Anterior pituitary cells defective in the cell-autonomous factor, df, undergo cell lineage specification but not expansion.

PubMed

Gage, P J; Roller, M L; Saunders, T L; Scarlett, L M; Camper, S A

1996-01-01

The Ames dwarf mouse transmits a recessive mutation (df) resulting in a profound anterior pituitary hypocellularity due to a general lack of thyrotropes, somatotropes and lactotropes. These cell types are also dependent on the pituitary-specific transcription factor, Pit-1. We present evidence that expression of Pit-1 and limited commitment to these cells lineages occurs in df/df pituitaries. Thus, the crucial role of df may be in lineage-specific proliferation, rather than cytodifferentiation. The presence of all three Pit-1-dependent cell types in clonally derived clusters provides compelling evidence that these three lineages share a common, pluripotent precursor cell. Clusters containing different combinations of Pit-1-dependent cell types suggests that the Pit-1+ precursor cells choose from multiple developmental options during ontogeny. Characterization of df/df<-->+/+ chimeric mice demonstrated that df functions by a cell-autonomous mechanism. Therefore, df and Pit-1 are both cell-autonomous factors required for thyrotrope, somatotrope and lactotrope ontogeny, but their relative roles are different.

From the cradle to the grave: activities of GATA-3 throughout T cell development and differentiation

PubMed Central

Hosoya, Tomonori; Maillard, Ivan; Engel, James Douglas

2010-01-01

Summary GATA family transcription factors play multiple vital roles in hematopoiesis in many cell lineages, and in particular, T cells require GATA-3 for execution of several developmental steps. Transcriptional activation of the Gata3 gene is observed throughout T-cell development and differentiation in stage-specific fashion. GATA-3 has been described as a master regulator of T-helper 2 (Th2) cell differentiation in mature CD4+ T cells. During T-cell development in the thymus, its roles in the CD4 vs. CD8 lineage choice and at the β-selection checkpoint are the best characterized. In contrast, its importance prior to β-selection has been obscured both by the developmental heterogeneity of double negative (DN) 1 thymocytes and the paucity of early T-lineage progenitors (ETPs), a subpopulation of DN1 cells that contains the most immature thymic progenitors that retain potent T-lineage developmental potential. By examining multiple lines of in vivo evidence procured through the analysis of Gata3 mutant mice, we have recently demonstrated that GATA-3 is additionally required at the earliest stage of thymopoiesis for the development of the ETP population. Here, we review the characterized functions of GATA-3 at each stage of T-cell development and discuss hypothetical molecular pathways that mediate these functions. PMID:20969588

Evolution of plant parasitism in the phylum Nematoda.

PubMed

Quist, Casper W; Smant, Geert; Helder, Johannes

2015-01-01

Within the species-rich and trophically diverse phylum Nematoda, at least four independent major lineages of plant parasites have evolved, and in at least one of these major lineages plant parasitism arose independently multiple times. Ribosomal DNA data, sequence information from nematode-produced, plant cell wall-modifying enzymes, and the morphology and origin of the style(t), a protrusible piercing device used to penetrate the plant cell wall, all suggest that facultative and obligate plant parasites originate from fungivorous ancestors. Data on the nature and diversification of plant cell wall-modifying enzymes point at multiple horizontal gene transfer events from soil bacteria to bacterivorous nematodes resulting in several distinct lineages of fungal or oomycete-feeding nematodes. Ribosomal DNA frameworks with sequence data from more than 2,700 nematode taxa combined with detailed morphological information allow for explicit hypotheses on the origin of agronomically important plant parasites, such as root-knot, cyst, and lesion nematodes.

Dissecting Embryonic Stem Cell Self-Renewal and Differentiation Commitment from Quantitative Models.

PubMed

Hu, Rong; Dai, Xianhua; Dai, Zhiming; Xiang, Qian; Cai, Yanning

2016-10-01

To model quantitatively embryonic stem cell (ESC) self-renewal and differentiation by computational approaches, we developed a unified mathematical model for gene expression involved in cell fate choices. Our quantitative model comprised ESC master regulators and lineage-specific pivotal genes. It took the factors of multiple pathways as input and computed expression as a function of intrinsic transcription factors, extrinsic cues, epigenetic modifications, and antagonism between ESC master regulators and lineage-specific pivotal genes. In the model, the differential equations of expression of genes involved in cell fate choices from regulation relationship were established according to the transcription and degradation rates. We applied this model to the Murine ESC self-renewal and differentiation commitment and found that it modeled the expression patterns with good accuracy. Our model analysis revealed that Murine ESC was an attractor state in culture and differentiation was predominantly caused by antagonism between ESC master regulators and lineage-specific pivotal genes. Moreover, antagonism among lineages played a critical role in lineage reprogramming. Our results also uncovered that the ordered expression alteration of ESC master regulators over time had a central role in ESC differentiation fates. Our computational framework was generally applicable to most cell-type maintenance and lineage reprogramming.

Lineage tracing of genome-edited alleles reveals high fidelity axolotl limb regeneration.

PubMed

Flowers, Grant Parker; Sanor, Lucas D; Crews, Craig M

2017-09-16

Salamanders are unparalleled among tetrapods in their ability to regenerate many structures, including entire limbs, and the study of this ability may provide insights into human regenerative therapies. The complex structure of the limb poses challenges to the investigation of the cellular and molecular basis of its regeneration. Using CRISPR/Cas, we genetically labelled unique cell lineages within the developing axolotl embryo and tracked the frequency of each lineage within amputated and fully regenerated limbs. This allowed us, for the first time, to assess the contributions of multiple low frequency cell lineages to the regenerating limb at once. Our comparisons reveal that regenerated limbs are high fidelity replicas of the originals even after repeated amputations.

Pericytes of Multiple Organs Do Not Behave as Mesenchymal Stem Cells In Vivo.

PubMed

Guimarães-Camboa, Nuno; Cattaneo, Paola; Sun, Yunfu; Moore-Morris, Thomas; Gu, Yusu; Dalton, Nancy D; Rockenstein, Edward; Masliah, Eliezer; Peterson, Kirk L; Stallcup, William B; Chen, Ju; Evans, Sylvia M

2017-03-02

Pericytes are widely believed to function as mesenchymal stem cells (MSCs), multipotent tissue-resident progenitors with great potential for regenerative medicine. Cultured pericytes isolated from distinct tissues can differentiate into multiple cell types in vitro or following transplantation in vivo. However, the cell fate plasticity of endogenous pericytes in vivo remains unclear. Here, we show that the transcription factor Tbx18 selectively marks pericytes and vascular smooth muscle cells in multiple organs of adult mouse. Fluorescence-activated cell sorting (FACS)-purified Tbx18-expressing cells behaved as MSCs in vitro. However, lineage-tracing experiments using an inducible Tbx18-CreERT2 line revealed that pericytes and vascular smooth muscle cells maintained their identity in aging and diverse pathological settings and did not significantly contribute to other cell lineages. These results challenge the current view of endogenous pericytes as multipotent tissue-resident progenitors and suggest that the plasticity observed in vitro or following transplantation in vivo arises from artificial cell manipulations ex vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Lineage plasticity-mediated therapy resistance in prostate cancer.

PubMed

Blee, Alexandra M; Huang, Haojie

2018-06-12

Therapy resistance is a significant challenge for prostate cancer treatment in clinic. Although targeted therapies such as androgen deprivation and androgen receptor (AR) inhibition are effective initially, tumor cells eventually evade these strategies through multiple mechanisms. Lineage reprogramming in response to hormone therapy represents a key mechanism that is increasingly observed. The studies in this area have revealed specific combinations of alterations present in adenocarcinomas that provide cells with the ability to transdifferentiate and perpetuate AR-independent tumor growth after androgen-based therapies. Interestingly, several master regulators have been identified that drive plasticity, some of which also play key roles during development and differentiation of the cell lineages in the normal prostate. Thus, further study of each AR-independent tumor type and understanding underlying mechanisms are warranted to develop combinational therapies that combat lineage plasticity in prostate cancer.

Multiple origins of interdependent endosymbiotic complexes in a genus of cicadas.

PubMed

Łukasik, Piotr; Nazario, Katherine; Van Leuven, James T; Campbell, Matthew A; Meyer, Mariah; Michalik, Anna; Pessacq, Pablo; Simon, Chris; Veloso, Claudio; McCutcheon, John P

2018-01-09

Bacterial endosymbionts that provide nutrients to hosts often have genomes that are extremely stable in structure and gene content. In contrast, the genome of the endosymbiont Hodgkinia cicadicola has fractured into multiple distinct lineages in some species of the cicada genus Tettigades To better understand the frequency, timing, and outcomes of Hodgkinia lineage splitting throughout this cicada genus, we sampled cicadas over three field seasons in Chile and performed genomics and microscopy on representative samples. We found that a single ancestral Hodgkinia lineage has split at least six independent times in Tettigades over the last 4 million years, resulting in complexes of between two and six distinct Hodgkinia lineages per host. Individual genomes in these symbiotic complexes differ dramatically in relative abundance, genome size, organization, and gene content. Each Hodgkinia lineage retains a small set of core genes involved in genetic information processing, but the high level of gene loss experienced by all genomes suggests that extensive sharing of gene products among symbiont cells must occur. In total, Hodgkinia complexes that consist of multiple lineages encode nearly complete sets of genes present on the ancestral single lineage and presumably perform the same functions as symbionts that have not undergone splitting. However, differences in the timing of the splits, along with dissimilar gene loss patterns on the resulting genomes, have led to very different outcomes of lineage splitting in extant cicadas.

Novel Bruton’s tyrosine kinase inhibitors currently in development

PubMed Central

D’Cruz, Osmond J; Uckun, Fatih M

2013-01-01

Bruton’s tyrosine kinase (Btk) is intimately involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is overexpressed and constitutively active in several B-lineage lymphoid malignancies. Btk has emerged as a new antiapoptotic molecular target for treatment of B-lineage leukemias and lymphomas. Preclinical and early clinical results indicate that Btk inhibitors may be useful in the treatment of leukemias and lymphomas. PMID:23493945

Sca-1 Identifies a Distinct Androgen-Independent Murine Prostatic Luminal Cell Lineage with Bipotent Potential

PubMed Central

Kwon, Oh-Joon; Zhang, Li; Xin, Li

2016-01-01

Recent lineage tracing studies support the existence of prostate luminal progenitors that possess extensive regenerative capacity, but their identity remains unknown. We show that Sca-1 (Stem Cell Antigen-1) identifies a small population of murine prostate luminal cells that reside in the proximal prostatic ducts adjacent to the urethra. Sca-1+ luminal cells do not express Nkx3.1. They do not carry the secretory function, although they express the androgen receptor. These cells are enriched in the prostates of castrated mice. In the in vitro prostate organoid assay, a small fraction of the Sca-1+ luminal cells are capable of generating budding organoids that are morphologically distinct from those derived from other cell lineages. Histologically, this type of organoid is composed of multiple inner layers of luminal cells surrounded by multiple outer layers of basal cells. When passaged, these organoids retain their morphological and histological features. Finally, the Sca-1+ luminal cells are capable of forming small prostate glands containing both basal and luminal cells in an in vivo prostate regeneration assay. Collectively, our study establishes the androgen-independent and bipotent organoid-forming Sca-1+ luminal cells as a functionally distinct cellular entity. These cells may represent a putative luminal progenitor population and serve as a cellular origin for castration resistant prostate cancer. PMID:26418304

Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum

PubMed Central

Choi, Eunyoung; Roland, Joseph T.; Barlow, Brittney J.; O’Neal, Ryan; Rich, Amy E.; Nam, Ki Taek; Shi, Chanjuan; Goldenring, James R.

