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PubMed2005

[New understanding and development of medical treatments for ocular diseases based upon molecular mechanisms].

A number of genes associated with life phenomena have been identified by the achievement of genome projects. As both comprehensive analysis and methods for investigation of specific genes have been developed, we can understand the pathogenesis of ocular diseases and develop novel medical treatments based upon detailed information on molecular mechanisms. In our review article, we focused on three vision-threatening ocular diseases; glaucoma, age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR), and discussed the potential and problems related to retinal regenerative therapy. Regarding glaucoma, we investigated the relationships between aqueous humor and cell components in the aqueous outflow route. We have revealed that the Rho-Rho-associated coiled-coil-forming protein kinase (ROCK) signal transduction pathway participates in regulation of the aqueous outflow route, and that ROCK inhibitors and several protein kinase inhibitors exert intraocular pressure-lowering effects. Also, we conducted a series of investigations on familial amyloidotic polyneuropathy (FAP), as a representative secondary glaucoma caused by genetic mutations in a single gene. We reviewed the clinical features of ocular complications derived from FAP, their molecular mechanisms and possibilities for the development of novel medical treatments. In addition, we discussed a novel therapeutic concept, "neuroprotection", and showed the potential of some drugs as candidates for the neuroprotective treatment of glaucoma. Against AMD, we have performed a series of experiments from the viewpoint of similarity with atherosclerotic lesions. We have shown the molecular mechanisms of AMD associated with up-regulated expression of scavenger receptors and the interaction between leukocytes and vascular endothelial cells. Furthermore, in the pathogenesis of PVR, we described the role of epithelial-mesenchymal transition in retinal pigment epithelial cells and demonstrated the usefulness of enzymatic vitrectomy. Although retinal regenerative therapy has attracted much attention from global investigators, we pointed out its limitation for clinical application, and developed researches on efficient culture method using physiologically active factors for proliferating retinal stem cells with multi-potentiality, differentiation of the transplanted progenitor cells, and axon guidance of neurons by extracellular matrices.

Animals↗
PubMed2001

From stem cells towards neural layers: a lesson from re-aggregated embryonic retinal cells.

Cells from dissociated embryonic avian retinae have the capacity to re-aggregate in rotation culture and form cellular spheres reconstituting a complete arrangement of all retinal layers. This exquisite phenomenon is based upon in vitro proliferation of multipotent precursor stem cells and spatial organization of their differentiating descendants. The addition of soluble factors from cultured retinal pigmented epithelial (RPE) or radial glial cells is essential to revert inside-out spheres (rosetted retinal spheres) into correctly laminated outside-out spheres (stratified spheres). Such complete restoration of a laminated brain tissue by cell re-aggregation has been achieved only for the embryonic avian retina, but not the mammalian retina, nor for other brain parts. This review summarises the history of the re-aggregation approach, presents avian retinal re-aggregate models, and analyses roles of the RPE and Müller cells for successful retinal tissue regeneration. It is predicted that these results will become biomedically relevant, as stem cell biology will soon open ways to produce large amounts of human retinal precursors.

Animals↗
PubMed2006

[Differentiation of transplanted mouse c-Kit+lin- bone marrow cells into hepatocytes in vitro].

OBJECTIVE: To ascertain whether mouse c-Kit(+)Lin- bone marrow cells have the potential of hepatic stem cells. METHODS: c-Kit(+)lin- bone marrow cells were isolated and purified by magnetic-activated cell sorting (MACS) from BALB/C male donor mice, and immediately transplanted into age-matched BALB/C syngeneic female mice with 35-Gy total liver irradiation. The recipients were sacrificed 1 month after the transplantation for pathological observation of the liver morphology. The presence of Y-chromosome was examined in the liver cells of the recipient by in situ hybridization (ISH), and alpha-fetoprotein (AFP) and albumin in the cells were detected by immunohistochemistry. RESULTS: The hepatocytes positive for Sry gene on Y-chromosome were identified 1 month after transplantation, and immunohistochemistry for AFP and albumin confirmed that the donor mice-derived cells were hepatocytes. CONCLUSION: c-Kit(+)lin- bone marrow cells have the potential of hepatic stem cells, which can reside and differentiate into hepatocytes in the liver after transplantation. c-Kit(+)lin- bone marrow cells can be used as the source cells of cell transplantation for liver disease.

Animals↗
PubMed2005

Bone marrow as a source of circulating CXCR4+ tissue-committed stem cells.

