Yumi Matsuzaki

Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury

Various types of induced pluripotent stem (iPS) cells have been established by different methods, and each type exhibits different biological properties. Before iPS cell-based clinical applications can be initiated, detailed evaluations of the cells, including their differentiation potentials and tumorigenic activities in different contexts, should be investigated to establish their safety and effectiveness for cell transplantation therapies. Here we show the directed neural differentiation of murine iPS cells and examine their therapeutic potential in a mouse spinal cord injury (SCI) model. “Safe” iPS-derived neurospheres, which had been pre-evaluated as nontumorigenic by their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse brain, produced electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro. Furthermore, when the safe iPS-derived neurospheres were transplanted into the spinal cord 9 d after contusive injury, they differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in remyelination and induced the axonal regrowth of host 5HT+ serotonergic fibers, promoting locomotor function recovery. However, the transplantation of iPS-derived neurospheres pre-evaluated as “unsafe” showed robust teratoma formation and sudden locomotor functional loss after functional recovery in the SCI model. These findings suggest that pre-evaluated safe iPS clone-derived neural stem/progenitor cells may be a promising cell source for transplantation therapy for SCI.

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell–, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0–Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells—and under the right conditions—differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.

Ontogeny and Multipotency of Neural Crest-Derived Stem Cells in Mouse Bone Marrow, Dorsal Root Ganglia, and Whisker Pad

Although recent reports have described multipotent, self-renewing, neural crest-derived stem cells (NCSCs), the NCSCs in various adult rodent tissues have not been well characterized or compared. Here we identified NCSCs in the bone marrow (BM), dorsal root ganglia, and whisker pad and prospectively isolated them from adult transgenic mice encoding neural crest-specific P0-Cre/Floxed-EGFP and Wnt1-Cre/Floxed-EGFP. Cultured EGFP-positive cells formed neurosphere-like structures that expressed NCSC genes and could differentiate into neurons, glial cells, and myofibroblasts, but the frequency of the cell types was tissue source dependent. Interestingly, we observed NCSCs in the aorta-gonad-mesonephros region, circulating blood, and liver at the embryonic stage, suggesting that NCSCs migrate through the bloodstream to the BM and providing an explanation for how neural cells are generated from the BM. The identification of NCSCs in accessible adult tissue provides a new potential source for autologous cell therapy after nerve injury or disease.

Bone marrow–derived cells express matrix metalloproteinases and contribute to regression of liver fibrosis in mice

Liver fibrosis is usually progressive, but it can occasionally be reversible if the causative agents are adequately removed or if patients are treated effectively. However, molecular mechanisms responsible for this reversibility of liver fibrosis have been poorly understood. To reveal the contribution of bone marrow (BM)‐derived cells to the spontaneous regression of liver fibrosis, mice were treated with repeated carbon tetrachloride injections after hematopoietic reconstitution with enhanced green fluorescent protein (EGFP)‐expressing BM cells. The distribution and characteristics of EGFP‐positive (EGFP+) cells present in fibrotic liver tissue were examined at different time points after cessation of carbon tetrachloride intoxication. A large number of EGFP+ cells were observed in liver tissue at peak fibrosis, which decreased during the recovery from liver fibrosis. Some of them, as well as EGFP‐negative (EGFP−) liver resident cells, expressed matrix metalloproteinase (MMP)‐13 and MMP‐9. Whereas MMP‐13 was transiently expressed mainly in the cells clustering in the periportal areas, MMP‐9 expression and enzymatic activity were detected over the resolution process in several different kinds of cells located in the portal areas and along the fibrous septa. Therapeutic recruitment of BM cells by granulocyte colony‐stimulating factor (G‐CSF) treatment significantly enhanced migration of BM‐derived cells into fibrotic liver and accelerated the regression of liver fibrosis. Experiments using transgenic mice overexpressing hepatocyte growth factor (HGF) indicated that G‐CSF and HGF synergistically increased MMP‐9 expression along the fibrous septa. Conclusion: Autologous BM cells contribute to the spontaneous regression of liver fibrosis, and their therapeutic derivation could be a new treatment strategy for intractable liver fibrosis. (HEPATOLOGY; 2007:213–222.)