2014-01-01

Objective The glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical regions within the stomach. We examined in detail the distribution of the full range of cell lineages within the human stomach. Design We determined the distribution of gastric gland cell lineages with specific immunocytochemical markers in entire stomach specimens from three non-obese organ donors. Results The anatomical body and antrum of the human stomach were defined by the presence of ghrelin and gastrin cells, respectively. Concentrations of somatostatin cells were observed in the proximal stomach. Parietal cells were seen in all glands of the body of stomach as well as in over 50% of antral glands. MIST1-expressing chief cells were predominantly observed in the body, although individual glands of the antrum also showed MIST1-expressing chief cells. While classically-described antral glands were observed with gastrin cells and deep antral mucous cells without any parietal cells, we also observed a substantial population of mixed-type glands containing both parietal cells and G cells throughout the antrum. Conclusions Enteroendocrine cells show distinct patterns of localization in the human stomach. The existence of antral glands with mixed cell lineages indicates that human antral glands may be functionally chimeric with glands assembled from multiple distinct stem cell populations. PMID:24488499

Multiple inductive signals are involved in the development of the ctenophore Mnemiopsis leidyi

NASA Technical Reports Server (NTRS)

Henry, J. Q.; Martindale, M. Q.

2001-01-01

Ctenophores possess eight longitudinally arrayed rows of comb plate cilia. Previous intracellular cell lineage analysis has shown that these comb rows are derived from two embryonic lineages, both daughters of the four e(1) micromeres (e(11) and e(12)) and a single daughter of the four m(1) micromeres (the m(12) micromeres). Although isolated e(1) micromeres will spontaneously generate comb plates, cell deletion experiments have shown that no comb plates appear during embryogenesis following the removal of e(1) descendents. Thus, the m(1) lineage requires the inductive interaction of the e(1) lineage to contribute to comb plate formation. Here we show that, although m(12) cells are normally the only m(1) derivatives to contribute to comb plate formation, m(11) cells are capable of generating comb plates in the absence m(12) cells. The reason that m(11) cells do not normally make comb rows may be attributable either to their more remote location relative to critical signaling centers (e.g., e(1) descendants) or to inhibitory signals that may be provided by other nearby cells such as sister cells m(12). In addition, we show that the signals provided by the e(1) lineage are not sufficient for m(1)-derived comb plate formation. Signals provided by endomesodermal progeny of either the E or the M lineages (the 3E or 2M macromeres) are also required. Copyright 2001 Academic Press.

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway* | Office of Cancer Genomics

Cancer.gov

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumors and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood.

Adipocyte Metabolic Pathways Regulated by Diet Control the Female Germline Stem Cell Lineage in Drosophila melanogaster

PubMed Central

Matsuoka, Shinya; Armstrong, Alissa R.; Sampson, Leesa L.; Laws, Kaitlin M.; Drummond-Barbosa, Daniela

2017-01-01

Nutrients affect adult stem cells through complex mechanisms involving multiple organs. Adipocytes are highly sensitive to diet and have key metabolic roles, and obesity increases the risk for many cancers. How diet-regulated adipocyte metabolic pathways influence normal stem cell lineages, however, remains unclear. Drosophila melanogaster has highly conserved adipocyte metabolism and a well-characterized female germline stem cell (GSC) lineage response to diet. Here, we conducted an isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to identify diet-regulated adipocyte metabolic pathways that control the female GSC lineage. On a rich (relative to poor) diet, adipocyte Hexokinase-C and metabolic enzymes involved in pyruvate/acetyl-CoA production are upregulated, promoting a shift of glucose metabolism toward macromolecule biosynthesis. Adipocyte-specific knockdown shows that these enzymes support early GSC progeny survival. Further, enzymes catalyzing fatty acid oxidation and phosphatidylethanolamine synthesis in adipocytes promote GSC maintenance, whereas lipid and iron transport from adipocytes controls vitellogenesis and GSC number, respectively. These results show a functional relationship between specific metabolic pathways in adipocytes and distinct processes in the GSC lineage, suggesting the adipocyte metabolism–stem cell link as an important area of investigation in other stem cell systems. PMID:28396508

Adipocyte Metabolic Pathways Regulated by Diet Control the Female Germline Stem Cell Lineage in Drosophila melanogaster.

PubMed

Matsuoka, Shinya; Armstrong, Alissa R; Sampson, Leesa L; Laws, Kaitlin M; Drummond-Barbosa, Daniela

2017-06-01

Nutrients affect adult stem cells through complex mechanisms involving multiple organs. Adipocytes are highly sensitive to diet and have key metabolic roles, and obesity increases the risk for many cancers. How diet-regulated adipocyte metabolic pathways influence normal stem cell lineages, however, remains unclear. Drosophila melanogaster has highly conserved adipocyte metabolism and a well-characterized female germline stem cell (GSC) lineage response to diet. Here, we conducted an isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to identify diet-regulated adipocyte metabolic pathways that control the female GSC lineage. On a rich (relative to poor) diet, adipocyte Hexokinase-C and metabolic enzymes involved in pyruvate/acetyl-CoA production are upregulated, promoting a shift of glucose metabolism toward macromolecule biosynthesis. Adipocyte-specific knockdown shows that these enzymes support early GSC progeny survival. Further, enzymes catalyzing fatty acid oxidation and phosphatidylethanolamine synthesis in adipocytes promote GSC maintenance, whereas lipid and iron transport from adipocytes controls vitellogenesis and GSC number, respectively. These results show a functional relationship between specific metabolic pathways in adipocytes and distinct processes in the GSC lineage, suggesting the adipocyte metabolism-stem cell link as an important area of investigation in other stem cell systems. Copyright © 2017 by the Genetics Society of America.

Concurrent detection of secreted products from human lymphocytes by microengraving: cytokines and antigen-reactive antibodies

PubMed Central

Bradshaw, Elizabeth M.; Kent, Sally C.; Tripuraneni, Vinay; Orban, Tihamer; Ploegh, Hidde L.; Hafler, David A.; Love, J. Christopher

2008-01-01

Cell surface determinants, cytokines and antibodies secreted by hematopoietic cells are used to classify their lineage and function. Currently available techniques are unable to elucidate multiple secreted proteins while also assigning phenotypic surface-displayed markers to the individual living cells. Here, a soft lithographic method, microengraving, was adapted for the multiplexed interrogation of populations of individual human peripheral blood mononuclear cells for secreted cytokines (IFN-γ and IL-6), antigen-specific antibodies, and lineage-specific surface-expressed markers. Application of the method to a clinical sample from a recent onset Type 1 diabetic subject with a positive titer of anti-insulin antibodies showed that ~0.58% of circulating CD19+ B cells secreted proinsulin-reactive antibodies of the IgG isotype and 2–3% of circulating cells secreted IL-6. These data demonstrate the utility of microengraving for interrogating multiple phenotypes of single human cells concurrently and for detecting rare populations of cells by their secreted products. PMID:18675591

Two hemocyte lineages exist in silkworm larval hematopoietic organ.

PubMed

Nakahara, Yuichi; Kanamori, Yasushi; Kiuchi, Makoto; Kamimura, Manabu

2010-07-28

Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster. To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids. From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori.

Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum.

PubMed

Choi, Eunyoung; Roland, Joseph T; Barlow, Brittney J; O'Neal, Ryan; Rich, Amy E; Nam, Ki Taek; Shi, Chanjuan; Goldenring, James R

2014-11-01

The glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical regions within the stomach. We examined in detail the distribution of the full range of cell lineages within the human stomach. We determined the distribution of gastric gland cell lineages with specific immunocytochemical markers in entire stomach specimens from three non-obese organ donors. The anatomical body and antrum of the human stomach were defined by the presence of ghrelin and gastrin cells, respectively. Concentrations of somatostatin cells were observed in the proximal stomach. Parietal cells were seen in all glands of the body of the stomach as well as in over 50% of antral glands. MIST1 expressing chief cells were predominantly observed in the body although individual glands of the antrum also showed MIST1 expressing chief cells. While classically described antral glands were observed with gastrin cells and deep antral mucous cells without any parietal cells, we also observed a substantial population of mixed type glands containing both parietal cells and G cells throughout the antrum. Enteroendocrine cells show distinct patterns of localisation in the human stomach. The existence of antral glands with mixed cell lineages indicates that human antral glands may be functionally chimeric with glands assembled from multiple distinct stem cell populations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Loss of Asxl1 leads to myelodysplastic syndrome-like disease in mice.

PubMed

Wang, Jiapeng; Li, Zhaomin; He, Yongzheng; Pan, Feng; Chen, Shi; Rhodes, Steven; Nguyen, Lihn; Yuan, Jin; Jiang, Li; Yang, Xianlin; Weeks, Ophelia; Liu, Ziyue; Zhou, Jiehao; Ni, Hongyu; Cai, Chen-Leng; Xu, Mingjiang; Yang, Feng-Chun

2014-01-23

ASXL1 is mutated/deleted with high frequencies in multiple forms of myeloid malignancies, and its alterations are associated with poor prognosis. De novo ASXL1 mutations cause Bohring-Opitz syndrome characterized by multiple congenital malformations. We show that Asxl1 deletion in mice led to developmental abnormalities including dwarfism, anophthalmia, and 80% embryonic lethality. Surviving Asxl1(-/-) mice lived for up to 42 days and developed features of myelodysplastic syndrome (MDS), including dysplastic neutrophils and multiple lineage cytopenia. Asxl1(-/-) mice had a reduced hematopoietic stem cell (HSC) pool, and Asxl1(-/-) HSCs exhibited decreased hematopoietic repopulating capacity, with skewed cell differentiation favoring granulocytic lineage. Asxl1(+/-) mice also developed mild MDS-like disease, which could progress to MDS/myeloproliferative neoplasm, demonstrating a haploinsufficient effect of Asxl1 in the pathogenesis of myeloid malignancies. Asxl1 loss led to an increased apoptosis and mitosis in Lineage(-)c-Kit(+) (Lin(-)c-Kit(+)) cells, consistent with human MDS. Furthermore, Asxl1(-/-) Lin(-)c-Kit(+) cells exhibited decreased global levels of H3K27me3 and H3K4me3 and altered expression of genes regulating apoptosis (Bcl2, Bcl2l12, Bcl2l13). Collectively, we report a novel ASXL1 murine model that recapitulates human myeloid malignancies, implying that Asxl1 functions as a tumor suppressor to maintain hematopoietic cell homeostasis. Future work is necessary to clarify the contribution of microenvironment to the hematopoietic phenotypes observed in the constitutional Asxl1(-/-) mice.

Conditional Creation and Rescue of Nipbl-Deficiency in Mice Reveals Multiple Determinants of Risk for Congenital Heart Defects

PubMed Central

Jacobs, Russell E.; Lopez-Burks, Martha E.; Choi, Hojae; Wikenheiser, Jamie; Hallgrimsson, Benedikt; Jamniczky, Heather A.; Fraser, Scott E.; Lander, Arthur D.; Calof, Anne L.

