerism and, consequently, of tolerance to pig organs and tissues.

Transfer of swine major histocompatibility complex class II genes into autologous bone marrow cells of baboons for the induction of tolerance across xenogeneic barriers.

BACKGROUND: The present study examined the potential role of gene therapy in the induction of tolerance to anti-porcine major histocompatibility complex (SLA) class II-mediated responses after porcine renal or skin xenografts. METHODS: Baboons were treated with a non-myeloablative or a myeloablative preparative regimen before bone marrow transplantation with autologous bone marrow cells retrovirally transduced to express both SLA class II DR and neomycin phosphotransferase (NeoR) genes, or the NeoR gene alone. Four months or more after bone marrow transplantation, the immunological response to a porcine kidney or skin xenograft was examined. Both the renal and skin xenografts were SLA DR-matched to the transgene, and recipients were conditioned by combinations of complement inhibitors, adsorption of natural antibodies, immunosuppressive therapy, and splenectomy. RESULTS: Although the long-term presence of the SLA transgene was detected in the peripheral blood and/or bone marrow cells of all baboons, the transcription of the transgene was transient. Autopsy tissues were available from one animal and demonstrated expression of the SLA DR transgene in lymphohematopoietic tissues. After kidney and skin transplantation, xenografts were rejected after 8-22 days. Long-term follow-up of control animals demonstrated that high levels of induced IgG antibodies to new non-alphaGal epitopes developed after organ rejection. In contrast, induced non-alphaGal IgG antibody responses were minimal in the SLA DR-transduced baboons. CONCLUSIONS: Transfer and expression of xenogeneic class II DR transgenes can be achieved in baboons. This therapy may prevent late T cell-dependent responses to porcine xenografts, which include induced non-alphaGal IgG antibody responses.

Transplantation of genetically marked cardiac muscle cells.

We examined the possibility that cardiomyocytes could be genetically marked or modified before being grafted to the heart under conditions applicable to the clinical setting. We used a replication-defective recombinant adenovirus carrying the beta-galactosidase reporter gene, and delivered it to cultured murine fetal cardiac myocytes. Virtually all fetal cardiomyocytes in a primary culture expressed beta-galactosidase 24 hours after recombinant adenovirus infection. These cells were transplanted to the hearts of syngenic adult recipient mice. Expression of the beta-galactosidase gene in the grafted cells was demonstrated by staining with 5-bromo-4-chloro-3-indoyl-beta-D-galactosidase, resulting in a blue color at the histochemical level and an electron-dense deposit on transmission electron microscopic analysis. Gene expression was recognized from 7 days to 12 weeks after transplantation. Implanted cardiomyocytes aligned themselves along the layers of the host myocardium. Formation of gap junctions was demonstrated by transmission electron microscopy. Neither inflammation nor fibrous scar tissue was detectable by histologic analysis. This study demonstrates that ex vivo gene transfer to the heart by means of the adenoviral vector is possible.

Epithelial regeneration and preservation of tracheal cartilage after tracheal replacement with cryopreserved allograft in the rat.

OBJECTIVE: We investigated the origin of the epithelium in transplanted cryopreserved tracheal allografts in rats and tried to clarify the mechanism by which immunogenicity is reduced in this procedure. METHODS: Tracheal transplantation was performed with PVG rats (allele at the RT1 locus: c) used as donors and ACI rats (allele at the RT1 locus: a) as recipients. After resection of a 5-ring segment of the cervical trachea of an ACI rat, the trachea was reconstructed with the cryopreserved tracheal segment of a PVG rat (n = 6). No immunosuppressive agents or steroids were given. Histologic changes were determined and immunohistochemical staining was performed to investigate major histocompatibility complex class I antigens of the transplanted tracheal segment. RESULTS: Two months after tracheal transplantation, 6 surviving ACI rats were killed. Histologically, the epithelium and tracheal cartilage of the transplanted cryopreserved segment displayed normal structure. Immunohistochemical staining showed that the major histocompatibility complex class I antigen of the ACI rat was expressed in the epithelium of the transplanted segment and that the class I antigen of the PVG rat was expressed in the cartilage of the transplanted segment. CONCLUSIONS: After transplantation of the cryopreserved trachea, the epithelium of the transplanted cryopreserved segment originated from the recipient epithelium whereas the cartilage retained the structure of the donor trachea. We hypothesize that transplantation of a cryopreserved trachea leads to the growth of the recipient's epithelium over the donor trachea, thereby reducing the antigenicity of the transplant.

Preservation of myocardial function and metabolism at subzero nonfreezing temperature storage of the heart.

