Koichi Gonda

Characterization of Freshly Isolated and Cultured Cells Derived From the Fatty and Fluid Portions of Liposuction Aspirates

Liposuction aspirates (primarily saline solution, blood, and adipose tissue fragments) separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and contain adipose‐derived adherent stromal cells (ASCs). Here we define cells isolated from the fluid portion of liposuction aspirates as liposuction aspirate fluid (LAF) cells. Stromal vascular fractions (SVF) were isolated separately from both portions and characterized under cultured and non‐cultured conditions. A comparable number of LAF and PLA cells were freshly isolated, but fewer LAF cells were adherent. CD34+CD45− cells from fresh LAF isolates were expanded by adherent culture, suggesting that LAF cells contain ASCs. Although freshly isolated PLA and LAF cells have distinct cell surface marker profiles, adherent PLA and LAF cells have quite similar characteristics with regard to growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed significant increased expression of CD29, CD44, CD49d, CD73, CD90, CD105, and CD151 and decreased expression of CD31 and CD45. Multicolor FACS analysis revealed that SVF are composed of heterogeneous cell populations including blood‐derived cells (CD45+), ASCs (CD31−CD34+CD45−CD90+CD105−CD146−), endothelial (progenitor) cells (CD31+CD34+CD45−CD90+CD105lowCD146+), pericytes (CD31−CD34−CD45−CD90+CD105−CD146+), and other cells. After plating, ASCs showed a dramatic increase in CD105 expression. Although some adherent ASCs lost CD34 expression with increasing culture time, our culture method maintained CD34 expression in ASCs for at least 10–20 weeks. These results suggest that liposuction‐derived cells may be useful and valuable for cell‐based therapies. J. Cell. Physiol.

EDG1 Is a Functional Sphingosine-1-phosphate Receptor That Is Linked via a Gi/o to Multiple Signaling Pathways, Including Phospholipase C Activation, Ca2+Mobilization, Ras-Mitogen-activated Protein Kinase Activation, and Adenylate Cyclase Inhibition

In Chinese hamster ovary (CHO) cells transiently transfected with an expression vector for EDG1, but not an empty vector, sphingosine-1-phosphate (SP) at a concentration as low as 10−10 m caused an increase in the intracellular free Ca2+ concentration ([Ca2+] i ) as a result of mobilization of Ca2+ from both intracellular and extracellular pools. In a CHO clone stably expressing EDG1 receptor (CHO-EDG1 cells), SP induced increases in the production of inositol phosphates and the [Ca2+] i and inhibited forskolin-induced increase in the cellular cAMP content, all in a manner sensitive to pertussis toxin. SP also activated mitogen-activated protein kinase in CHO-EDG1 cells in pertussis toxin-sensitive and Ras-dependent manners. To evaluate the spectrum of agonists for EDG1, we used human erythroleukemia (HEL) cells, which at naive state do not respond to SP or structurally related lipids with an increase in the [Ca2+] i . In HEL cells stably expressing EDG1 receptor (HEL-EDG1 cells), SP dose-dependently increased the [Ca2+] i with half-maximal and maximal concentration values of 10−9 and 3 × 10−7 m, respectively; sphingosylphosphorylcholine at exclusively high concentrations, but not sphingosine at all, also increased the [Ca2+] i . HEL-EDG1 cells bound32P-labeled SP, which was displaced dose dependently by unlabeled SP. These results indicate that EDG1, a member of the EDG family G protein-coupled receptors, is a specific, high-affinity SP receptor.

Inhibition of rat vascular smooth muscle proliferation in vitro and in vivo by bone morphogenetic protein-2.