2016-01-01

Elucidating the causes of congenital heart defects is made difficult by the complex morphogenesis of the mammalian heart, which takes place early in development, involves contributions from multiple germ layers, and is controlled by many genes. Here, we use a conditional/invertible genetic strategy to identify the cell lineage(s) responsible for the development of heart defects in a Nipbl-deficient mouse model of Cornelia de Lange Syndrome, in which global yet subtle transcriptional dysregulation leads to development of atrial septal defects (ASDs) at high frequency. Using an approach that allows for recombinase-mediated creation or rescue of Nipbl deficiency in different lineages, we uncover complex interactions between the cardiac mesoderm, endoderm, and the rest of the embryo, whereby the risk conferred by genetic abnormality in any one lineage is modified, in a surprisingly non-additive way, by the status of others. We argue that these results are best understood in the context of a model in which the risk of heart defects is associated with the adequacy of early progenitor cell populations relative to the sizes of the structures they must eventually form. PMID:27606604

Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ

PubMed Central

Nakahara, Yuichi; Kanamori, Yasushi; Kiuchi, Makoto; Kamimura, Manabu

2010-01-01

Background Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster. Methodology/Principal Findings To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids. Conclusions/Significance From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori. PMID:20676370

The Immunomodulatory and Neuroprotective Effects of Mesenchymal Stem Cells (MSCs) in Experimental Autoimmune Encephalomyelitis (EAE): A Model of Multiple Sclerosis (MS)

PubMed Central

Al Jumah, Mohammed A.; Abumaree, Mohamed H.

2012-01-01

Mesenchymal stem cells (MSCs) are multipotent cells that differentiate into the mesenchymal lineages of adipocytes, osteocytes and chondrocytes. MSCs can also transdifferentiate and thereby cross lineage barriers, differentiating for example into neurons under certain experimental conditions. MSCs have anti-proliferative, anti-inflammatory and anti-apoptotic effects on neurons. Therefore, MSCs were tested in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), for their effectiveness in modulating the pathogenic process in EAE to develop effective therapies for MS. The data in the literature have shown that MSCs can inhibit the functions of autoreactive T cells in EAE and that this immunomodulation can be neuroprotective. In addition, MSCs can rescue neural cells via a mechanism that is mediated by soluble factors, which provide a suitable environment for neuron regeneration, remyelination and cerebral blood flow improvement. In this review, we discuss the effectiveness of MSCs in modulating the immunopathogenic process and in providing neuroprotection in EAE. PMID:22942767

Prenatal Ablation of Nicotinic Receptor alpha7 Cell Lineages Produces Lumbosacral Spina Bifida the Severity of Which is Modified by Choline and Nicotine Exposure

PubMed Central

Rogers, Scott W; Tvrdik, Petr; Capecchi, Mario R; Gahring, Lorise C

2012-01-01

Lumbosacral spina bifida is a common debilitating birth defect whose multiple causes are poorly understood. Here, we provide the first genetic delineation of cholinergic nicotinic receptor alpha7 (Chrna7) expression and link the ablation of the Chrna7 cell lineage to this condition in the mouse. Using homologous recombination, an IRES-Cre bi-cistronic cassette was introduced into the 3′ noncoding region of Chrna7 (Chrna7:Cre) for identifying cell lineages expressing this gene. This lineage first appears at embryonic day E9.0 in rhombomeres 3 and 5 of the neural tube and extends to cell subsets in most tissues by E14.5. Ablation of the Chrna7:Cre cell lineage in embryos from crosses with conditionally expressed attenuated diphtheria toxin results in precise developmental defects including omphalocele (89%) and open spina bifida (SB; 80%). We hypothesized that like humans, this defect would be modified by environmental compounds not only folic acid or choline but also nicotine. Prenatal chronic oral nicotine administration substantially worsened the defect to often include the rostral neural tube. In contrast, supplementation of the maternal diet with 2% choline decreased SB prevalence to 38% and dramatically reduced the defect severity. Folic acid supplementation only trended towards a reduced SB frequency. The omphalocele was unaffected by these interventions. These studies identify the Chrna7 cell lineage as participating in posterior neuropore closure and present a novel model of lower SB that can be substantially modified by the prenatal environment. © 2012 Wiley Periodicals, Inc. PMID:22473653

Human and murine very small embryonic-like cells represent multipotent tissue progenitors, in vitro and in vivo.

PubMed

Havens, Aaron M; Sun, Hongli; Shiozawa, Yusuke; Jung, Younghun; Wang, Jingcheng; Mishra, Anjali; Jiang, Yajuan; O'Neill, David W; Krebsbach, Paul H; Rodgerson, Denis O; Taichman, Russell S

2014-04-01

The purpose of this study was to determine the lineage progression of human and murine very small embryonic-like (HuVSEL or MuVSEL) cells in vitro and in vivo. In vitro, HuVSEL and MuVSEL cells differentiated into cells of all three embryonic germ layers. HuVSEL cells produced robust mineralized tissue of human origin compared with controls in calvarial defects. Immunohistochemistry demonstrated that the HuVSEL cells gave rise to neurons, adipocytes, chondrocytes, and osteoblasts within the calvarial defects. MuVSEL cells were also able to differentiate into similar lineages. First round serial transplants of MuVSEL cells into irradiated osseous sites demonstrated that ∼60% of the cells maintained their VSEL cell phenotype while other cells differentiated into multiple tissues at 3 months. Secondary transplants did not identify donor VSEL cells, suggesting limited self renewal but did demonstrate VSEL cell derivatives in situ for up to 1 year. At no point were teratomas identified. These studies show that VSEL cells produce multiple cellular structures in vivo and in vitro and lay the foundation for future cell-based regenerative therapies for osseous, neural, and connective tissue disorders.

Concise classification of the genomic porcine endogenous retroviral gamma1 load to defined lineages.

PubMed

Klymiuk, Nikolai; Wolf, Eckhard; Aigner, Bernhard

2008-02-05

We investigated the infection history of porcine endogenous retroviruses (PERV) gamma1 by analyzing published env and LTR sequences. PERV sequences from various breeds, porcine cell lines and infected human primary cells were included in the study. We identified a considerable number of retroviral lineages indicating multiple independent colonization events of the porcine genome. A recent boost of the proviral load in an isolated pig herd and exclusive occurrence of distinct lineages in single studies indicated the ongoing colonization of the porcine genome with endogenous retroviruses. Retroviral recombination between co-packaged genomes was a general factor for PERV gamma1 diversity which indicated the simultaneous expression of different proviral loci over a period of time. In total, our detailed description of endogenous retroviral lineages is the prerequisite for breeding approaches to minimize the infectious potential of porcine tissues for the subsequent use in xenotransplantation.

Tracing cell lineages in videos of lens-free microscopy.

PubMed

Rempfler, Markus; Stierle, Valentin; Ditzel, Konstantin; Kumar, Sanjeev; Paulitschke, Philipp; Andres, Bjoern; Menze, Bjoern H

2018-06-05

In vitro experiments with cultured cells are essential for studying their growth and migration pattern and thus, for gaining a better understanding of cancer progression and its treatment. Recent progress in lens-free microscopy (LFM) has rendered it an inexpensive tool for label-free, continuous live cell imaging, yet there is only little work on analysing such time-lapse image sequences. We propose (1) a cell detector for LFM images based on fully convolutional networks and residual learning, and (2) a probabilistic model based on moral lineage tracing that explicitly handles multiple detections and temporal successor hypotheses by clustering and tracking simultaneously. (3) We benchmark our method in terms of detection and tracking scores on a dataset of three annotated sequences of several hours of LFM, where we demonstrate our method to produce high quality lineages. (4) We evaluate its performance on a somewhat more challenging problem: estimating cell lineages from the LFM sequence as would be possible from a corresponding fluorescence microscopy sequence. We present experiments on 16 LFM sequences for which we acquired fluorescence microscopy in parallel and generated annotations from them. Finally, (5) we showcase our methods effectiveness for quantifying cell dynamics in an experiment with skin cancer cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Temporal Transcriptional Profiling of Somatic and Germ Cells Reveals Biased Lineage Priming of Sexual Fate in the Fetal Mouse Gonad

PubMed Central

Jameson, Samantha A.; Natarajan, Anirudh; Cool, Jonah; DeFalco, Tony; Maatouk, Danielle M.; Mork, Lindsey; Munger, Steven C.; Capel, Blanche

2012-01-01

The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates. PMID:22438826

Mapping human pluripotent stem cell differentiation pathways using high throughput single-cell RNA-sequencing.

PubMed

Han, Xiaoping; Chen, Haide; Huang, Daosheng; Chen, Huidong; Fei, Lijiang; Cheng, Chen; Huang, He; Yuan, Guo-Cheng; Guo, Guoji

2018-04-05

Human pluripotent stem cells (hPSCs) provide powerful models for studying cellular differentiations and unlimited sources of cells for regenerative medicine. However, a comprehensive single-cell level differentiation roadmap for hPSCs has not been achieved. We use high throughput single-cell RNA-sequencing (scRNA-seq), based on optimized microfluidic circuits, to profile early differentiation lineages in the human embryoid body system. We present a cellular-state landscape for hPSC early differentiation that covers multiple cellular lineages, including neural, muscle, endothelial, stromal, liver, and epithelial cells. Through pseudotime analysis, we construct the developmental trajectories of these progenitor cells and reveal the gene expression dynamics in the process of cell differentiation. We further reprogram primed H9 cells into naïve-like H9 cells to study the cellular-state transition process. We find that genes related to hemogenic endothelium development are enriched in naïve-like H9. Functionally, naïve-like H9 show higher potency for differentiation into hematopoietic lineages than primed cells. Our single-cell analysis reveals the cellular-state landscape of hPSC early differentiation, offering new insights that can be harnessed for optimization of differentiation protocols.

A mex3 homolog is required for differentiation during planarian stem cell lineage development

PubMed Central

Zhu, Shu Jun; Hallows, Stephanie E; Currie, Ko W; Xu, ChangJiang; Pearson, Bret J

2015-01-01

Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment. DOI: http://dx.doi.org/10.7554/eLife.07025.001 PMID:26114597

Mesenchymal Stem Cell Therapy for Nerve Regeneration and Immunomodulation after Composite Tissue Allotransplantation

DTIC Science & Technology

2012-02-01

10-1-0927 TITLE: Mesenchymal Stem Cell Therapy for Nerve Regeneration and Immunomodulation after Composite Tissue Allotransplantation...immunosuppression. Bone Marrow Derived Mesenchymal stem cells (BM-MSCs) are pluripotent cells, capable of differentiation along multiple mesenchymal lineages into...As part of implemented transition from University of Pittsburgh to Johns Hopkins University, we optimized our mesenchymal stem cell (MSC) isolation

A minimal spatial cell lineage model of epithelium: tissue stratification and multi-stability

NASA Astrophysics Data System (ADS)

Yeh, Wei-Ting; Chen, Hsuan-Yi

2018-05-01

A minimal model which includes spatial and cell lineage dynamics for stratified epithelia is presented. The dependence of tissue steady state on cell differentiation models, cell proliferation rate, cell differentiation rate, and other parameters are studied numerically and analytically. Our minimal model shows some important features. First, we find that morphogen or mechanical stress mediated interaction is necessary to maintain a healthy stratified epithelium. Furthermore, comparing with tissues in which cell differentiation can take place only during cell division, tissues in which cell division and cell differentiation are decoupled can achieve relatively higher degree of stratification. Finally, our model also shows that in the presence of short-range interactions, it is possible for a tissue to have multiple steady states. The relation between our results and tissue morphogenesis or lesion is discussed.

Expression of different functional isoforms in haematopoiesis.