Several studies have suggested that adult haematopoietic stem cells (HSCs) may be capable of transdifferentiating across tissue-lineage boundaries, giving rise to the concept that these stem cells are plastic in their differentiative capacity. This topic created much excitement in the scientific community, with the prospect of employing HSCs in tissue/organ regeneration (e.g. heart infarct, stroke, liver damage) as an alternative to multipotent embryonic stem cells. However, recent observations, and several alternative explanations of previously published data (e.g. cell fusion, epigenetic changes), do not support the concept of HSC plasticity. Our recent studies, in which we employed chemotactic isolation to a stromal-cell-derived-factor-1 (SDF-1) gradient combined with real-time reverse transcriptase (RT)-PCR/immuno-histochemical analyses, revealed that bone marrow (BM) contains a highly mobile population of CXCR4+ cells that express mRNA/proteins for various markers of early tissue-committed stem cells (TCSCs). Based on this we postulate that the BM is not only a home for HSCs, but also a 'hideout' for non-haematopoietic CXCR4+ TCSCs, and we suggest that their presence in BM tissue should be considered before experimental evidence is interpreted simply as transdifferentiation/plasticity of HSCs. Furthermore, our observation that the number of TCSCs is the highest in BM of young animals and decreases with age provides a novel insight into aging, and may explain why the regeneration process becomes less effective in older individuals.

Aging↗
PubMed2002

Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts.

Stem cells and neuroblasts derived from mouse embryos undergo repeated asymmetric cell divisions, generating neural lineage trees similar to those of invertebrates. In Drosophila, unequal distribution of Numb protein during mitosis produces asymmetric cell divisions and consequently diverse neural cell fates. We investigated whether a mouse homologue m-numb had a similar role during mouse cortical development. Progenitor cells isolated from the embryonic mouse cortex were followed as they underwent their next cell division in vitro. Numb distribution was predominantly asymmetric during asymmetric cell divisions yielding a beta-tubulin III(-) progenitor and a beta-tubulin III(+) neuronal cell (P/N divisions) and predominantly symmetric during divisions producing two neurons (N/N divisions). Cells from the numb knockout mouse underwent significantly fewer asymmetric P/N divisions compared to wild type, indicating a causal role for Numb. When progenitor cells derived from early (E10) cortex undergo P/N divisions, both daughters express the progenitor marker Nestin, indicating their immature state, and Numb segregates into the P or N daughter with similar frequency. In contrast, when progenitor cells derived from later E13 cortex (during active neurogenesis in vivo) undergo P/N divisions they produce a Nestin(+) progenitor and a Nestin(-) neuronal daughter, and Numb segregates preferentially into the neuronal daughter. Thus during mouse cortical neurogenesis, as in Drosophila neurogenesis, asymmetric segregation of Numb could inhibit Notch activity in one daughter to induce neuronal differentiation. At terminal divisions generating two neurons, Numb was symmetrically distributed in approximately 80% of pairs and asymmetrically in 20%. We found a significant association between Numb distribution and morphology: most sisters of neuron pairs with symmetric Numb were similar and most with asymmetric Numb were different. Developing cortical neurons with Numb had longer processes than those without. Numb is expressed by neuroblasts and stem cells and can be asymmetrically segregated by both. These data indicate Numb has an important role in generating asymmetric cell divisions and diverse cell fates during mouse cortical development.

Animals↗
PubMed2006

Low extracellular calcium is sufficient for survival and proliferation of murine mesencephalic neural precursor cells.

Various media and Ca2+ concentrations are employed to culture neural progenitor cells (NPCs). We have therefore explored the effects of extracellular calcium concentrations on the survival, proliferation, spontaneous apoptosis and self-renewal capacity of mesencephalic NPCs grown adherently and as free-floating neurospheres. We employed EMEM supplemented with various concentrations of extracellular CaCl2 (0.1-1 mM). Raising the calcium concentration from 0.1 mM to 0.6 mM resulted in an increased number of NPCs growing as a monolayer and increased the protein yield of cells growing in neurospheres (24+/-3 microg total proteins in 0.1 mM Ca2+ medium vs. 316+/-34 microg proteins in 1 mM Ca2+ medium). Concentrations more than 0.6 mM did not result in a further improvement of proliferation or survival. Elimination of calcium from our control medium by 1 mM EGTA resulted in a decrease in cell number from 82+/-2 x 10(4) NPCs/ml observed in control medium to 62+/-2 x 10(4) NPCs/ml observed in calcium-free media. Protein yield dropped significantly in calcium-free media, accompanied by the decreased expression of the proliferation marker PCNA and the pro-survival marker Bcl-2. Two weeks of expansion as neurospheres caused spontaneous cell death in more than 90% of NPCs grown in 0.1 mM CaCl2 EMEM compared with 42% in 1 mM CaCl2 EMEM. Although the action of Ca2+ on NPCs appears to be complex, the presented data strongly suggest that extracellular calcium plays a crucial role in the maintenance of NPCs in a healthy and proliferating state; physiological concentrations (>1.0 mM) are not required, a concentration of 0.5 mM being adequate for cell maintenance.

