Naoki Nakayama | The University of Texas Health Science Center at Houston (original) (raw)
Papers by Naoki Nakayama
Journal of Biological Chemistry, 1987
The nucleotide sequence of the Escherichia coli dnaC gene and the primary structure of the dnaC p... more The nucleotide sequence of the Escherichia coli dnaC gene and the primary structure of the dnaC protein were determined. The NH2-terminal amino acid sequence of the dnaC protein matched that predicted from the nucleotide sequence of the 735-base pair coding region. The dnaC gene lacks characteristic promoter structures; neither the "Pribnow box" nor the "-35 sequence" was detected within 222 base pairs upstream from the initiator ATG codon. There is, however, a typical Shine-Dalgarno sequence 7-10 base pairs before the ATG codon. An upstream open reading frame, separated by just 2 base pairs from the coding region of dnaC, encodes the COOH-terminal half of the dnaT product (protein i; Masai, H., Bond, M. W., and Arai, K. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1256-1260). The dnaC protein contains 245 amino acids with a calculated molecular weight of 27,894 consistent with the observed value (29,000). Similar to dnaG and dnaT, dnaC uses several minor codons; the significance of these minor codons to the low level expression of the protein product in E. coli cells remains to be determined. The in vitro site-directed mutagenesis method was employed to determine the functional region involved in interaction with dnaB protein. The first cysteine residue located in the NH2-terminal region of the dnaC protein (Cys69) was shown to be important for this activity. Overall sequence homology between dnaC protein and lambda P protein, functionally analogous to the dnaC protein in the lambda phage DNA replication, is not extensive. There are, however, several short stretches of homologous regions including the NH2-terminal eight amino acids and the Cys78 region of dnaC protein.
Journal of Cell Science, 2002
Throughout life, the hematopoietic system requires a supportive microenvironment that allows for ... more Throughout life, the hematopoietic system requires a supportive microenvironment that allows for the maintenance and differentiation of hematopoietic stem cells (HSC). To understand the cellular interactions and molecules that provide these functions, investigators have previously established stromal cell lines from the late gestational stage and adult murine hematopoietic microenvironments. However, the stromal cell microenvironment that supports the emergence, expansion and maintenance of HSCs during mid-gestational stages has been largely unexplored. Since several tissues within the mouse embryo are known to harbor HSCs (i.e. aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal cell clones from these mid-gestational sites. Owing to the limited cell numbers,isolations were performed with tissues from transgenic embryos containing the ts SV40 Tag gene (tsA58) under the transcriptional control of constitutive and ubiquitously expressing promoters. We report here ...
Pluripotent Stem Cells - From the Bench to the Clinic, 2016
Development, 2014
Pluripotent embryonic stem cells (ESCs) generate rostral paraxial mesoderm-like progeny in 5-6 da... more Pluripotent embryonic stem cells (ESCs) generate rostral paraxial mesoderm-like progeny in 5-6 days of differentiation induced by Wnt3a and Noggin (Nog). We report that canonical Wnt signaling introduced either by forced expression of activated β-catenin, or the small-molecule inhibitor of Gsk3, CHIR99021, satisfied the need for Wnt3a signaling, and that the small-molecule inhibitor of BMP type I receptors, LDN193189, was able to replace Nog. Mesodermal progeny generated using such small molecules were chondrogenic in vitro, and expressed trunk paraxial mesoderm markers such as Tcf15 and Meox1, and somite markers such as Uncx, but failed to express sclerotome markers such as Pax1. Induction of the osteochondrogenically committed sclerotome from somite requires sonic hedgehog and Nog. Consistently, Pax1 and Bapx1 expression was induced when the isolated paraxial mesodermal progeny were treated with SAG1 (a hedgehog receptor agonist) and LDN193189, then Sox9 expression was induced, le...