PubMed

Grech, Godfrey; Pollacco, Joel; Portelli, Mark; Sacco, Keith; Baldacchino, Shawn; Grixti, Justine; Saliba, Christian

2014-01-01

Haematopoiesis is a complex process regulated at various levels facilitating rapid responses to external factors including stress, modulation of lineage commitment and terminal differentiation of progenitors. Although the transcription program determines the RNA pool of a cell, various mRNA strands can be obtained from the same template, giving rise to multiple protein isoforms. The majority of variants and isoforms co-occur in normal haematopoietic cells or are differentially expressed at various maturity stages of progenitor maturation and cellular differentiation within the same lineage or across lineages. Genetic aberrations or specific cellular states result in the predominant expression of abnormal isoforms leading to deregulation and disease. The presence of upstream open reading frames (uORF) in 5' untranslated regions (UTRs) of a transcript, couples the utilization of start codons with the cellular status and availability of translation initiation factors (eIFs). In addition, tissue-specific and cell lineage-specific alternative promoter use, regulates several transcription factors producing transcript variants with variable 5' exons. In this review, we propose to give a detailed account of the differential isoform formation, causing haematological malignancies.

Cochlear hair cell regeneration after noise-induced hearing loss: does regeneration follow development?

PubMed Central

Zheng, Fei; Zuo, Jian

2017-01-01

Noise-induced hearing loss (NIHL) affects a large number of military personnel and civilians. Regenerating inner-ear cochlear hair cells (HCs) is a promising strategy to restore hearing after NIHL. In this review, we first summarize recent transcriptome profile analysis of zebrafish lateral lines and chick utricles where spontaneous HC regeneration occurs after HC damage. We then discuss recent studies in other mammalian regenerative systems such as pancreas, heart and central nervous system. Both spontaneous and forced HC regeneration occurs in mammalian cochleae in vivo involving proliferation and direct lineage conversion. However, both processes are inefficient and incomplete, and decline with age. For direct lineage conversion in vivo in cochleae and in other systems, further improvement requires multiple factors, including transcription, epigenetic and trophic factors, with appropriate stoichiometry in appropriate architectural niche. Increasing evidence from other systems indicates that the molecular paths of direct lineage conversion may be different from those of normal developmental lineages. We therefore hypothesize that HC regeneration does not have to follow HC development and that epigenetic memory of supporting cells influences the HC regeneration, which may be a key to successful cochlear HC regeneration. Finally, we discuss recent efforts in viral gene therapy and drug discovery for HC regeneration. We hope that combination therapy targeting multiple factors and epigenetic signaling pathways will provide promising avenues for HC regeneration in humans with NIHL and other types of hearing loss. PMID:28034617

A Quantitative Proteomic Analysis of Hemogenic Endothelium Reveals Differential Regulation of Hematopoiesis by SOX17

PubMed Central

Clarke, Raedun L.; Robitaille, Aaron M.; Moon, Randall T.; Keller, Gordon

2015-01-01

Summary The in vitro derivation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is complicated by the existence of multiple overlapping embryonic blood cell programs called primitive, erythromyeloid progenitor (EMP), and definitive. As HSCs are only generated during the definitive stage of hematopoiesis, deciphering the regulatory pathways that control the emergence of this program and identifying markers that distinguish it from the other programs are essential. To identify definitive specific pathways and marker sets, we used label-free proteomics to determine the proteome of embryo-derived and mouse embryonic stem cell-derived VE-CADHERIN+CD45− definitive hematopoietic progenitors. With this approach, we identified Stat1 as a marker that distinguishes the definitive erythroid lineage from the primitive- and EMP-derived lineages. Additionally, we provide evidence that the generation of the Stat1+ definitive lineage is dependent on Sox17. These findings establish an approach for monitoring the emergence of definitive hematopoiesis in the PSC differentiation cultures. PMID:26267830

Multihormonal pituitary adenoma concomitant with Pit-1 and Tpit lineage cells causing acromegaly associated with subclinical Cushing's disease: a case report.

PubMed

Takiguchi, Tomoko; Koide, Hisashi; Nagano, Hidekazu; Nakayama, Akitoshi; Fujimoto, Masanori; Tamura, Ai; Komai, Eri; Shiga, Akina; Kono, Takashi; Higuchi, Seiichiro; Sakuma, Ikki; Hashimoto, Naoko; Suzuki, Sawako; Miyabayashi, Yui; Ishiwatari, Norio; Horiguchi, Kentaro; Nakatani, Yukio; Yokote, Koutaro; Tanaka, Tomoaki

2017-09-02

A functional pituitary adenoma can produce multiple anterior-pituitary hormones, such as growth hormone (GH) -producing adenomas (GHoma) with prolactin or thyrotropin stimulating hormone production in the same lineage. However, it is very rare that acromegaly shows subclinical Cushing's disease (SCD) beyond the lineage. Here we describe the involvement of intratumoral coexistence with 2 types of hormone-producing cells associated with different lineage in acromegaly concomitant with SCD. In our study, we performed clinical evaluation of the patient showing acromegaly with SCD. To elucidate the mechanisms of this pathology, we analyzed immunohistochemistry and gene expression of anterior-pituitary hormones and transcriptional factors in the resected pituitary tumor. On immunohistochemical staining, most of the tumor cells were strongly stained for GH antibody, while some cells were strongly positive for adrenocorticotropic hormone (ACTH). Gene expression analysis of a transsphenoidal surgery sample of the pituitary gland revealed that ACTH-related genes, such as POMC, Tpit, and NeuroD1 mRNA, had higher expression in the tumor tissue than the nonfunctional adenoma but lower expression compared to an adenoma of typical Cushing's disease. Further, double-labeling detection methods with a fluorescent stain for ACTH and GH demonstrated the coexistence of ACTH-positive cells (GH-negative) among the GH-positive cells in the tumor. Additionally, Pit-1 expression was reduced in the ACTH-positive cells from tumor tissue primary culture. Here we described a case of a pituitary tumor diagnosed with acromegaly associated with SCD. We performed quantitative-expression analyses of transcriptional factors of the tumor tissue and immunohistochemistry analysis of tumor-derived primary culture cells, which suggested that the multihormonal pituitary adenoma concomitant with Pit-1 and Tpit lineage cells caused acromegaly associated with SCD.

Human immunodeficiency viruses appear compartmentalized to the female genital tract in cross-sectional analyses but genital lineages do not persist over time.

PubMed

Bull, Marta E; Heath, Laura M; McKernan-Mullin, Jennifer L; Kraft, Kelli M; Acevedo, Luis; Hitti, Jane E; Cohn, Susan E; Tapia, Kenneth A; Holte, Sarah E; Dragavon, Joan A; Coombs, Robert W; Mullins, James I; Frenkel, Lisa M

2013-04-15

Whether unique human immunodeficiency type 1 (HIV) genotypes occur in the genital tract is important for vaccine development and management of drug resistant viruses. Multiple cross-sectional studies suggest HIV is compartmentalized within the female genital tract. We hypothesize that bursts of HIV replication and/or proliferation of infected cells captured in cross-sectional analyses drive compartmentalization but over time genital-specific viral lineages do not form; rather viruses mix between genital tract and blood. Eight women with ongoing HIV replication were studied during a period of 1.5 to 4.5 years. Multiple viral sequences were derived by single-genome amplification of the HIV C2-V5 region of env from genital secretions and blood plasma. Maximum likelihood phylogenies were evaluated for compartmentalization using 4 statistical tests. In cross-sectional analyses compartmentalization of genital from blood viruses was detected in three of eight women by all tests; this was associated with tissue specific clades containing multiple monotypic sequences. In longitudinal analysis, the tissues-specific clades did not persist to form viral lineages. Rather, across women, HIV lineages were comprised of both genital tract and blood sequences. The observation of genital-specific HIV clades only in cross-sectional analysis and an absence of genital-specific lineages in longitudinal analyses suggest a dynamic interchange of HIV variants between the female genital tract and blood.

Reciprocal Interactions between Multiple Myeloma Cells and Osteoprogenitor Cells Affect Bone Formation and Tumor Growth

DTIC Science & Technology

2015-12-01

cells (HSCs) are multipotent cells that differentiate into myeloid, lymphoid and erythroid lineages, and have short-term or long-term regenerative...All rights reserved Nature Reviews | Rheumatology a b MPP CMP CLP Lymphoid cells NK cellB cell T cell Megakaryocyte and erythrocytes Macrophage and...into other cell types. CLP, common lymphoid progenitor; CMP, common myeloid progenitor; MPP, multipotent progenitor; NK cell , natural killer cell . R E

The 4-Celled Tetrabaena socialis Nuclear Genome Reveals the Essential Components for Genetic Control of Cell Number at the Origin of Multicellularity in the Volvocine Lineage.

PubMed

Featherston, Jonathan; Arakaki, Yoko; Hanschen, Erik R; Ferris, Patrick J; Michod, Richard E; Olson, Bradley J S C; Nozaki, Hisayoshi; Durand, Pierre M

2018-04-01

Multicellularity is the premier example of a major evolutionary transition in individuality and was a foundational event in the evolution of macroscopic biodiversity. The volvocine chlorophyte lineage is well suited for studying this process. Extant members span unicellular, simple colonial, and obligate multicellular taxa with germ-soma differentiation. Here, we report the nuclear genome sequence of one of the most morphologically simple organisms in this lineage-the 4-celled colonial Tetrabaena socialis and compare this to the three other complete volvocine nuclear genomes. Using conservative estimates of gene family expansions a minimal set of expanded gene families was identified that associate with the origin of multicellularity. These families are rich in genes related to developmental processes. A subset of these families is lineage specific, which suggests that at a genomic level the evolution of multicellularity also includes lineage-specific molecular developments. Multiple points of evidence associate modifications to the ubiquitin proteasomal pathway (UPP) with the beginning of coloniality. Genes undergoing positive or accelerating selection in the multicellular volvocines were found to be enriched in components of the UPP and gene families gained at the origin of multicellularity include components of the UPP. A defining feature of colonial/multicellular life cycles is the genetic control of cell number. The genomic data presented here, which includes diversification of cell cycle genes and modifications to the UPP, align the genetic components with the evolution of this trait.

GATA-3 is required for early T lineage progenitor development

PubMed Central

Hosoya, Tomonori; Kuroha, Takashi; Moriguchi, Takashi; Cummings, Dustin; Maillard, Ivan; Lim, Kim-Chew

2009-01-01

Most T lymphocytes appear to arise from very rare early T lineage progenitors (ETPs) in the thymus, but the transcriptional programs that specify ETP generation are not completely known. The transcription factor GATA-3 is required for the development of T lymphocytes at multiple late differentiation steps as well as for the development of thymic natural killer cells. However, a role for GATA-3 before the double-negative (DN) 3 stage of T cell development has to date been obscured both by the developmental heterogeneity of DN1 thymocytes and the paucity of ETPs. We provide multiple lines of in vivo evidence through the analysis of T cell development in Gata3 hypomorphic mutant embryos, in irradiated mice reconstituted with Gata3 mutant hematopoietic cells, and in mice conditionally ablated for the Gata3 gene to show that GATA-3 is required for ETP generation. We further show that Gata3 loss does not affect hematopoietic stem cells or multipotent hematopoietic progenitors. Finally, we demonstrate that Gata3 mutant lymphoid progenitors exhibit neither increased apoptosis nor diminished cell-cycle progression. Thus, GATA-3 is required for the cell-autonomous development of the earliest characterized thymic T cell progenitors. PMID:19934022

Lineage fate of ductular reactions in liver injury and carcinogenesis.