Vascular proliferative disorders are characterized by the proliferation of vascular smooth muscle cells (SMCs) and excessive extracellular matrix synthesis. We found that bone morphogenetic protein-2 (BMP-2) inhibited serum-stimulated increases in DNA synthesis and cell number of cultured rat arterial SMCs in a fashion quite different from that in the case of transforming growth factor-beta1 (TGF-beta1). In addition, TGF-beta1 stimulated collagen synthesis in SMCs, whereas BMP-2 did not. In an in vivo rat carotid artery balloon injury model, the adenovirus-mediated transfer of the BMP-2 gene inhibited injury-induced intimal hyperplasia. These results indicate that BMP-2 has the ability to inhibit SMC proliferation without stimulating extracellular matrix synthesis, and suggest the possibility of therapeutic application of BMP-2 for the prevention of vascular proliferative disorders.

Influences of centrifugation on cells and tissues in liposuction aspirates: optimized centrifugation for lipotransfer and cell isolation.

Background: Although injective autologous fat transplantation is one of the most attractive options for soft-tissue augmentation, problems such as unpredictability and fibrosis resulting from fat necrosis limit its universal acceptance. Centrifugation is one of most common methods for overcoming these difficulties. This study was performed to investigate quantitatively the effects of centrifugation on liposuction aspirates to optimize centrifugal conditions for fat transplantation and isolation of adipose-derived stem cells. Methods: Liposuction aspirates, obtained from eight healthy female donors, were either not centrifuged or centrifuged at 400, 700, 1200, 3000, or 4200 g for 3 minutes. The volumes of the oil, adipose, and fluid portions and numbers of blood cells and adipose-derived cells in each portion were examined. The processed adipose tissues (1 ml) were injected into athymic mice, and grafts were harvested and weighed at 4 weeks. Morphologic alterations were observed using light and scanning electron microscopy. Results: Centrifugation concentrated adipose tissues and adipose-derived stem cells in the adipose portion and partly removed red blood cells from the adipose portion. Centrifugation at more than 3000 g significantly damaged adipose-derived stem cells. Centrifugation enhanced graft take per 1 ml centrifuged adipose but reduced calculated graft take per 1 ml adipose before centrifugation. Conclusions: Excessive centrifugation can destroy adipocytes and adipose-derived stem cells, but appropriate centrifugation concentrates them, resulting in enhanced graft take. The authors tentatively recommend 1200 g as an optimized centrifugal force for obtaining good short- and long-term results in adipose transplantation.

Preserved Proliferative Capacity and Multipotency of Human Adipose-Derived Stem Cells after Long-Term Cryopreservation

Background: Human adipose-derived stem (stromal) cells are promising as a regenerative therapy tool for defective tissues of mesenchymal lineage, including fat, bone, and cartilage, and blood vessels. In potential future clinical applications, adipose-derived stem cell cryopreservation could be an indispensable fundamental technology, as has occurred in other fields involving cell-based therapies using hematopoietic stem cells and umbilical cord blood cells. Methods: The authors examined the proliferative capacity and multipotency of human adipose-derived stem cells isolated from lipoaspirates of 18 patients in total before and after a 6-month cryopreservation following their defined protocol. Proliferative capacity was quantified by measuring doubling time in cell culture, and multipotency was examined with differentiation assays for chondrogenic, osteogenic, and adipogenic lineages. In addition, expression profiles of cell surface markers were determined by flow cytometry and compared between fresh and cryopreserved adipose-derived stem cells. Results: Cryopreserved adipose-derived stem cells fully retained the potential for differentiation into adipocytes, osteoblasts, and chondrocytes and for proliferative capacity. Flow cytometric analyses revealed that surface marker expression profiles remained constant before and after storage. Conclusions: Adipose-derived stem cells can be cryopreserved at least for up to 6 months under the present protocol without any loss of proliferative or differentiation potential. These results ensure the availability of autologous banked adipose-derived stem cells for clinical applications in the future.

IFATS collection: Fibroblast growth factor-2-induced hepatocyte growth factor secretion by adipose-derived stromal cells inhibits postinjury fibrogenesis through a c-Jun N-terminal kinase-dependent mechanism.