Bioengineering, 2021
It is widely accepted that chondral defects in articular cartilage of adult joints are never repa... more It is widely accepted that chondral defects in articular cartilage of adult joints are never repaired spontaneously, which is considered to be one of the major causes of age-related degenerative joint disorders, such as osteoarthritis. Since mobilization of subchondral bone (marrow) cells and addition of chondrocytes or mesenchymal stromal cells into full-thickness defects show some degrees of repair, the lack of self-repair activity in adult articular cartilage can be attributed to lack of reparative cells in adult joints. In contrast, during a fetal or embryonic stage, joint articular cartilage has a scar-less repair activity, suggesting that embryonic joints may contain cells responsible for such activity, which can be chondrocytes, chondroprogenitors, or other cell types such as skeletal stem cells. In this respect, the tendency of pluripotent stem cells (PSCs) to give rise to cells of embryonic characteristics will provide opportunity, especially for humans, to obtain cells car...
Developmental Biology, 2001
Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dors... more Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dorsalizing the Xenopus embryo. A novel secreted protein, CHL (for chordin-like), with significant homology to chordin, was isolated from mouse bone marrow stromal cells. Injection of CHL RNA into Xenopus embryos induced a secondary axis. Recombinant CHL protein inhibited the BMP4-dependent differentiation of embryonic stem
Journal of cell …, 2003
The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. ... more The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. Among the resulting cells, those expressing flk-1 and/or PDGFRα displayed chondrogenic activity in the presence of TGFβ3 and expressed cartilage-specific genes in 7 to 16 day ...
Immunity, 1995
The receptors for IL-3, GM-CSF, and IL-5 share a common beta subunit (beta c), and mice have an a... more The receptors for IL-3, GM-CSF, and IL-5 share a common beta subunit (beta c), and mice have an additional IL-3 beta subunit (beta IL3). We have independently generated mice carrying null mutations of each molecule. beta c mutant bone marrow showed no response to GM-CSF or IL-5, whereas IL-3 stimulation of beta c or beta IL3 mutant bone marrow was normal. beta c mutant mice showed lung pathology consisting of lymphocytic infiltration and areas resembling alveolar proteinosis, and also exhibited low basal numbers of eosinophils. Infection of beta c mutant mice by Nippostrongylus brasiliensis resulted in the absence of blood and lung eosinophilia. Animals repopulated with beta c mutant bone marrow cells showed slower leukocyte recovery and reduced eosinophil numbers. These data define the role of beta c in vivo, and show a phenotype that is likely to be the cumulative effect of loss of GM-CSF and IL-5 stimulation.
Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF) β super family.... more Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF) β super family. These molecules play important roles during many organogenic processes, even though they were originally identified as factors promoting the ectopic formation of cartilage and bone. The concentration of active BMP is controlled in part by inhibitors from three BMP binding protein families: short gastrulation/chordin, noggin and cerberus (reviewed by Balemans and Van Hul, 2002). Among these, chordin and noggin were the first proteins found to inhibit the activity of bound BMPs by preventing interactions with their BMP receptors (Piccolo et al., 1996; Zimmerman et al., 1996). Although noggin is encoded by a single gene in mammals, chordin belongs to a family of proteins that share a cysteine-rich pro-collagen repeat [or chordin-like cysteine-rich repeat (CR)], which is also found in various extracellular matrix proteins (reviewed by Garcia Abreu et al., 2002). Without exception, the homol...
Stem cell reports, Jan 19, 2018
Cartilage pellets generated from ectomesenchymal progeny of human pluripotent stem cells (hPSCs) ... more Cartilage pellets generated from ectomesenchymal progeny of human pluripotent stem cells (hPSCs) in vitro eventually show signs of commitment of chondrocytes to hypertrophic differentiation. When transplanted subcutaneously, most of the surviving pellets were fully mineralized by 8 weeks. In contrast, treatment with the adenylyl cyclase activator, forskolin, in vitro resulted in slightly enlarged cartilage pellets containing an increased proportion of proliferating immature chondrocytes that expressed very low levels of hypertrophic/terminally matured chondrocyte-specific genes. Forskolin treatment also enhanced hyaline cartilage formation by reducing type I collagen gene expression and increasing sulfated glycosaminoglycan accumulation in the developed cartilage. Chondrogenic mesoderm from hPSCs and dedifferentiated nasal chondrocytes responded similarly to forskolin. Furthermore, forskolin treatment in vitro increased the frequency at which the cartilage pellets maintained unminer...