PubMed

Jörs, Simone; Jeliazkova, Petia; Ringelhan, Marc; Thalhammer, Julian; Dürl, Stephanie; Ferrer, Jorge; Sander, Maike; Heikenwalder, Mathias; Schmid, Roland M; Siveke, Jens T; Geisler, Fabian

2015-06-01

Ductular reactions (DRs) are observed in virtually all forms of human liver disease; however, the histogenesis and function of DRs in liver injury are not entirely understood. It is widely believed that DRs contain bipotential liver progenitor cells (LPCs) that serve as an emergency cell pool to regenerate both cholangiocytes and hepatocytes and may eventually give rise to hepatocellular carcinoma (HCC). Here, we used a murine model that allows highly efficient and specific lineage labeling of the biliary compartment to analyze the histogenesis of DRs and their potential contribution to liver regeneration and carcinogenesis. In multiple experimental and genetic liver injury models, biliary cells were the predominant precursors of DRs but lacked substantial capacity to produce new hepatocytes, even when liver injuries were prolonged up to 12 months. Genetic modulation of NOTCH and/or WNT/β-catenin signaling within lineage-tagged DRs impaired DR expansion but failed to redirect DRs from biliary differentiation toward the hepatocyte lineage. Further, lineage-labeled DRs did not produce tumors in genetic and chemical HCC mouse models. In summary, we found no evidence in our system to support mouse biliary-derived DRs as an LPC pool to replenish hepatocytes in a quantitatively relevant way in injury or evidence that DRs give rise to HCCs.

The "Yin" and "Yang" of Cell Cycle Progression and Differentiation in the Oligodendroglial Lineage

ERIC Educational Resources Information Center

Nguyen, Laurent; Borgs, Laurence; Vandenbosch, Renaud; Mangin, Jean-Marie; Beukelaers, Pierre; Moonen, Gustave; Gallo, Vittorio; Malgrange, Brigitte; Belachew, Shibeshih

2006-01-01

In white matter disorders such as leukodystrophies (LD), periventricular leucomalacia (PVL), or multiple sclerosis (MS), the hypomyelination or the remyelination failure by oligodendrocyte progenitor cells involves errors in the sequence of events that normally occur during development when progenitors proliferate, migrate through the white…

IRF-8 extinguishes neutrophil production and promotes dendritic cell lineage commitment in both myeloid and lymphoid mouse progenitors

PubMed Central

Becker, Amy M.; Michael, Drew G.; Satpathy, Ansuman T.; Sciammas, Roger; Singh, Harinder

2012-01-01

While most blood lineages are assumed to mature through a single cellular and developmental route downstream of HSCs, dendritic cells (DCs) can be derived from both myeloid and lymphoid progenitors in vivo. To determine how distinct progenitors can generate similar downstream lineages, we examined the transcriptional changes that accompany loss of in vivo myeloid potential as common myeloid progenitors differentiate into common DC progenitors (CDPs), and as lymphoid-primed multipotent progenitors (LMPPs) differentiate into all lymphoid progenitors (ALPs). Microarray studies revealed that IFN regulatory factor 8 (IRF-8) expression increased during each of these transitions. Competitive reconstitutions using Irf8−/− BM demonstrated cell-intrinsic defects in the formation of CDPs and all splenic DC subsets. Irf8−/− common myeloid progenitors and, unexpectedly, Irf8−/− ALPs produced more neutrophils in vivo than their wild-type counterparts at the expense of DCs. Retroviral expression of IRF-8 in multiple progenitors led to reduced neutrophil production and increased numbers of DCs, even in the granulocyte-macrophage progenitor (GMP), which does not normally possess conventional DC potential. These data suggest that IRF-8 represses a neutrophil module of development and promotes convergent DC development from multiple lymphoid and myeloid progenitors autonomously of cellular context. PMID:22238324

SHIP1-expressing mesenchymal stem cells regulate hematopoietic stem cell homeostasis and lineage commitment during aging.

PubMed

Iyer, Sonia; Brooks, Robert; Gumbleton, Matthew; Kerr, William G

2015-05-01

Hematopoietic stem cell (HSC) self-renewal and lineage choice are subject to intrinsic control. However, this intrinsic regulation is also impacted by external cues provided by niche cells. There are multiple cellular components that participate in HSC support with the mesenchymal stem cell (MSC) playing a pivotal role. We had previously identified a role for SH2 domain-containing inositol 5'-phosphatase-1 (SHIP1) in HSC niche function through analysis of mice with germline or induced SHIP1 deficiency. In this study, we show that the HSC compartment expands significantly when aged in a niche that contains SHIP1-deficient MSC; however, this expanded HSC compartment exhibits a strong bias toward myeloid differentiation. In addition, we show that SHIP1 prevents chronic G-CSF production by the aging MSC compartment. These findings demonstrate that intracellular signaling by SHIP1 in MSC is critical for the control of HSC output and lineage commitment during aging. These studies increase our understanding of how myeloid bias occurs in aging and thus could have implications for the development of myeloproliferative disease in aging.

Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

PubMed Central

Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

2014-01-01

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary. PMID:24658126

Comprehensive single cell-resolution analysis of the role of chromatin regulators in early C. elegans embryogenesis.

PubMed

Krüger, Angela V; Jelier, Rob; Dzyubachyk, Oleh; Zimmerman, Timo; Meijering, Erik; Lehner, Ben

2015-02-15

Chromatin regulators are widely expressed proteins with diverse roles in gene expression, nuclear organization, cell cycle regulation, pluripotency, physiology and development, and are frequently mutated in human diseases such as cancer. Their inhibition often results in pleiotropic effects that are difficult to study using conventional approaches. We have developed a semi-automated nuclear tracking algorithm to quantify the divisions, movements and positions of all nuclei during the early development of Caenorhabditis elegans and have used it to systematically study the effects of inhibiting chromatin regulators. The resulting high dimensional datasets revealed that inhibition of multiple regulators, including F55A3.3 (encoding FACT subunit SUPT16H), lin-53 (RBBP4/7), rba-1 (RBBP4/7), set-16 (MLL2/3), hda-1 (HDAC1/2), swsn-7 (ARID2), and let-526 (ARID1A/1B) affected cell cycle progression and caused chromosome segregation defects. In contrast, inhibition of cir-1 (CIR1) accelerated cell division timing in specific cells of the AB lineage. The inhibition of RNA polymerase II also accelerated these division timings, suggesting that normal gene expression is required to delay cell cycle progression in multiple lineages in the early embryo. Quantitative analyses of the dataset suggested the existence of at least two functionally distinct SWI/SNF chromatin remodeling complex activities in the early embryo, and identified a redundant requirement for the egl-27 and lin-40 MTA orthologs in the development of endoderm and mesoderm lineages. Moreover, our dataset also revealed a characteristic rearrangement of chromatin to the nuclear periphery upon the inhibition of multiple general regulators of gene expression. Our systematic, comprehensive and quantitative datasets illustrate the power of single cell-resolution quantitative tracking and high dimensional phenotyping to investigate gene function. Furthermore, the results provide an overview of the functions of essential chromatin regulators during the early development of an animal. Copyright © 2014 Elsevier Inc. All rights reserved.

STATs and macrophage fusion.

PubMed

Miyamoto, Takeshi

2013-07-01

Macrophages play a pivotal role in host defense against multiple foreign materials such as bacteria, parasites and artificial devices. Some macrophage lineage cells, namely osteoclasts and foreign body giant cells (FBGCs), form multi-nuclear giant cells by the cell-cell fusion of mono-nuclear cells. Osteoclasts are bone-resorbing cells, and are formed in the presence of RANKL on the surface of bones, while FBGCs are formed in the presence of IL-4 or IL-13 on foreign materials such as artificial joints, catheters and parasites. Recently, fusiogenic mechanisms and the molecules required for the cell-cell fusion of these macrophage lineage cells were, at least in part, clarified. Dendritic cell specific transmembrane protein (DC-STAMP) and osteoclast stimulatory transmembrane protein (OC-STAMP), both of which comprise seven transmembrane domains, are required for both osteoclast and FBGC cell-cell fusion. STAT6 was demonstrated to be required for the cell-cell fusion of FBGCs but not osteoclasts. In this review, advances in macrophage cell-cell fusion are discussed.

A unique stylopod patterning mechanism by Shox2-controlled osteogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Wenduo; Song, Yingnan; Huang, Zhen

Here, vertebrate appendage patterning is programmed by Hox-TALE factorbound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clusteringmore » around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.« less

A unique stylopod patterning mechanism by Shox2-controlled osteogenesis

DOE PAGES

Ye, Wenduo; Song, Yingnan; Huang, Zhen; ...

2016-06-10

Here, vertebrate appendage patterning is programmed by Hox-TALE factorbound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clusteringmore » around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.« less

Pharmacogenomic identification of small molecules for lineage specific manipulation of subventricular zone germinal activity.

PubMed

Azim, Kasum; Angonin, Diane; Marcy, Guillaume; Pieropan, Francesca; Rivera, Andrea; Donega, Vanessa; Cantù, Claudio; Williams, Gareth; Berninger, Benedikt; Butt, Arthur M; Raineteau, Olivier

2017-03-01

Strategies for promoting neural regeneration are hindered by the difficulty of manipulating desired neural fates in the brain without complex genetic methods. The subventricular zone (SVZ) is the largest germinal zone of the forebrain and is responsible for the lifelong generation of interneuron subtypes and oligodendrocytes. Here, we have performed a bioinformatics analysis of the transcriptome of dorsal and lateral SVZ in early postnatal mice, including neural stem cells (NSCs) and their immediate progenies, which generate distinct neural lineages. We identified multiple signaling pathways that trigger distinct downstream transcriptional networks to regulate the diversity of neural cells originating from the SVZ. Next, we used a novel in silico genomic analysis, searchable platform-independent expression database/connectivity map (SPIED/CMAP), to generate a catalogue of small molecules that can be used to manipulate SVZ microdomain-specific lineages. Finally, we demonstrate that compounds identified in this analysis promote the generation of specific cell lineages from NSCs in vivo, during postnatal life and adulthood, as well as in regenerative contexts. This study unravels new strategies for using small bioactive molecules to direct germinal activity in the SVZ, which has therapeutic potential in neurodegenerative diseases.

Disruption of Hox9,10,11 function results in cellular level lineage infidelity in the kidney.

PubMed

Drake, Keri A; Adam, Mike; Mahoney, Robert; Potter, S Steven

2018-04-20

Hox genes are important regulators of development. The 39 mammalian Hox genes have considerable functional overlap, greatly confounding their study. In this report, we generated mice with multiple combinations of paralogous and flanking Abd-B Hox gene mutations to investigate functional redundancies in kidney development. The resulting mice developed a number of kidney abnormalities, including hypoplasia, agenesis, and severe cysts, with distinct Hox functions observed in early metanephric kidney formation and nephron progenitor maintenance. Most surprising, however, was that extensive removal of Hox shared function in these kidneys resulted in cellular level lineage infidelity. Strikingly, mutant nephron tubules consisted of intermixed cells with proximal tubule, loop of Henle, and collecting duct identities, with some single cells expressing markers associated with more than one nephron segment. These results indicate that Hox genes are required for proper lineage selection/maintenance and full repression of genes involved in cell fate restriction in the developing kidney.