Animals↗
PubMed2003

In vivo and in vitro differentiation of myocytes from human bone marrow-derived multipotent progenitor cells.

OBJECTIVE: Recent studies have shown that bone marrow (BM) contains cells capable of differentiating into myocytes in vivo. However, addition of demethylation drugs has been necessary to induce myocyte differentiation from BM cells in vitro, and precise mechanisms of BM cells' conversion to myocytes and the origin of those cells have not been established. We investigated the expression of myogenic markers during differentiation and maturation of myocytes from BM-derived multipotent adult progenitor cells (MAPC) under physiological culture condition. MATERIALS AND METHODS: Frozen BM samples from 21 healthy donors were used as a source of MAPC. To induce myocyte differentiation MAPC was cultured in the presence of 5% FCS, VEGF, bFGF, and IGF-1, and the expressions of myocyte markers were examined at various time points. We also investigated engraftment and differentiation of MAPC-derived myocytes in vivo. RESULTS: Frozen BM-derived MAPC, cultured under the physiological myogenic condition, demonstrated spatial expression patterns of several myocyte markers similar to that of authentic myocyte differentiation. When injected into murine muscles, MAPC treated with the myogenic condition engrafted and differentiated into myocyte marker-positive cells and myotubes in vivo. CONCLUSION: For the first time, we were able to induce myocyte formation from BM cells under the physiological condition in vitro and demonstrated that treating cells with this condition prior to intramuscular injection increased efficiency of engraftment and differentiation in vivo.

Adolescent↗
PubMed2004

Enhanced engraftment of human cells in RAG2/gammac double-knockout mice after treatment with CL2MDP liposomes.

OBJECTIVE: The ability of human cells to repopulate the bone marrow of nonobese diabetic immunodeficient mice (NOD/SCID) is commonly used as a standard assay to quantify the primitive human hematopoietic stem cell population. We studied the applicability of the immunodeficient RAG2(-/-)gammac(-/-) double-knockout mouse for this purpose. METHODS: RAG2(-/-)gammac(-/-) mice and NOD/SCID mice were injected intravenously (i.v.) with umbilical cord blood-derived CD34(+) cells and engraftment was quantified by determining the human CD45+ cell chimerism in bone marrow at several time points. RAG2(-/-)gammac(-/-) were pretreated with total-body irradiation and depleted of macrophages in liver, spleen, and bone marrow by i.v. injection of clodronate diphosphonate containing liposomes. RESULTS: We demonstrated that the frequency of chimerism and the level of engraftment in macrophage-depleted RAG2(-/-)gammac(-/-) largely resemble that in NOD/SCID mice. Also similar is the multilineage differentiation pattern in the two mouse strains at 7 weeks after transplantation, with a prominent outgrowth in RAG2(-/-)gammac(-/-) of CD19+ cells (88% +/- 10%). Cells of other lineages were clearly less frequent: 9% +/- 2% myeloid cells and 0.1% +/- 0.1% erythroid cells. As for immature progenitors, 6% +/- 1% of the human cells express the CD34 antigen and 0.4% +/- 0.1% have the CD34+,CD33,38,71(-) phenotype. The presence of human committed progenitors (i.e., CFU-GM/BFU-E) was evident. The persistence of human cells at 4 months after transplantation shows that the RAG2(-/-)gammac(-/-) support long-term maintenance of human hematopoiesis. CONCLUSION: Our findings indicate that macrophage-depleted RAG2(-/-)gammac(-/-) are a suitable model for studying human hematopoiesis including multipotential stem cells, and long-term repopulation.

Animals↗
PubMed2005

The activin signaling pathway promotes differentiation of dI3 interneurons in the spinal neural tube.