BACKGROUND: The hypothermic simple immersion technique has been widely used to preserve the donor heart for transplantation. However, there is still controversy as to which temperature provides the best protection against prolonged ischemia. The low molecular weight solutes within cells depress the freezing point to -0.6 degree C. In practice, however, most cells do not freeze internally unless they are cooled below -10 degrees C. We investigated the effects of subzero nonfreezing storage at -1 degree C on the preservation of myocardial metabolism and function. METHODS: Isolated Wistar rat hearts were subjected to 6 hours of preservation with the intracellular type University of Wisconsin solution; the hearts of the subzero group were preserved at subzero nonfreezing (-1 degree C) temperature, and the hearts of the control group were at 4 degrees C. Recovery of cardiac function, myocardial adenine nucleotides content, and myocardial water content were evaluated after preservation. RESULTS: Subzero group resulted in significantly better aortic flow, cardiac output, and aortic systolic pressure than in the control group. Myocardial adenosine triphosphate, adenosine diphosphate, and total adenine nucleotides at end-storage were significantly better preserved in subzero group when compared with the control group. Myocardial water content at reperfusion significantly increased in the control group compared with the subzero group. CONCLUSIONS: Storage in the intracellular type solution at subzero nonfreezing (-1 degree C) temperature as compared with 4 degrees C appears to prolong myocardial preservation with respect to the enhancement of postischemic functional recovery, preservation of myocardial adenine nucleotides during ischemia, and prevention of myocardial edema at reperfusion.

Porcine hematopoietic cell xenotransplantation in nonhuman primates is complicated by thrombotic microangiopathy.

Thrombotic microangiopathy (TM) is a serious complication of bone marrow transplantation (BMT) that resembles thrombotic thrombocytopenic purpura (TTP). In attempting to achieve hematopoietic cell chimerism in the pig-to-baboon model, we have observed TM following infusion of high doses (>10(10) cells/kg) of porcine peripheral blood mobilized progenitor cells (PBPC) into baboons. We performed investigations to analyze the pathobiology of this TM and to test therapeutic interventions to ameliorate it. PBPC were obtained by leukapheresis of cytokine-stimulated swine. The initial observations were made in two baboons that underwent a non-myeloablative regimen (NMR) prior to PBPC transplantation (TX) (group 1). We then studied three experimental groups. Group 2 (n = 2) received NMR without PBPC TX. Group 3 (n = 2) received PBPC TX alone. Group 4 (n = 6) received NMR + PBPC TX combined with prostacyclin, low-dose heparin, methylprednisolone, and cyclosporine was replaced by anti-CD40L mAb in five cases. Baboons in groups 1 and 3 developed severe thrombocytopenia (<10,000/mm3), intravascular hemolysis with schistocytosis (>10/high powered field (hpf)), increase in plasma lactate dehydrogenase (LDH) (2500-9000 U/l), transient neurologic changes, renal insufficiency, and purpura. Autopsy on two baboons confirmed extensive platelet thrombi in the microcirculation, and, similar to clinical BMT-associated TM/TTP, no unusually large vWF multimers or changes in vWF protease activity were observed in the plasma of baboons with TM. In group 2, self-limited thrombocytopenia occurred for 10-15 days following NMR. Group 4 baboons developed thrombocytopenia (<20,000/mm3) rarely requiring platelet transfusion, minimal schistocytosis (<3/hpf), minor increase in LDH (<1000 U/l), with no clinical sequelae. We conclude that high-dose porcine PBPC infusion into baboons induces a microangiopathic state with vWF biochemical parameters resembling clinical BMT-associated TM/TTP and that administration of antithrombotic and anti-inflammatory agents can ameliorate this complication.

Identification of the indoleamine 2,3-dioxygenase nucleotide sequence in a rat liver transplant model.

A tryptophan catabolizer, indoleamine 2,3-dioxygenase (IDO) is highly expressed in the placenta and plays an essential role in maternal tolerance. Recent data have shown that the administration of an IDO inhibitor blocked not only maternal tolerance but also liver allograft tolerance. However, little is known about the induction of IDO in liver allografts, although a gene specific for tryptophan 2,3-dioxygenase (TDO) is believed to be expressed in the liver. In the present study, we investigated whether IDO is induced in liver allografts. Synthetic oligonucleotide primers based on the mouse IDO cDNA sequence were used to amplify RNA derived from livers of donor, syngeneic or allogeneic OLT rats. RNA encoding IDO was induced in the rat allogeneic liver after orthotopic liver transplantation (OLT), but not in syngeneic OLT. The rat nucleotide sequence of the RT-PCR products obtained from OLT livers revealed identities of 89% homology to the mouse IDO and of 68% to the human IDO. This study demonstrated the presence of RNA encoding IDO in allogeneic OLT livers, which may be involved in the immune response after liver transplantation.