Stem cell-like properties of the endometrial side population: Implication in endometrial regeneration

Background The human endometrium undergoes cyclical regeneration throughout a womans reproductive life. Ectopic implantation of endometrial cells through retrograde menstruation gives rise to endometriotic lesions which affect approximately 10% of reproductive-aged women. The high regenerative capacity of the human endometrium at eutopic and ectopic sites suggests the existence of stem/progenitor cells and a unique angiogenic system. The objective of this study was to isolate and characterize putative endometrial stem/progenitor cells and to address how they might be involved in the physiology of endometrium. Methodology/Principal Findings We found that approximately 2% of the total cells obtained from human endometrium displayed a side population (SP) phenotype, as determined by flow cytometric analysis of Hoechst-stained cells. The endometrial SP (ESP) cells exhibited preferential expression of several endothelial cell markers compared to endometrial main population (EMP) cells. A medium specific for endothelial cell culture enabled ESP cells to proliferate and differentiate into various types of endometrial cells, including glandular epithelial, stromal and endothelial cells in vitro, whereas in the same medium, EMP cells differentiated only into stromal cells. Furthermore, ESP cells, but not EMP cells, reconstituted organized endometrial tissue with well-delineated glandular structures when transplanted under the kidney capsule of severely immunodeficient mice. Notably, ESP cells generated endothelial cells that migrated into the mouse kidney parenchyma and formed mature blood vessels. This potential for in vivo angiogenesis and endometrial cell regeneration was more prominent in the ESP fraction than in the EMP fraction, as the latter mainly gave rise to stromal cells in vivo. Conclusions/Significance These results indicate that putative endometrial stem cells are highly enriched in the ESP cells. These unique characteristics suggest that ESP cells might drive physiological endometrial regeneration and be involved in the pathogenesis of endometriosis.

Isolation of Multipotent Neural Crest‐Derived Stem Cells from the Adult Mouse Cornea

We report the presence of neural crest‐derived corneal precursors (COPs) that initiate spheres by clonal expansion from a single cell. COPs expressed the stem cell markers nestin, Notch1, Musashi‐1, and ABCG2 and showed the side population cell phenotype. COPs were multipotent with the ability to differentiate into adipocytes, chondrocytes, as well as neural cells, as shown by the expression of β‐III‐tubulin, glial fibrillary acidic protein, and neurofilament‐M. COP spheres prepared from E/nestin‐enhanced green fluorescent protein (EGFP) mice showed induction of EGFP expression that was not originally observed in the cornea, indicating activation of the neural‐specific nestin second intronic enhancer in culture. COPs were Sca‐1+, CD34+, CD45−, and c‐kit−. Numerous GFP+ cells were observed in the corneas of mice transplanted with whole bone marrow of transgenic mice ubiquitously expressing GFP; however, no GFP+ COP spheres were initiated from these mice. On the other hand, COP spheres from transgenic mice encoding P0‐Cre/Floxed‐EGFP as well as Wnt1‐Cre/Floxed‐EGFP were GFP+, indicating the neural crest origin of COPs, which was confirmed by the expression of the embryonic neural crest markers Twist, Snail, Slug, and Sox9. Taken together, these data indicate the existence of neural crest‐derived, multipotent stem cells in the adult cornea.

Side population in human uterine myometrium displays phenotypic and functional characteristics of myometrial stem cells

Over the course of pregnancy, the human uterus undergoes a 500- to 1,000-fold increase in volume and a 24-fold increase in weight. The uterine smooth muscle layer or myometrium is remodeled, and both cell hypertrophy and hyperplasia are evident. The origin of the new smooth muscle cells, however, is unclear. They may arise from existing smooth muscle cells, or they may be the product of stem cell differentiation. This study describes a subset of myometrial cells isolated from nonpregnant human myometrium that represents the myometrial stem cell population. This was characterized as side population of myometrial cells (myoSP) by a distinct Hoechst dye efflux pattern. In contrast to the main population of myometrial cells (myoMP), myoSP resided in quiescence, underexpressed or lacked myometrial cell markers, and could proliferate and eventually differentiate into mature myometrial cells in vitro only under low oxygen concentration. Although myoMP displayed mature myometrial phenotypes before and after in vitro cultivation, only myoSP, not myoMP, generated functional human myometrial tissues efficiently when transplanted into the uteri of severely immunodeficient mice. Finally, myoSP were multipotent and made to differentiate into osteocytes and adipocytes in vitro under the appropriate differentiation-inducing conditions. Thus, myoSP exhibited phenotypic and functional characteristics of myometrial stem cells. Study of myoSP will improve the understanding of myometrial physiology and the pathogenesis of myometrium-derived diseases such as leiomyoma. myoSP may also represent a novel source of biological material that could be used in the reconstruction of not only the human uterus but also other organs as well.