Human Immunodeficiency Viruses Appear Compartmentalized to the Female Genital Tract in Cross-Sectional Analyses but Genital Lineages Do Not Persist Over Time

PubMed Central

Bull, Marta E.; Heath, Laura M.; McKernan-Mullin, Jennifer L.; Kraft, Kelli M.; Acevedo, Luis; Hitti, Jane E.; Cohn, Susan E.; Tapia, Kenneth A.; Holte, Sarah E.; Dragavon, Joan A.; Coombs, Robert W.; Mullins, James I.; Frenkel, Lisa M.

2013-01-01

Background. Whether unique human immunodeficiency type 1 (HIV) genotypes occur in the genital tract is important for vaccine development and management of drug resistant viruses. Multiple cross-sectional studies suggest HIV is compartmentalized within the female genital tract. We hypothesize that bursts of HIV replication and/or proliferation of infected cells captured in cross-sectional analyses drive compartmentalization but over time genital-specific viral lineages do not form; rather viruses mix between genital tract and blood. Methods. Eight women with ongoing HIV replication were studied during a period of 1.5 to 4.5 years. Multiple viral sequences were derived by single-genome amplification of the HIV C2-V5 region of env from genital secretions and blood plasma. Maximum likelihood phylogenies were evaluated for compartmentalization using 4 statistical tests. Results. In cross-sectional analyses compartmentalization of genital from blood viruses was detected in three of eight women by all tests; this was associated with tissue specific clades containing multiple monotypic sequences. In longitudinal analysis, the tissues-specific clades did not persist to form viral lineages. Rather, across women, HIV lineages were comprised of both genital tract and blood sequences. Conclusions. The observation of genital-specific HIV clades only in cross-sectional analysis and an absence of genital-specific lineages in longitudinal analyses suggest a dynamic interchange of HIV variants between the female genital tract and blood. PMID:23315326

Isolation and characterization of porcine adipose tissue-derived adult stem cells.

PubMed

Williams, Kellie J; Picou, Alicia A; Kish, Sharon L; Giraldo, Angelica M; Godke, Robert A; Bondioli, Kenneth R

2008-01-01

Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34. Copyright 2008 S. Karger AG, Basel.

Myeloid transformation of plasma cell myeloma: molecular evidence of clonal evolution revealed by next generation sequencing.

PubMed

Gralewski, Jonathon H; Post, Ginell R; van Rhee, Frits; Yuan, Youzhong

2018-02-20

Plasma cell myeloma (PCM) is a neoplasm of terminally differentiated B lymphocytes with molecular heterogeneity. Although therapy-related myeloid neoplasms are common in plasma cell myeloma patients after chemotherapy, transdifferentiation of plasma cell myeloma into myeloid neoplasms has not been reported in literature. Here we report a very rare case of myeloid neoplasm transformed from plasma cell myeloma. A 60-year-old man with a history of plasma cell myeloma with IGH-MAF gene rearrangement and RAS/RAF mutations developed multiple soft tissue lesions one year following melphalan-based chemotherapy and autologous stem cell transplant. Morphological and immunohistochemical characterization of the extramedullary disease demonstrated that the tumor cells were derived from the monocyte-macrophage lineage. Next generation sequencing (NGS) studies detected similar clonal aberrations in the diagnostic plasma cell population and post-therapy neoplastic cells, including IGH-MAF rearrangement, multiple genetic mutations in RAS signaling pathway proteins, and loss of tumor suppressor genes. Molecular genetic analysis also revealed unique genomic alterations in the transformed tumor cells, including gain of NF1 and loss of TRAF3. To our knowledge, this is the first case of myeloid sarcoma transdifferentiated from plasma cell neoplasm. Our findings in this unique case suggest clonal evolution of plasma cell myeloma to myeloma neoplasm and the potential roles of abnormal RAS/RAF signaling pathway in lineage switch or transdifferentiation.

Human bone marrow-derived mesenchymal cells differentiate and mature into endocrine pancreatic lineage in vivo.

PubMed

Phadnis, Smruti M; Joglekar, Mugdha V; Dalvi, Maithili P; Muthyala, Sudhakar; Nair, Prabha D; Ghaskadbi, Surendra M; Bhonde, Ramesh R; Hardikar, Anandwardhan A

2011-03-01

The scarcity of human islets for transplantation remains a major limitation of cell replacement therapy for diabetes. Bone marrow-derived progenitor cells are of interest because they can be isolated, expanded and offered for such therapy under autologous/allogeneic settings. We characterized and compared human bone marrow-derived mesenchymal cells (hBMC) obtained from (second trimester), young (1-24 years) and adult (34-81 years) donors. We propose a novel protocol that involves assessment of paracrine factors from regenerating pancreas in differentiation and maturation of hBMC into endocrine pancreatic lineage in vivo. We observed that donor age was inversely related to growth potential of hBMC. Following in vitro expansion and exposure to specific growth factors involved in pancreatic development, hBMC migrated and formed islet-like cell aggregates (ICA). ICA show increased abundance of pancreatic transcription factors (Ngn3, Brn4, Nkx6.1, Pax6 and Isl1). Although efficient differentiation was not achieved in vitro, we observed significant maturation and secretion of human c-peptide (insulin) upon transplantation into pancreactomized and Streptozotocin (STZ)-induced diabetic mice. Transplanted ICA responded to glucose and maintained normoglycemia in diabetic mice. Our data demonstrate that hBMC have tremendous in vitro expansion potential and can be differentiated into multiple lineages, including the endocrine pancreatic lineage. Paracrine factors secreted from regenerating pancreas help in efficient differentiation and maturation of hBMC, possibly via recruiting chromatin modulators, to generate glucose-responsive insulin-secreting cells.

Transcription factor AP-2γ induces early Cdx2 expression and represses HIPPO signaling to specify the trophectoderm lineage

PubMed Central

Cao, Zubing; Carey, Timothy S.; Ganguly, Avishek; Wilson, Catherine A.; Paul, Soumen; Knott, Jason G.

2015-01-01

Cell fate decisions are fundamental to the development of multicellular organisms. In mammals the first cell fate decision involves segregation of the pluripotent inner cell mass and the trophectoderm, a process regulated by cell polarity proteins, HIPPO signaling and lineage-specific transcription factors such as CDX2. However, the regulatory mechanisms that operate upstream to specify the trophectoderm lineage have not been established. Here we report that transcription factor AP-2γ (TFAP2C) functions as a novel upstream regulator of Cdx2 expression and position-dependent HIPPO signaling in mice. Loss- and gain-of-function studies and promoter analysis revealed that TFAP2C binding to an intronic enhancer is required for activation of Cdx2 expression during early development. During the 8-cell to morula transition TFAP2C potentiates cell polarity to suppress HIPPO signaling in the outside blastomeres. TFAP2C depletion triggered downregulation of PARD6B, loss of apical cell polarity, disorganization of F-actin, and activation of HIPPO signaling in the outside blastomeres. Rescue experiments using Pard6b mRNA restored cell polarity but only partially corrected position-dependent HIPPO signaling, suggesting that TFAP2C negatively regulates HIPPO signaling via multiple pathways. Several genes involved in regulation of the actin cytoskeleton (including Rock1, Rock2) were downregulated in TFAP2C-depleted embryos. Inhibition of ROCK1 and ROCK2 activity during the 8-cell to morula transition phenocopied TFAP2C knockdown, triggering a loss of position-dependent HIPPO signaling and decrease in Cdx2 expression. Altogether, these results demonstrate that TFAP2C facilitates trophectoderm lineage specification by functioning as a key regulator of Cdx2 transcription, cell polarity and position-dependent HIPPO signaling. PMID:25858457

Staged induction of HIV-1 glycan–dependent broadly neutralizing antibodies

DOE PAGES

Bonsignori, Mattia; Kreider, Edward F.; Fera, Daniela; ...

2017-03-15

A preventive HIV-1 vaccine should induce HIV-1–specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3- glycan bnAb. Two autologousmore » neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3- glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs.« less

Staged induction of HIV-1 glycan–dependent broadly neutralizing antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bonsignori, Mattia; Kreider, Edward F.; Fera, Daniela

A preventive HIV-1 vaccine should induce HIV-1–specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3- glycan bnAb. Two autologousmore » neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3- glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs.« less

Intra-genomic variation in symbiotic dinoflagellates: recent divergence or recombination between lineages?

PubMed

Wilkinson, Shaun P; Fisher, Paul L; van Oppen, Madeleine J H; Davy, Simon K

2015-03-14

The symbiosis between corals and the dinoflagellate alga Symbiodinium is essential for the development and survival of coral reefs. Yet this fragile association is highly vulnerable to environmental disturbance. A coral's ability to tolerate temperature stress depends on the fitness of its resident symbionts, whose thermal optima vary extensively between lineages. However, the in hospite population genetic structure of Symbiodinium is poorly understood and mostly based on analysis of bulk DNA extracted from thousands to millions of cells. Using quantitative single-cell PCR, we enumerated DNA polymorphisms in the symbionts of the reef-building coral Pocillopora damicornis, and applied a model selection approach to explore the potential for recombination between coexisting Symbiodinium populations. Two distinct Symbiodinium ITS2 sequences (denoted C100 and C109) were retrieved from all P. damicornis colonies analysed. However, the symbiont assemblage consisted of three distinct Symbiodinium populations: cells featuring pure arrays of ITS2 type C109, near-homogeneous cells of type C100 (with trace ITS2 copies of type C109), and those with co-dominant C100 and C109 ITS2 repeats. The symbiont consortia of some colonies consisted almost entirely of these putative C100 × C109 recombinants. Our results are consistent with the occurrence of sexual recombination between Symbiodinium types C100 and C109. While the multiple-copy nature of the ITS2 dictates that the observed pattern of intra-genomic co-dominance may be a result of incomplete concerted evolution of intra-genomic polymorphisms, this is a less likely explanation given the occurrence of homogeneous cells of the C109 type. Conclusive evidence for inter-lineage recombination and introgression in this genus will require either direct observational evidence or a single-cell genotyping approach targeting multiple, single-copy loci.

Mesenchymal stem cells: biological characteristics and potential clinical applications.

PubMed

Kassem, Moustapha

2004-01-01

Mesenchymal stem cells (MSC) are clonogenic, non-hematpoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages, for example, osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages, for example, neuronal-like cells. Several methods are currently available for isolation of the MSC based on their physical and physico-chemical characteristics, for example, adherence to plastics or other extracellular matrix components. Because of the ease of their isolation and their extensive differentiation potential, MSC are among the first stem cell types to be introduced in the clinic. Several studies have demonstrated the possible use of MSC in systemic transplantation for systemic diseases, local implantation for local tissue defects, as a vehicle for genes in gene therapy protocols or to generate transplantable tissues and organs in tissue engineering protocols. Before their widespread use in therapy, methods allowing the generation of large number of cells without affecting their differentiation potential as well as technologies that overcome immunological rejection (in case allogenic transplantation) must be developed.

Flow cytometric discrimination of seven lineage markers by using two fluorochromes

PubMed Central

Boin, Francesco; Giardino Torchia, Maria Letizia; Borrello, Ivan; Noonan, Kimberly A.; Neil, Matthew; Soloski, Mark J.