The generation of the appropriate types and numbers of mature neurons during the development of the spinal cord requires the careful coordination of patterning, proliferation, and differentiation. In the dorsal neural tube, this coordination is achieved by the combined action of multiple ligands of both the Wnt and TGF-beta families, and their effectors, such as the bHLH proteins. TGF-beta signaling acting through the BMP receptors is necessary for the generation of several dorsal interneuron types. Other TGF-beta ligands expressed in the dorsal neural tube interact with the Activin receptors, which signal via a different set of SMAD proteins than BMPs. The effects of Activin signaling on the developing neural tube have not been described. Here we have activated the Activin signal transduction pathway in a cell-autonomous manner in the developing chick neural tube. We find that a constitutively active Activin receptor promotes differentiation throughout the neural tube. Although most differentiated cell populations are unaffected by Activin signaling, the number of dorsal interneuron 3 (dI3) cells is specifically increased. Our data suggest that Activin signaling may promote the formation of the dI3 precursor cells within a region circumscribed by BMP signaling and that this function is not dependent upon BMP signaling.

Activin Receptors, Type I↗
PubMed2006

BMP and FGF regulate the differentiation of multipotential pericardial mesoderm into the myocardial or epicardial lineage.

Proepicardial cells give rise to epicardium, coronary vasculature and cardiac fibroblasts. The proepicardium is derived from the mesodermal lining of the prospective pericardial cavity that simultaneously contributes myocardium to the venous pole of the elongating primitive heart tube. Using proepicardial explant cultures, we show that proepicardial cells have the potential to differentiate into cardiac muscle cells, reflecting the multipotency of this pericardial mesoderm. The differentiation into the myocardial or epicardial lineage is mediated by the cooperative action of BMP and FGF signaling. BMP2 is expressed in the distal IFT myocardium and stimulates cardiomyocyte formation. FGF2 is expressed in the proepicardium and stimulates differentiation into the epicardial lineage. In the base of the proepicardium, coexpression of BMP2 and FGF2 inhibits both myocardial and epicardial differentiation. We conclude that the epicardial/myocardial lineage decisions are mediated by an extrinsic, inductive mechanism, which is determined by the position of the cells in the pericardial mesoderm.

Animals↗
PubMed2002

Cell-type-specific expression of protein tyrosine kinase-related receptor RYK in the central nervous system of the rat.

The mammalian RYK is an orphan receptor that contains a catalytically inactive tyrosine-kinase-related domain. Its Drosophila homolog, Lio/Drl, is required for axon pathfinding in developing brain. Our previous study suggested that RYK mRNA is expressed in nestin-positive progenitor cells and neurons. In the present study, immunohistochemistry has been used to further localize RYK in the central nervous system of rats to identify the lineage of the RYK-expressing cells. In the embryonic forebrain, RYK colocalized with nestin in the ventricular zone and with MAP2 in the cortical plate, suggesting that RYK is expressed in neural progenitor cells and neurons. Localization of RYK in embryonic spinal cord also suggested its expression in both cell types. In primary cultures of rat cerebrum, RYK expression was observed in all neurons, as well as in a significant population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes. However, no RYK expression was detected in type-1 astrocytes or microglia. Multipotent neural stem cell line MNS-70 was also analyzed for expression of RYK, and most of the cells were positive for both RYK and nestin in the undifferentiated stage. In the differentiated stage, expression of RYK was detected in the neurons, but not in type-1 astrocytes. In conclusion, RYK is expressed in nestin-positive progenitor cells and neurons, and in a certain population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes in developing CNS. These findings show that expression of RYK in rat CNS is tightly regulated in a cell-type-specific manner.

Animals↗
PubMed2003

Murine serpin 2A is a redox-sensitive intracellular protein.

Murine serpin 2A is expressed at high levels in haemopoietic progenitors and down-regulated on differentiation. When it is constitutively expressed in the multipotent haemopoietic cell line, FDCP-Mix, it causes a delay in differentiation and increased clonogenic potential. The serpin is also dramatically up-regulated on T-cell activation. It has an unusual reactive site Cys-Cys sequence, a unique C-terminal extension and lacks a typical cleavable N-terminal signal sequence. In spite of these features, the protein is not a member of the ovalbumin-serpin family, but is instead most closely related to human antichymotrypsin. We have shown that the serpin is intracellular with prominent nuclear localization. Transverse urea gradient gels and CD studies show that the protein undergoes the stressed-relaxed conformational change typical of inhibitory serpins. However, we have not detected complex-forming activity with a set of proteases. Thermal denaturation studies also show that the protein has decreased structural stability under reducing conditions, although it lacks disulphide bonds within the core of the molecule. Our results show that serpin 2A is an intracellular protein with the potential to mediate its biological effects via interaction with non-protease intracellular targets. Furthermore, the results presented suggest a model whereby the serpin interactions could be modulated by redox conditions or conformational change induced by cleavage of the reactive-site loop.