Adipose‐derived stem/stromal cells (ASCs) not only function as tissue‐specific progenitor cells but also are multipotent and secrete angiogenic growth factors, such as hepatocyte growth factor (HGF), under certain circumstances. However, the biological role and regulatory mechanism of this secretion have not been well studied. We focused on the role of ASCs in the process of adipose tissue injury and repair and found that among injury‐associated growth factors, fibroblast growth factor‐2 (FGF‐2) strongly promoted ASC proliferation and HGF secretion through a c‐Jun N‐terminal kinase (JNK) signaling pathway. In a mouse model of ischemia‐reperfusion injury of adipose tissue, regenerative changes following necrotic and apoptotic changes were seen for 2 weeks. Acute release of FGF‐2 by injured adipose tissue was followed by upregulation of HGF. During the adipose tissue remodeling process, adipose‐derived 5‐bromo‐2‐deoxyuridine‐positive cells were shown to be ASCs (CD31−CD34+). Inhibition of JNK signaling inhibited the activation of ASCs and delayed the remodeling process. In addition, inhibition of FGF‐2 or JNK signaling prevented postinjury upregulation of HGF and led to increased fibrogenesis in the injured adipose tissue. Increased fibrogenesis also followed the administration of a neutralizing antibody against HGF. FGF‐2 released from injured tissue acts through a JNK signaling pathway to stimulate ASCs to proliferate and secrete HGF, contributing to the regeneration of adipose tissue and suppression of fibrogenesis after injury. This study revealed a functional role for ASCs in the response to injury and provides new insight into the therapeutic potential of ASCs. STEM CELLS 2009;27:238–249

Subtype-specific, differential activities of the EDG family receptors for sphingosine-1-phosphate, a novel lysophospholipid mediator.

The lysosphingolipid sphingosine-1-phosphate (S1P) and the structurally related lipid lysophosphatidic acid (LPA) elicit a wide spectrum of biological responses in a variety of cell types, including mitogenesis, cell-shape changes, migration and contraction. Recent studies have unveiled the existence of the G protein-coupled heptahelical receptor subfamily for the biologically active lysophospholipids, which consists of the two receptor subgroups specific for S1P and LPA, respectively. The S1P receptor subgroup comprises four members, i.e. EDG-1, EDG-3, EDG-5/AGR16 and EDG-6, with considerable amino acid similarity among them. The S1P receptor subtypes are coupled to different heterotrimeric G proteins, leading to the activation of a unique set of multiple intracellular signaling pathways. The expression of transcripts of the S1P receptor subtypes is wide-spread, except for EDG-6 which exhibits lymphoid tissue-specific expression. Plasma contains substantial concentrations of S1P as well as LPA. Activated platelets appear to be a major source of S1P and LPA in blood. In addition, accumulating evidence demonstrates that S1P and LPA are released from a variety of cell types in response to various extracellular stimuli. These observations demonstrate the existence of the novel signaling system comprising the lysosphingolipids and their cognate receptors, suggesting physiological and pathological roles.

S1P2, the G Protein–Coupled Receptor for Sphingosine-1-Phosphate, Negatively Regulates Tumor Angiogenesis and Tumor Growth In vivo in Mice

Sphingosine-1-phosphate (S1P) has been implicated in tumor angiogenesis by acting through the G(i)-coupled chemotactic receptor S1P(1). Here, we report that the distinct receptor S1P(2) is responsible for mediating the G(12/13)/Rho-dependent inhibitory effects of S1P on Akt, Rac, and cell migration, thereby negatively regulating tumor angiogenesis and tumor growth. By using S1P(2)(LacZ/+) mice, we found that S1P(2) was expressed in both tumor and normal blood vessels in many organs, in both endothelial cells (EC) and vascular smooth muscle cells, as well as in tumor-associated, CD11b-positive bone marrow-derived cells (BMDC). Lewis lung carcinoma or B16 melanoma cells implanted in S1P(2)-deficient (S1P(2)(-/-)) mice displayed accelerated tumor growth and angiogenesis with enhanced association of vascular smooth muscle cells and pericytes. S1P(2)(-/-) ECs exhibited enhanced Rac activity, Akt phosphorylation, cell migration, proliferation, and tube formation in vitro. Coinjection of S1P(2)(-/-) ECs and tumor cells into wild-type mice also produced a relative enhancement of tumor growth and angiogenesis in vivo. S1P(2)(-/-) mice were also more efficient at recruiting CD11b-positive BMDCs into tumors compared with wild-type siblings. Bone marrow chimera experiments revealed that S1P(2) acted in BMDCs to promote tumor growth and angiogenesis. Our results indicate that, in contrast to endothelial S1P(1), which stimulates tumor angiogenesis, S1P(2) on ECs and BMDCs mediates a potent inhibition of tumor angiogenesis, suggesting a novel therapeutic tactic for anticancer treatment.