2017-01-01

Flow cytometry is the primary immunological technique used to analyze multiple parameters on complex cell populations. We present a staining method that identifies major human mononuclear lymphoid and myeloid populations (CD4+ and CD8+ T cells, γδ T cells, B cells, NK cells and monocytes), using only two fluorochromes and a minimal number of cells. Our approach increases the number of markers recordable on most flow cytometers allowing for a deeper and more comprehensive immunophenotyping. PMID:29190813

NG2-expressing cells as oligodendrocyte progenitors in the normal and demyelinated adult central nervous system

PubMed Central

Polito, Annabella; Reynolds, Richard

2005-01-01

The mammalian adult central nervous system (CNS) is known to respond rapidly to demyelinating insults by regenerating oligodendrocytes for remyelination from a dividing precursor population. A widespread population of cells exists within the adult CNS that is thought to belong to the oligodendrocyte lineage, but which do not express proteins characteristic of mature myelinating oligodendrocytes, such as myelin basic protein (MBP) and 2,3-cyclic nucleotide 3-phosphodiesterase (CNP). Instead, these cells have phenotypic characteristics of a more immature stage of the oligodendrocyte lineage. They express the NG2 chondroitin sulphate proteoglycan, in addition to O4 and the platelet-derived growth factor α-receptor, all widely accepted as markers for oligodendrocyte progenitor cells (OPCs) throughout development. However, NG2+ cells residing in the adult CNS do not resemble embryonic or neonatal NG2+ cells in terms of their morphology or proliferation characteristics, but instead represent a unique type of glial cell that has the ability to react rapidly to CNS damage. In this review, we present the evidence that adult NG2+ cells are part of the oligodendrocyte lineage and are capable of giving rise to new oligodendrocytes under both normal and demyelinating conditions. We also review the literature that these cells may have multiple functional roles within the adult CNS, notwithstanding their primary role as OPCs. PMID:16367798

In silico lineage tracing through single cell transcriptomics identifies a neural stem cell population in planarians.

PubMed

Molinaro, Alyssa M; Pearson, Bret J

2016-04-27

The planarian Schmidtea mediterranea is a master regenerator with a large adult stem cell compartment. The lack of transgenic labeling techniques in this animal has hindered the study of lineage progression and has made understanding the mechanisms of tissue regeneration a challenge. However, recent advances in single-cell transcriptomics and analysis methods allow for the discovery of novel cell lineages as differentiation progresses from stem cell to terminally differentiated cell. Here we apply pseudotime analysis and single-cell transcriptomics to identify adult stem cells belonging to specific cellular lineages and identify novel candidate genes for future in vivo lineage studies. We purify 168 single stem and progeny cells from the planarian head, which were subjected to single-cell RNA sequencing (scRNAseq). Pseudotime analysis with Waterfall and gene set enrichment analysis predicts a molecularly distinct neoblast sub-population with neural character (νNeoblasts) as well as a novel alternative lineage. Using the predicted νNeoblast markers, we demonstrate that a novel proliferative stem cell population exists adjacent to the brain. scRNAseq coupled with in silico lineage analysis offers a new approach for studying lineage progression in planarians. The lineages identified here are extracted from a highly heterogeneous dataset with minimal prior knowledge of planarian lineages, demonstrating that lineage purification by transgenic labeling is not a prerequisite for this approach. The identification of the νNeoblast lineage demonstrates the usefulness of the planarian system for computationally predicting cellular lineages in an adult context coupled with in vivo verification.

Antibody producing B lineage cells invade the central nervous system predominantly at the time of and triggered by acute Epstein-Barr virus infection: A hypothesis on the origin of intrathecal immunoglobulin synthesis in multiple sclerosis.

PubMed

Otto, Carolin; Hofmann, Jörg; Ruprecht, Klemens

2016-06-01

Patients with multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), typically have an intrathecal synthesis of immunoglobulin (Ig)G. Intrathecal IgG is produced by B lineage cells that entered the CNS, but why and when these cells invade the CNS of patients with MS is unknown. The intrathecal IgG response in patients with MS is polyspecific and part of it is directed against different common viruses (e.g. measles virus, rubella virus, varicella zoster virus). Strong and consistent evidence suggests an association of MS and Epstein-Barr virus (EBV) infection and EBV seroprevalence in patients with MS is practically 100%. However, intriguingly, despite of the universal EBV seroprevalence, the frequency of intrathecally produced IgG to EBV in patients with MS is much lower than that of intrathecally produced IgG to other common viruses. The acute phase of primary EBV infection is characterized by a strong polyclonal B cell activation. As typical for humoral immune responses against viruses, EBV specific IgG is produced only with a temporal delay after acute EBV infection. Aiming to put the above facts into a logical structure, we here propose the hypothesis that in individuals going on to develop MS antibody producing B lineage cells invade the CNS predominantly at the time of and triggered by acute primary EBV infection. Because at the time of acute EBV infection EBV IgG producing B lineage cells have not yet occurred, the hypothesis could explain the universal EBV seroprevalence and the low frequency of intrathecally produced IgG to EBV in patients with MS. Evidence supporting the hypothesis could be provided by large prospective follow-up studies of individuals with symptomatic primary EBV infection (infectious mononucleosis). Furthermore, the clarification of the molecular mechanism underlying an EBV induced invasion of B lineage cells into the CNS of individuals going on to develop MS could corroborate it, too. If true, our hypothesis would link EBV infection, the most important environmental risk factor for MS, with intrathecal IgG synthesis, the most characteristic laboratory feature of MS. Besides explaining the origin of intrathecal IgG synthesis in patients with MS, the hypothesis could thus also provide a conceptual framework for clarifying the mechanism through which EBV contributes to the development of MS. Copyright © 2016 Elsevier Ltd. All rights reserved.

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations.

PubMed

Gómez-Villafuertes, Rosa; Paniagua-Herranz, Lucía; Gascon, Sergio; de Agustín-Durán, David; Ferreras, María de la O; Gil-Redondo, Juan Carlos; Queipo, María José; Menendez-Mendez, Aida; Pérez-Sen, Ráquel; Delicado, Esmerilda G; Gualix, Javier; Costa, Marcos R; Schroeder, Timm; Miras-Portugal, María Teresa; Ortega, Felipe

2017-12-16

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

Notch3 marks clonogenic mammary luminal progenitor cells in vivo.

PubMed

Lafkas, Daniel; Rodilla, Veronica; Huyghe, Mathilde; Mourao, Larissa; Kiaris, Hippokratis; Fre, Silvia

2013-10-14

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive "triple negative" human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2(SAT) transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells.

Notch3 marks clonogenic mammary luminal progenitor cells in vivo

PubMed Central

Lafkas, Daniel; Rodilla, Veronica; Huyghe, Mathilde; Mourao, Larissa; Kiaris, Hippokratis

2013-01-01

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive “triple negative” human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2SAT transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells. PMID:24100291

Concepts of Cell Lineage in Mammalian Embryos.

PubMed

Papaioannou, Virginia E

2016-01-01

Cell lineage is the framework for understanding cellular diversity, stability of differentiation, and its relationship to pluripotency. The special condition of in utero development in mammals has presented challenges to developmental biologists in tracing cell lineages but modern imaging and cell marking techniques have allowed the gradual elucidation of lineage relationships. Early experimental embryology approaches had limited resolution and relied of suboptimal cell markers and considerable disturbance to the embryos. Transgenic technology introduced genetic markers, particularly fluorescent proteins that, combined with sophisticated imaging modalities, greatly increase resolution and allow clonal analysis within lineages. The concept of cell lineage has also undergone evolution as it became possible to trace the lineage of cells based not only on their physical location or attributes but also on their gene expression pattern, thus opening up mechanistic lines of investigation into the determinants of cell lineage. © 2016 Elsevier Inc. All rights reserved.

Nitric Oxide Modulates TGF-β–Directive Signals To Suppress Foxp3+ Regulatory T Cell Differentiation and Potentiate Th1 Development

PubMed Central

Lee, Seung-Woo; Choi, Heonsik; Eun, So-Young; Fukuyama, Satoshi; Croft, Michael

2011-01-01

TGF-β can induce Foxp3+ inducible regulatory T cells (Treg) and also synergize with IL-6 and IL-4 to induce Th17 and Th9 cells. We now report that NO modulates TGF-β activity away from Treg but toward the Th1 lineage. NO potentiated Th1 differentiation in the presence of TGF-β in both IL-12–independent and –dependent fashions by augmenting IFN-γ–activated STAT-1 and T-bet. Differentiation into Treg, Th1, and Th17 lineages could be modulated by NO competing with other cofactors, such as IL-6 and retinoic acid. NO antagonized IL-6 to block TGF-β–directed Th17 differentiation, and together with IL-6, NO suppressed Treg development induced by TGF-β and retinoic acid. Furthermore, we show that physiologically produced NO from TNF and inducible NO synthase-producing dendritic cells can contribute to Th1 development predominating over Treg development through a synergistic activity induced when these cells cocluster with conventional dendritic cells presenting Ag to naive Th cells. This illustrates that NO is another cofactor allowing TGF-β to participate in development of multiple Th lineages and suggests a new mechanism by which NO, which is associated with protection against intracellular pathogens, might maintain effective Th1 immunity. PMID:21555530

The Mediator complex: a master coordinator of transcription and cell lineage development.

PubMed

Yin, Jing-wen; Wang, Gang

2014-03-01

Mediator is a multiprotein complex that is required for gene transcription by RNA polymerase II. Multiple subunits of the complex show specificity in relaying information from signals and transcription factors to the RNA polymerase II machinery, thus enabling control of the expression of specific genes. Recent studies have also provided novel mechanistic insights into the roles of Mediator in epigenetic regulation, transcriptional elongation, termination, mRNA processing, noncoding RNA activation and super enhancer formation. Based on these specific roles in gene regulation, Mediator has emerged as a master coordinator of development and cell lineage determination. Here, we describe the most recent advances in understanding the mechanisms of Mediator function, with an emphasis on its role during development and disease.

A generic, cost-effective, and scalable cell lineage analysis platform

PubMed Central

Biezuner, Tamir; Spiro, Adam; Raz, Ofir; Amir, Shiran; Milo, Lilach; Adar, Rivka; Chapal-Ilani, Noa; Berman, Veronika; Fried, Yael; Ainbinder, Elena; Cohen, Galit; Barr, Haim M.; Halaban, Ruth; Shapiro, Ehud

2016-01-01

Advances in single-cell genomics enable commensurate improvements in methods for uncovering lineage relations among individual cells. Current sequencing-based methods for cell lineage analysis depend on low-resolution bulk analysis or rely on extensive single-cell sequencing, which is not scalable and could be biased by functional dependencies. Here we show an integrated biochemical-computational platform for generic single-cell lineage analysis that is retrospective, cost-effective, and scalable. It consists of a biochemical-computational pipeline that inputs individual cells, produces targeted single-cell sequencing data, and uses it to generate a lineage tree of the input cells. We validated the platform by applying it to cells sampled from an ex vivo grown tree and analyzed its feasibility landscape by computer simulations. We conclude that the platform may serve as a generic tool for lineage analysis and thus pave the way toward large-scale human cell lineage discovery. PMID:27558250

Mucopolysaccharidosis enzyme production by bone marrow and dental pulp derived human mesenchymal stem cells.