Animals↗
PubMed2006

Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. METHODS: As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. RESULTS: We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP). DISCUSSION: Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.

Antigens, Differentiation↗
PubMed2001

Acute erythremic myelosis (true erythroleukaemia): a variant of AML FAB-M6.

AIMS: Classic erythroleukaemia (acute myeloid leukaemia M6, or M6 AML) is defined as an excess of myeloblasts in an erythroid predominant background. Leukaemia variants in which the primitive blast cells are demonstrably erythroid are extremely rare and poorly characterised. Variably referred to as "true erythroleukaemia" or "acute erythremic myelosis", they are often included within the M6 AML category even though they do not meet strict criteria for this type of AML. METHODS: Two cases of acute erythroid neoplasia are presented with clinical, morphological, immunophenotypic, and cytogenetic analysis. RESULTS: Both patients presented with profound anaemia, one in a setting of long standing myelodysplasia. Bone marrow examination revealed a predominant population of highly dysplastic erythroid cells in both cases. In one case, the liver was infiltrated by neoplastic erythroid cells. Both patients died within four months of diagnosis. CONCLUSIONS: This report illustrates that cases of acute leukaemia occur in which the dominant neoplastic cell is a primitive erythroid cell without an accompanying increase in myeloblasts. This does not preclude the neoplastic clone originating in a multipotent haemopoietic stem cell, as suggested by cases arising in patients with myelodysplasia. Acute erythremic myelosis should be recognised as a distinct variant of M6 AML.

Adult↗
PubMed2006

Oligodendrocytes and radial glia derived from adult rat spinal cord progenitors: morphological and immunocytochemical characterization.

Self-renewing, multipotent neural progenitor cells (NPCs) reside in the adult mammalian spinal cord ependymal region. The current study characterized, in vitro, the native differentiation potential of spinal cord NPCs isolated from adult enhanced green fluorescence protein rats. Neurospheres were differentiated, immunocytochemistry (ICC) was performed, and the positive cells were counted as a percentage of Hoescht+ nuclei in 10 random fields. Oligodendrocytes constituted most of the NPC progeny (58.0% of differentiated cells; 23.4% in undifferentiated spheres). ICC and electron microscopy (EM) showed intense myelin production by neurospheres and progeny. The number of differentiated astrocytes was 18.0%, but only 2.8% in undifferentiated spheres. The number of differentiated neurons was 7.4%, but only 0.85% in undifferentiated spheres. The number of differentiated radial glia (RG) was 73.0% and in undifferentiated spheres 80.9%. EM showed an in vitro phagocytic capability of NPCs. The number of undifferentiated NPCs was 32.8% under differentiation conditions and 78.9% in undifferentiated spheres. Compared with ependymal region spheres, the spheres derived from the peripheral white matter of the spinal cord produced glial-restricted precursors. These findings indicate that adult rat spinal cord ependymal NPCs differentiate preferentially into oligodendrocytes and RG, which may support axonal regeneration in future trials of transplant therapy for spinal cord injury.

Animals↗
PubMed2002

Hematopoietic potential of neural stem cells: plasticity versus heterogeneity.

Organ-specific stem cells have been identified in a variety of mammalian tissues. These cells hold great promise for cellular therapy if they can reliably produce functional progeny of specific lineages. A central dogma in development has been that organ-specific stem cells are restricted to making the differentiated cell types of the tissue from which they are isolated. However, a substantial body of evidence exists that stem-cell populations from neural and hematopoietic tissues can generate the other cell types, suggesting that adult organ-specific stem cells may have a broader differentiation potential than originally thought. It remains unclear whether this apparent stem cell plasticity is attributable to transdifferentiation of tissue specific stem cells, the co-existence of multiple stem cells with different potentials, or resident totipotent stem cells in these tissues. Recent evidence, in fact, indicates that there may be a fourth explanation for the "apparent" plasticity of stem cells: cell fusion. Here, the authors critically examine the existing data to assess the extent of phenotypic conversion of bone marrow-to-brain and brain-to-blood and discuss some of the contentious issues surrounding these studies. We conclude that there is strong evidence for a multipotent neurohematopoietic stem-cell population in human and mouse brain, although further characterization of these cells will be required if the goal of engineering tissues for therapeutic applications is to be realized.

Animals↗