PloS one
Moyamoya disease (MMD) is a slow, progressive steno-occlusive disease, arising in the terminal po... more Moyamoya disease (MMD) is a slow, progressive steno-occlusive disease, arising in the terminal portions of the cerebral internal carotid artery. However, the functions and characteristics of the endothelial cells (ECs) in MMD are unknown. We analyzed these features using induced pluripotent stem cell (iPSC)-derived ECs. iPSC lines were established from the peripheral blood of three patients with MMD carrying the variant RNF213 R4810K, and three healthy persons used as controls. After the endothelial differentiation of iPSCs, CD31+CD144+ cells were purified as ECs using a cell sorter. We analyzed their proliferation, angiogenesis, and responses to some angiogenic factors, namely VEGF, bFGF, TGF-β, and BMP4. The ECs were also analyzed using DNA microarray and proteomics to perform comprehensive gene and protein expression analysis. Angiogenesis was significantly impaired in MMD regardless of the presence of any angiogenic factor. On the contrary, endothelial proliferation was not sign...
Molecular therapy. Methods & clinical development, 2016
Three populations of muscle-derived cells (PP1, PP3, and PP6) were isolated from mouse skeletal m... more Three populations of muscle-derived cells (PP1, PP3, and PP6) were isolated from mouse skeletal muscle using modified preplate technique and retrovirally transduced with BMP4/GFP. In vitro, the PP1 cells (fibroblasts) proliferated significantly slower than the PP3 (myoblasts) and PP6 cells (muscle-derived stem cells); the PP1 and PP6 cells showed a superior rate of survival compared with PP3 cells under oxidative stress; and the PP6 cells showed significantly superior chondrogenic capabilities than PP1 and PP3 cells. In vivo, the PP6 cells promoted superior cartilage regeneration compared with the other muscle-derived cell populations. The cartilage defects in the PP6 group had significantly higher histological scores than those of the other muscle-derived cell groups, and GFP detection revealed that the transplanted PP6 cells showed superior in vivo cell survival and chondrogenic capabilities compared with the PP1 and PP3 cells. PP6 cells (muscle-derived stem cells) are superior t...
Journal of Biological Chemistry, 1987
The nucleotide sequence of the Escherichia coli dnaC gene and the primary structure of the dnaC p... more The nucleotide sequence of the Escherichia coli dnaC gene and the primary structure of the dnaC protein were determined. The NH2-terminal amino acid sequence of the dnaC protein matched that predicted from the nucleotide sequence of the 735-base pair coding region. The dnaC gene lacks characteristic promoter structures; neither the "Pribnow box" nor the "-35 sequence" was detected within 222 base pairs upstream from the initiator ATG codon. There is, however, a typical Shine-Dalgarno sequence 7-10 base pairs before the ATG codon. An upstream open reading frame, separated by just 2 base pairs from the coding region of dnaC, encodes the COOH-terminal half of the dnaT product (protein i; Masai, H., Bond, M. W., and Arai, K. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1256-1260). The dnaC protein contains 245 amino acids with a calculated molecular weight of 27,894 consistent with the observed value (29,000). Similar to dnaG and dnaT, dnaC uses several minor codons; the significance of these minor codons to the low level expression of the protein product in E. coli cells remains to be determined. The in vitro site-directed mutagenesis method was employed to determine the functional region involved in interaction with dnaB protein. The first cysteine residue located in the NH2-terminal region of the dnaC protein (Cys69) was shown to be important for this activity. Overall sequence homology between dnaC protein and lambda P protein, functionally analogous to the dnaC protein in the lambda phage DNA replication, is not extensive. There are, however, several short stretches of homologous regions including the NH2-terminal eight amino acids and the Cys78 region of dnaC protein.