Ex vivo gene transfer into myocardium using replication-defective retrovirus.

Heart transplantation is the most effective therapy for chronic severe heart failure, but there is an extreme shortage of hearts available. We examined the possibility that cardiomyocytes can be modified genetically prior to being grafted to the heart. We used a replication-defective retrovirus carrying the beta-galactosidase (beta-gal) reporter gene. The beta-gal gene was transduced into murine fetal cardiac myocytes by culturing a recombinant retrovirus-producing cell line in a Transwell plate hung into the primary cardiomyocyte culture. The cultured cells were stained with the di-beta-D-galactopyranoside (FDG) and were sorted by fluorescence-activated cell sorting (FACS). FACS analysis showed that 25.5 +/- 4.3% of the cardiomyocytes in a primary culture were positive for beta-gal activity. These cells were transplanted into the hearts of syngeneic adult mice. Expression of the beta-gal gene in the grafted cells was demonstrated by staining with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside (X-gal). Gene expression was recognized as long as 6 mo after cell transplantation. Histologic analysis showed neither inflammation nor fibrous scar tissue on the host myocardium. This study demonstrated that genetically modified cardiac cells were transplantable to the heart.

Gene transfer into the donor heart during cold preservation for heart transplantation.

BACKGROUND: Ex vivo gene transfer to heart grafts may hold promise as a means of changing alloreactivity or xenoreactivity after transplantation. However, it remains to be determined how effectively gene transfer can be accomplished within a short time in cold-stored grafts that are ready to be transplanted. METHODS: We performed an experimental study using a replication-defective adenovirus (Adex1CALacZ) encoding the Escherichia coli beta-galactosidase (beta-gal) gene to perform gene transfer to heart grafts awaiting transplantation. Thirty hearts of Wistar rats were removed and their coronary arteries were perfused with University of Wisconsin solution containing 1 x 10(9), 1 x 10(10), or 1 x 10(11) plaque-forming units of the recombinant adenovirus at 4 degrees C for 60 minutes. As a control, other hearts were perfused with University of Wisconsin solution with an adenoviral vector that did not contain the beta-gal gene (Adex1w1) for the same period. After perfusion, the grafts were implanted in the necks of syngeneic adult rats. The grafts were removed each week after transplantation and their expression of beta-gal was assessed by 5-bromo-4-chloro-3-indoyl-beta-D-galactoside staining. RESULTS: Successful gene transfer and expression of the beta-gal gene were demonstrated in adenovirus-perfused hearts. Gene transfer occurred preferentially in the cardiomyocytes over the endothelial cells and smooth muscle cells of the coronary vessels. In hearts perfused with 1 x 10(9) plaque-forming units of the adenovirus, gene expression persisted for 4 weeks after transfer, but it diminished gradually and was minimal by day 28. Histologic analyses revealed slight inflammatory reactions in the myocardium. In hearts perfused with 1 x 10(10) and 1 x 10(11) plaque-forming units of the adenovirus, beta-gal diminished 3 weeks after transplantation and a prominent infiltration of leukocytes was recognized in the myocardium. CONCLUSIONS: This study demonstrated that the cardiomyocytes of heart grafts express an exogenous gene product after adenovirus-mediated gene transfer under hypothermic preservation conditions. However, immune or inflammatory reactions to recombinant adenoviruses must be taken into account when a large number of adenoviruses are injected into the coronary arteries.

[A report of initial clinical use of left ventricular assist system with left ventricular inflow cannula for acute myocardial infarction].

Previous clinical results of left ventricular assist system (LVAS) therapy for cardiogenic shock due to acute myocardial infarction (AMI) are still unacceptable. Japanese LVAS was designed for left atrial inflow cannulation. However, to obtain higher initial LVAS flow and more decompression of left ventricular (LV) cavity and to avoid thromboembolic event, LV inflow cannulation is a preferable procedure. Therefore, LV inflow cannula for Japanese LVAS (Toyobo) was developed. We treated three AMI cases with Toyobo-LVAS using the new LV inflow cannula. All patients were in cardiogenic shock status since broad antero-septal AMI and treated with percutaneous cardio-pulmonary support before the LVAS installation. The LVAS was effective for recovery from cardiogenic shock status and multiple organ failure. Two patients died because of serious LVAS associated complications such as bleeding (case 1, 8 days) and cerebral thromboembolism (case 2, 45 days). One of them was assisted for 202 days and underwent second operation. Sixty days after removal of LVAS, the patient died due to sepsis. The technique of LV inflow cannulation is improved through our experiences. However, our result suggests that renovation of the regimen for anti-coagulation and anti-septic therapy are necessary.