Rapid expansion of human adipose-derived stromal cells preserving multipotency

BACKGROUND Adipose-derived stromal (stem) cells (ASC) have been shown to be of great therapeutic use in pre-clinical studies in diverse fields, but a standard expansion method has not been established. We investigated the effects of an endothelial growth medium (EGM-2) on ASC, focusing on proliferation and differentiation potentials. METHODS ASC were cultured in EGM-2 and DMEM. Doubling time and total cell number were compared between the two media. The proliferative effect of each growth factor supplemented in EGM-2 was also examined. Cultured cells in each medium were examined for surface marker expression using flow cytometry. Differentiation into the adipogenic, chondrogenic and osteogenic lineages was analyzed after culture in each medium. RESULTS ASC cultured with EGM-2 proliferated much more rapidly (10(5) times in 2 weeks) and reached the stationary phase earlier than those cultured with DMEM. Among the supplements contained in EGM-2, only fibroblast growth factor-2 (FGF-2) significantly promoted proliferation of ASC, although the proliferative effect of FGF-2 was much less than that of EGM-2, suggesting a synergism among other supplement factors. Flow cytometry and differentiation assays suggested that ASC cultured in EGM-2 preserved immunophenotype and differentiation capacity for at least three mesenchymal lineages (adipogenic, chondrogenic and osteogenic), similar to those cultured with DMEM. DISCUSSION The present expansion method markedly accelerates proliferation of ASC, preserving their multipotent differentiation capacities, and lays the groundwork for establishing a practical route to mega-expansion of ASC for clinical applications.

Sphingosine-1-phosphate receptor-2 deficiency leads to inhibition of macrophage proinflammatory activities and atherosclerosis in apoE-deficient mice.

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that has pleiotropic effects in a variety of cell types including ECs, SMCs, and macrophages, all of which are central to the development of atherosclerosis. It may therefore exert stimulatory and inhibitory effects on atherosclerosis. Here, we investigated the role of the S1P receptor S1PR2 in atherosclerosis by analyzing S1pr2-/- mice with an Apoe-/- background. S1PR2 was expressed in macrophages, ECs, and SMCs in atherosclerotic aortas. In S1pr2-/-Apoe-/- mice fed a high-cholesterol diet for 4 months, the area of the atherosclerotic plaque was markedly decreased, with reduced macrophage density, increased SMC density, increased eNOS phosphorylation, and downregulation of proinflammatory cytokines compared with S1pr2+/+Apoe-/- mice. Bone marrow chimera experiments indicated a major role for macrophage S1PR2 in atherogenesis. S1pr2-/-Apoe-/- macrophages showed diminished Rho/Rho kinase/NF-κB (ROCK/NF-κB) activity. Consequently, they also displayed reduced cytokine expression, reduced oxidized LDL uptake, and stimulated cholesterol efflux associated with decreased scavenger receptor expression and increased cholesterol efflux transporter expression. S1pr2-/-Apoe-/- ECs also showed reduced ROCK and NF-κB activities, with decreased MCP-1 expression and elevated eNOS phosphorylation. Pharmacologic S1PR2 blockade in S1pr2+/+Apoe-/- mice diminished the atherosclerotic plaque area in aortas and modified LDL accumulation in macrophages. We conclude therefore that S1PR2 plays a critical role in atherogenesis and may serve as a novel therapeutic target for atherosclerosis.