Journal of Cell Science, 2002
Throughout life, the hematopoietic system requires a supportive microenvironment that allows for ... more Throughout life, the hematopoietic system requires a supportive microenvironment that allows for the maintenance and differentiation of hematopoietic stem cells (HSC). To understand the cellular interactions and molecules that provide these functions, investigators have previously established stromal cell lines from the late gestational stage and adult murine hematopoietic microenvironments. However, the stromal cell microenvironment that supports the emergence, expansion and maintenance of HSCs during mid-gestational stages has been largely unexplored. Since several tissues within the mouse embryo are known to harbor HSCs (i.e. aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal cell clones from these mid-gestational sites. Owing to the limited cell numbers,isolations were performed with tissues from transgenic embryos containing the ts SV40 Tag gene (tsA58) under the transcriptional control of constitutive and ubiquitously expressing promoters. We report here ...
Pluripotent Stem Cells - From the Bench to the Clinic, 2016
Development, 2014
Pluripotent embryonic stem cells (ESCs) generate rostral paraxial mesoderm-like progeny in 5-6 da... more Pluripotent embryonic stem cells (ESCs) generate rostral paraxial mesoderm-like progeny in 5-6 days of differentiation induced by Wnt3a and Noggin (Nog). We report that canonical Wnt signaling introduced either by forced expression of activated β-catenin, or the small-molecule inhibitor of Gsk3, CHIR99021, satisfied the need for Wnt3a signaling, and that the small-molecule inhibitor of BMP type I receptors, LDN193189, was able to replace Nog. Mesodermal progeny generated using such small molecules were chondrogenic in vitro, and expressed trunk paraxial mesoderm markers such as Tcf15 and Meox1, and somite markers such as Uncx, but failed to express sclerotome markers such as Pax1. Induction of the osteochondrogenically committed sclerotome from somite requires sonic hedgehog and Nog. Consistently, Pax1 and Bapx1 expression was induced when the isolated paraxial mesodermal progeny were treated with SAG1 (a hedgehog receptor agonist) and LDN193189, then Sox9 expression was induced, le...
Bioengineering, 2021
It is widely accepted that chondral defects in articular cartilage of adult joints are never repa... more It is widely accepted that chondral defects in articular cartilage of adult joints are never repaired spontaneously, which is considered to be one of the major causes of age-related degenerative joint disorders, such as osteoarthritis. Since mobilization of subchondral bone (marrow) cells and addition of chondrocytes or mesenchymal stromal cells into full-thickness defects show some degrees of repair, the lack of self-repair activity in adult articular cartilage can be attributed to lack of reparative cells in adult joints. In contrast, during a fetal or embryonic stage, joint articular cartilage has a scar-less repair activity, suggesting that embryonic joints may contain cells responsible for such activity, which can be chondrocytes, chondroprogenitors, or other cell types such as skeletal stem cells. In this respect, the tendency of pluripotent stem cells (PSCs) to give rise to cells of embryonic characteristics will provide opportunity, especially for humans, to obtain cells car...
Developmental Biology, 2001
Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dors... more Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dorsalizing the Xenopus embryo. A novel secreted protein, CHL (for chordin-like), with significant homology to chordin, was isolated from mouse bone marrow stromal cells. Injection of CHL RNA into Xenopus embryos induced a secondary axis. Recombinant CHL protein inhibited the BMP4-dependent differentiation of embryonic stem
Journal of cell …, 2003
The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. ... more The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. Among the resulting cells, those expressing flk-1 and/or PDGFRα displayed chondrogenic activity in the presence of TGFβ3 and expressed cartilage-specific genes in 7 to 16 day ...
Immunity, 1995
The receptors for IL-3, GM-CSF, and IL-5 share a common beta subunit (beta c), and mice have an a... more The receptors for IL-3, GM-CSF, and IL-5 share a common beta subunit (beta c), and mice have an additional IL-3 beta subunit (beta IL3). We have independently generated mice carrying null mutations of each molecule. beta c mutant bone marrow showed no response to GM-CSF or IL-5, whereas IL-3 stimulation of beta c or beta IL3 mutant bone marrow was normal. beta c mutant mice showed lung pathology consisting of lymphocytic infiltration and areas resembling alveolar proteinosis, and also exhibited low basal numbers of eosinophils. Infection of beta c mutant mice by Nippostrongylus brasiliensis resulted in the absence of blood and lung eosinophilia. Animals repopulated with beta c mutant bone marrow cells showed slower leukocyte recovery and reduced eosinophil numbers. These data define the role of beta c in vivo, and show a phenotype that is likely to be the cumulative effect of loss of GM-CSF and IL-5 stimulation.
Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF) β super family.... more Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF) β super family. These molecules play important roles during many organogenic processes, even though they were originally identified as factors promoting the ectopic formation of cartilage and bone. The concentration of active BMP is controlled in part by inhibitors from three BMP binding protein families: short gastrulation/chordin, noggin and cerberus (reviewed by Balemans and Van Hul, 2002). Among these, chordin and noggin were the first proteins found to inhibit the activity of bound BMPs by preventing interactions with their BMP receptors (Piccolo et al., 1996; Zimmerman et al., 1996). Although noggin is encoded by a single gene in mammals, chordin belongs to a family of proteins that share a cysteine-rich pro-collagen repeat [or chordin-like cysteine-rich repeat (CR)], which is also found in various extracellular matrix proteins (reviewed by Garcia Abreu et al., 2002). Without exception, the homol...
Stem cell reports, Jan 19, 2018
Cartilage pellets generated from ectomesenchymal progeny of human pluripotent stem cells (hPSCs) ... more Cartilage pellets generated from ectomesenchymal progeny of human pluripotent stem cells (hPSCs) in vitro eventually show signs of commitment of chondrocytes to hypertrophic differentiation. When transplanted subcutaneously, most of the surviving pellets were fully mineralized by 8 weeks. In contrast, treatment with the adenylyl cyclase activator, forskolin, in vitro resulted in slightly enlarged cartilage pellets containing an increased proportion of proliferating immature chondrocytes that expressed very low levels of hypertrophic/terminally matured chondrocyte-specific genes. Forskolin treatment also enhanced hyaline cartilage formation by reducing type I collagen gene expression and increasing sulfated glycosaminoglycan accumulation in the developed cartilage. Chondrogenic mesoderm from hPSCs and dedifferentiated nasal chondrocytes responded similarly to forskolin. Furthermore, forskolin treatment in vitro increased the frequency at which the cartilage pellets maintained unminer...
PloS one
Moyamoya disease (MMD) is a slow, progressive steno-occlusive disease, arising in the terminal po... more Moyamoya disease (MMD) is a slow, progressive steno-occlusive disease, arising in the terminal portions of the cerebral internal carotid artery. However, the functions and characteristics of the endothelial cells (ECs) in MMD are unknown. We analyzed these features using induced pluripotent stem cell (iPSC)-derived ECs. iPSC lines were established from the peripheral blood of three patients with MMD carrying the variant RNF213 R4810K, and three healthy persons used as controls. After the endothelial differentiation of iPSCs, CD31+CD144+ cells were purified as ECs using a cell sorter. We analyzed their proliferation, angiogenesis, and responses to some angiogenic factors, namely VEGF, bFGF, TGF-β, and BMP4. The ECs were also analyzed using DNA microarray and proteomics to perform comprehensive gene and protein expression analysis. Angiogenesis was significantly impaired in MMD regardless of the presence of any angiogenic factor. On the contrary, endothelial proliferation was not sign...
Molecular therapy. Methods & clinical development, 2016
Three populations of muscle-derived cells (PP1, PP3, and PP6) were isolated from mouse skeletal m... more Three populations of muscle-derived cells (PP1, PP3, and PP6) were isolated from mouse skeletal muscle using modified preplate technique and retrovirally transduced with BMP4/GFP. In vitro, the PP1 cells (fibroblasts) proliferated significantly slower than the PP3 (myoblasts) and PP6 cells (muscle-derived stem cells); the PP1 and PP6 cells showed a superior rate of survival compared with PP3 cells under oxidative stress; and the PP6 cells showed significantly superior chondrogenic capabilities than PP1 and PP3 cells. In vivo, the PP6 cells promoted superior cartilage regeneration compared with the other muscle-derived cell populations. The cartilage defects in the PP6 group had significantly higher histological scores than those of the other muscle-derived cell groups, and GFP detection revealed that the transplanted PP6 cells showed superior in vivo cell survival and chondrogenic capabilities compared with the PP1 and PP3 cells. PP6 cells (muscle-derived stem cells) are superior t...