Cardiac mesenchymal progenitors from postmortem cardiac tissues retained cellular characterization.

BACKGROUND AND OBJECTIVE: Currently, cells for transplantation in regenerative medicine are derived from either autologous or allogeneic tissue. The former has the drawbacks that the quality of donor cells may depend on the condition of the patient, while the quantity of the cells may also be limited. To solve these problems, we investigated the potential of allogeneic cardiac mesenchymal progenitors (CMPs) derived from postmortem hearts, which may be immunologically privileged similar to bone marrow-derived mesenchymal progenitors. MATERIALS AND METHODS: We examined whether viable CMPs could be isolated from C57/B6 murine cardiac tissues harvested at 24 hours postmortem. After 2- to 3-week propagation with a high dose of basic fibroblast growth factor, we performed cellular characteristics analyses, which included proliferation and differentiation property flow cytometry and microarray analyses. RESULTS: Postmortem CMPs had a longer lag phase after seeding than CMPs obtained from living tissues, but otherwise had similar characteristics in all the analyses. In addition, global gene expression analysis by microarray showed that cells derived from postmortem and living tissues had similar characteristics. CONCLUSION: These results indicate that allogeneic postmortem CMPs have potential for cell transplantation because they circumvent the issue of both the quality and quantity of donor cells.

Direct human mitochondrial transfer: a novel concept based on the endosymbiotic theory.

Mitochondria play an essential role in eukaryotes, and mitochondrial dysfunction is implicated in several diseases. Therefore, intercellular mitochondrial transfer has been proposed as a mechanism for cell-based therapy. In addition, internalization of isolated mitochondria cells by simple coincubation was reported to improve mitochondrial function in the recipient cells. However, substantial evidence for internalization of isolated mitochondria is still lacking, and its precise mechanism remains elusive. We tested whether enriched mitochondria can be internalized into cultured human cells by simple coincubation using fluorescence microscopy and flow cytometry. Mitochondria were isolated from endometrial gland-derived mesenchymal cells (EMCs) or EMCs stably expressing mitochondrial-targeted red fluorescent protein (EMCs-DsRed-mito), and enriched by anti-mitochondrial antibody-conjugated microbeads. They were coincubated with isogeneic EMCs stably expressing green fluorescent protein (GFP). Live fluorescence imaging clearly showed that DsRed-labeled mitochondria accumulated in the cytoplasm of EMCs stably expressing GFP around the nucleus. Flow cytometry confirmed the presence of a distinct population of GFP and DsRed double-positive cells within the recipient cells. In addition, transfer efficiency depended on mitochondrial concentration, indicating that human cells may possess the inherent ability to internalize mitochondria. Therefore, this study supports the application of direct transfer of isogeneic mitochondria as a novel approach for the treatment of diseases associated with mitochondrial dysfunction.

Allogeneic amniotic membrane-derived mesenchymal stromal cell transplantation in a porcine model of chronic myocardial ischemia.

Introduction. Amniotic membrane contains a multipotential stem cell population and is expected to possess the machinery to regulate immunological reactions. We investigated the safety and efficacy of allogeneic amniotic membrane-derived mesenchymal stromal cell (AMSC) transplantation in a porcine model of chronic myocardial ischemia as a preclinical trial. Methods. Porcine AMSCs were isolated from amniotic membranes obtained by cesarean section just before delivery and were cultured to increase their numbers before transplantation. Chronic myocardial ischemia was induced by implantation of an ameroid constrictor around the left circumflex coronary artery. Four weeks after ischemia induction, nine swine were assigned to undergo either allogeneic AMSC transplantation or normal saline injection. Functional analysis was performed by echocardiography, and histological examinations were carried out by immunohistochemistry 4 weeks after AMSC transplantation. Results. Echocardiography demonstrated that left ventricular ejection fraction was significantly improved and left ventricular dilatation was well attenuated 4 weeks after AMSC transplantation. Histological assessment showed a significant reduction in percentage of fibrosis in the AMSC transplantation group. Injected allogeneic green fluorescent protein (GFP)-expressing AMSCs were identified in the immunocompetent host heart without the use of any immunosuppressants 4 weeks after transplantation. Immunohistochemistry revealed that GFP colocalized with cardiac troponin T and cardiac troponin I. Conclusions. We have demonstrated that allogeneic AMSC transplantation produced histological and functional improvement in the impaired myocardium in a porcine model of chronic myocardial ischemia. The transplanted allogeneic AMSCs survived without the use of any immunosuppressants and gained cardiac phenotype through either their transdifferentiation or cell fusion.