Musculin/MyoR is expressed in kidney side population cells and can regulate their function.

Musculin/MyoR is a new member of basic helix-loop-helix transcription factors, and its expression is limited to skeletal muscle precursors. Here, we report that musculin/MyoR is expressed in adult kidney side population (SP) cells and can regulate their function. SP phenotype can be used to purify stem cell–rich fractions. Microarray analysis clarified that musculin/MyoR was exclusively expressed in kidney SP cells, and the cells resided in the renal interstitial space. Musculin/MyoR-positive cells were decreased in acute renal failure, but infusion of kidney SP cells increased musculin/MyoR-positive cells and improved renal function. Kidney SP cells in reversible acute renal failure expressed a high level of renoprotective factors and leukemia inhibitory factor (LIF), but not in irreversible chronic renal failure. In cultured kidney SP cells, LIF stimulated gene expression of renoprotective factors, and down-regulation of musculin/MyoR augmented LIF-induced gene expression. Our results suggest that musculin/MyoR may play important roles not only in developmental processes but also in regenerative processes in adult tissue.

Flow cytometric analysis of neural stem cells in the developing and adult mouse brain

Despite recent progress in the neural stem cell biology, their cellular characteristics have not been described well. We investigated various characteristics of neural stem cells (NSCs) in vivo during CNS development, using FACS to identify the NSCs. We first examined stage‐dependent changes in the physical parameters, using forward scatter (FSC) and side scatter (SSC) profiles, of NSCs from the developing striatum, where they appear to be active throughout the life of mammals. NSCs were divided into several fractions according to their FSC/SSC profile. With development, their number decreased in the FSChigh fractions but increased in the FSClow/SSChigh fraction, whereas NSCs were significantly concentrated in the fraction containing the largest cells (about 20 μm in diameter) at any stage, which were mostly the cells with the highest nestin‐enhancer activity. Furthermore, we demonstrated that, at all stages examined, the “side population” (SP), defined as the Hoechst 33342 low/negative fraction, which is known to be a stem cell‐enriched population in bone marrow, was also enriched for Notch1‐positive immature neural cells (about 60%) from the developing striatum. However, these immature SP cells were not detected in the large‐cell fraction, however, but were concentrated instead in the FSClow/mid fractions. FACS analysis showed that SP cells from adults were included to some extent in the CD24low/PNAlow fraction, where NSCs were greatly concentrated. Collectively, the characteristics of NSCs were not uniform and changed developmentally.

Noninvasive and real-time assessment of reconstructed functional human endometrium in NOD/SCID/γcnull immunodeficient mice

Human uterine endometrium exhibits unique properties of cyclical regeneration and remodeling throughout reproductive life and also is subject to endometriosis through ectopic implantation of retrogradely shed endometrial fragments during menstruation. Here we show that functional endometrium can be regenerated from singly dispersed human endometrial cells transplanted beneath the kidney capsule of NOD/SCID/γcnull immunodeficient mice. In addition to the endometrium-like structure, hormone-dependent changes, including proliferation, differentiation, and tissue breakdown and shedding (menstruation), can be reproduced in the reconstructed endometrium, the blood to which is supplied predominantly by human vessels invading into the mouse kidney parenchyma. Furthermore, the hormone-dependent behavior of the endometrium regenerated from lentivirally engineered endometrial cells expressing a variant luciferase can be assessed noninvasively and quantitatively by in vivo bioluminescence imaging. These results indicate that singly dispersed endometrial cells have potential applications for tissue reconstitution, angiogenesis, and human–mouse chimeric vessel formation, providing implications for mechanisms underlying the physiological endometrial regeneration during the menstrual cycle and the establishment of endometriotic lesions. This animal system can be applied as the unique model of endometriosis or for other various types of neoplastic diseases with the capacity of noninvasive and real-time evaluation of the effect of therapeutic agents and gene targeting when the relevant cells are transplanted beneath the kidney capsule.