PubMed

Jackson, Matilda; Derrick Roberts, Ainslie; Martin, Ellenore; Rout-Pitt, Nathan; Gronthos, Stan; Byers, Sharon

2015-04-01

Mucopolysaccharidoses (MPS) are inherited metabolic disorders that arise from a complete loss or a reduction in one of eleven specific lysosomal enzymes. MPS children display pathology in multiple cell types leading to tissue and organ failure and early death. Mesenchymal stem cells (MSCs) give rise to many of the cell types affected in MPS, including those that are refractory to current treatment protocols such as hematopoietic stem cell (HSC) based therapy. In this study we compared multiple MPS enzyme production by bone marrow derived (hBM) and dental pulp derived (hDP) MSCs to enzyme production by HSCs. hBM MSCs produce significantly higher levels of MPS I, II, IIIA, IVA, VI and VII enzyme than HSCs, while hDP MSCs produce significantly higher levels of MPS I, IIIA, IVA, VI and VII enzymes. Higher transfection efficiency was observed in MSCs (89%) compared to HSCs (23%) using a lentiviral vector. Over-expression of four different lysosomal enzymes resulted in up to 9303-fold and up to 5559-fold greater levels in MSC cell layer and media respectively. Stable, persistent transduction of MSCs and sustained over-expression of MPS VII enzyme was observed in vitro. Transduction of MSCs did not affect the ability of the cells to differentiate down osteogenic, adipogenic or chondrogenic lineages, but did partially delay differentiation down the non-mesodermal neurogenic lineage. Copyright © 2015 Elsevier Inc. All rights reserved.

Multiple prethymic defects underlie age-related loss of T progenitor competence

PubMed Central

Zediak, Valerie P.; Maillard, Ivan

2007-01-01

Aging in mice and humans is characterized by declining T-lymphocyte production in the thymus, yet it is unclear whether aging impacts the T-lineage potential of hematopoietic progenitors. Although alterations in the lymphoid progenitor content of aged mouse bone marrow (BM) have been described, irradiation-reconstitution experiments have failed to reveal defects in T-lineage potential of BM hematopoietic progenitors or purified hematopoietic stem cells (HSCs) from aged mice. Here, we assessed T-progenitor potential in unmanipulated recipient mice without conditioning irradiation. T-progenitor potential was reduced in aged BM compared with young BM, and this reduction was apparent at the earliest stages of intrathymic differentiation. Further, enriched populations of aged HSCs or multipotent progenitors (MPPs) gave rise to fewer T-lineage cells than their young counterparts. Whereas the T-precursor frequency within the MPP pool was unchanged, there was a 4-fold decline in T-precursor frequency within the HSC pool. In addition, among the T-competent HSC clones, there were fewer highly proliferative clones in the aged HSC pool than in the young HSC pool. These results identify T-compromised aged HSCs and define the nature and cellular sites of prethymic, age-related defects in T-lineage differentiation potential. PMID:17456721

Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell–cell interaction

PubMed Central

Kwak, Minsuk; Mu, Luye; Lu, Yao; Chen, Jonathan J.; Brower, Kara; Fan, Rong

2013-01-01

Secreted proteins including cytokines, chemokines, and growth factors represent important functional regulators mediating a range of cellular behavior and cell–cell paracrine/autocrine signaling, e.g., in the immunological system (Rothenberg, 2007), tumor microenvironment (Hanahan and Weinberg, 2011), or stem cell niche (Gnecchi etal., 2008). Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically identical cell population can give rise to diverse phenotypic differences (Niepel etal., 2009). Non-genetic heterogeneity is also emerging as a potential barrier to accurate monitoring of cellular immunity and effective pharmacological therapies (Cohen etal., 2008; Gascoigne and Taylor, 2008), but can hardly assessed using conventional approaches that do not examine cellular phenotype at the functional level. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer. PMID:23390614

Directing lineage specification of human mesenchymal stem cells by decoupling electrical stimulation and physical patterning on unmodified graphene

NASA Astrophysics Data System (ADS)

Balikov, Daniel A.; Fang, Brian; Chun, Young Wook; Crowder, Spencer W.; Prasai, Dhiraj; Lee, Jung Bok; Bolotin, Kiril I.; Sung, Hak-Joon

2016-07-01

The organization and composition of the extracellular matrix (ECM) have been shown to impact the propagation of electrical signals in multiple tissue types. To date, many studies with electroactive biomaterial substrates have relied upon passive electrical stimulation of the ionic media to affect cell behavior. However, development of cell culture systems in which stimulation can be directly applied to the material - thereby isolating the signal to the cell-material interface and cell-cell contracts - would provide a more physiologically-relevant paradigm for investigating how electrical cues modulate lineage-specific stem cell differentiation. In the present study, we have employed unmodified, directly-stimulated, (un)patterned graphene as a cell culture substrate to investigate how extrinsic electrical cycling influences the differentiation of naïve human mesenchymal stem cells (hMSCs) without the bias of exogenous biochemicals. We first demonstrated that cyclic stimulation does not deteriorate the cell culture media or result in cytotoxic pH, which are critical experiments for correct interpretation of changes in cell behavior. We then measured how the expression of osteogenic and neurogenic lineage-specific markers were altered simply by exposure to electrical stimulation and/or physical patterns. Expression of the early osteogenic transcription factor RUNX2 was increased by electrical stimulation on all graphene substrates, but the mature marker osteopontin was only modulated when stimulation was combined with physical patterns. In contrast, the expression of the neurogenic markers MAP2 and β3-tubulin were enhanced in all electrical stimulation conditions, and were less responsive to the presence of patterns. These data indicate that specific combinations of non-biological inputs - material type, electrical stimulation, physical patterns - can regulate hMSC lineage specification. This study represents a substantial step in understanding how the interplay of electrophysical stimuli regulate stem cell behavior and helps to clarify the potential for graphene substrates in tissue engineering applications.

HLA Class I Depleted hESC as a Source of Hypoimmunogenic Cells for Tissue Engineering Applications.

PubMed

Karabekian, Zaruhi; Ding, Hao; Stybayeva, Gulnaz; Ivanova, Irina; Muselimyan, Narine; Haque, Amranul; Toma, Ian; Posnack, Nikki G; Revzin, Alexander; Leitenberg, David; Laflamme, Michael A; Sarvazyan, Narine

2015-10-01

Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells (ESCs) provide a unique opportunity to modify the original source of differentiated cells to minimize their rejection by nonautologous hosts. Immune rejection of nonautologous human embryonic stem cell (hESC) derivatives can be reduced by downregulating human leukocyte antigen (HLA) class I molecules, without affecting the ability of these cells to differentiate into specific lineages. Beta-2-microglobulin (B2M) expression was decreased by lentiviral transduction using human anti-HLA class I light-chain B2M short hairpin RNA. mRNA levels of B2M were decreased by 90% in a RUES2-modified hESC line, as determined by quantitative real time-polymerase chain reaction analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as target cells in a mixed lymphocyte reaction assay, transduced hESCs and their differentiated derivatives did not stimulate allogeneic T-cell proliferation. Using a cardiac differentiation protocol, transduced hESCs formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESCs were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages. HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from nonautologous hESC sources with improved immunocompatibility.

HLA Class I Depleted hESC as a Source of Hypoimmunogenic Cells for Tissue Engineering Applications

PubMed Central

Karabekian, Zaruhi; Ding, Hao; Stybayeva, Gulnaz; Ivanova, Irina; Muselimyan, Narine; Haque, Amranul; Toma, Ian; Posnack, Nikki G.; Revzin, Alexander; Leitenberg, David; Laflamme, Michael A.

2015-01-01

Background: Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells (ESCs) provide a unique opportunity to modify the original source of differentiated cells to minimize their rejection by nonautologous hosts. Hypothesis: Immune rejection of nonautologous human embryonic stem cell (hESC) derivatives can be reduced by downregulating human leukocyte antigen (HLA) class I molecules, without affecting the ability of these cells to differentiate into specific lineages. Methods and Results: Beta-2-microglobulin (B2M) expression was decreased by lentiviral transduction using human anti-HLA class I light-chain B2M short hairpin RNA. mRNA levels of B2M were decreased by 90% in a RUES2-modified hESC line, as determined by quantitative real time-polymerase chain reaction analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as target cells in a mixed lymphocyte reaction assay, transduced hESCs and their differentiated derivatives did not stimulate allogeneic T-cell proliferation. Using a cardiac differentiation protocol, transduced hESCs formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESCs were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages. Conclusions: HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from nonautologous hESC sources with improved immunocompatibility. PMID:26218149

Massive intracerebral Epstein-Barr virus reactivation in lethal multiple sclerosis relapse after natalizumab withdrawal.

PubMed

Serafini, Barbara; Scorsi, Eleonora; Rosicarelli, Barbara; Rigau, Valérie; Thouvenot, Eric; Aloisi, Francesca

2017-06-15

Rebound of disease activity in multiple sclerosis patients after natalizumab withdrawal is a potentially life-threatening event. To verify whether highly destructive inflammation after natalizumab withdrawal is associated with Epstein-Barr virus (EBV) reactivation in central nervous system infiltrating B-lineage cells and cytotoxic immunity, we analyzed post-mortem brain tissue from a patient who died during a fulminating MS relapse following natalizumab withdrawal. Numerous EBV infected B cells/plasma cells and CD8+ T cells infiltrated all white matter lesions; the highest frequency of EBV lytically infected cells and granzyme B+ CD8+ T cells was observed in actively demyelinating lesions. These results may encourage switching to B-cell depleting therapy after natalizumab discontinuation. Copyright © 2017 Elsevier B.V. All rights reserved.

Enteric nervous system specific deletion of Foxd3 disrupts glial cell differentiation and activates compensatory enteric progenitors.

PubMed

Mundell, Nathan A; Plank, Jennifer L; LeGrone, Alison W; Frist, Audrey Y; Zhu, Lei; Shin, Myung K; Southard-Smith, E Michelle; Labosky, Patricia A

2012-03-15

The enteric nervous system (ENS) arises from the coordinated migration, expansion and differentiation of vagal and sacral neural crest progenitor cells. During development, vagal neural crest cells enter the foregut and migrate in a rostro-to-caudal direction, colonizing the entire gastrointestinal tract and generating the majority of the ENS. Sacral neural crest contributes to a subset of enteric ganglia in the hindgut, colonizing the colon in a caudal-to-rostral wave. During this process, enteric neural crest-derived progenitors (ENPs) self-renew and begin expressing markers of neural and glial lineages as they populate the intestine. Our earlier work demonstrated that the transcription factor Foxd3 is required early in neural crest-derived progenitors for self-renewal, multipotency and establishment of multiple neural crest-derived cells and structures including the ENS. Here, we describe Foxd3 expression within the fetal and postnatal intestine: Foxd3 was strongly expressed in ENPs as they colonize the gastrointestinal tract and was progressively restricted to enteric glial cells. Using a novel Ednrb-iCre transgene to delete Foxd3 after vagal neural crest cells migrate into the midgut, we demonstrated a late temporal requirement for Foxd3 during ENS development. Lineage labeling of Ednrb-iCre expressing cells in Foxd3 mutant embryos revealed a reduction of ENPs throughout the gut and loss of Ednrb-iCre lineage cells in the distal colon. Although mutant mice were viable, defects in patterning and distribution of ENPs were associated with reduced proliferation and severe reduction of glial cells derived from the Ednrb-iCre lineage. Analyses of ENS-lineage and differentiation in mutant embryos suggested activation of a compensatory population of Foxd3-positive ENPs that did not express the Ednrb-iCre transgene. Our findings highlight the crucial roles played by Foxd3 during ENS development including progenitor proliferation, neural patterning, and glial differentiation and may help delineate distinct molecular programs controlling vagal versus sacral neural crest development. Copyright © 2012 Elsevier Inc. All rights reserved.

Integration of information and volume visualization for analysis of cell lineage and gene expression during embryogenesis

NASA Astrophysics Data System (ADS)

Cedilnik, Andrej; Baumes, Jeffrey; Ibanez, Luis; Megason, Sean; Wylie, Brian

2008-01-01

Dramatic technological advances in the field of genomics have made it possible to sequence the complete genomes of many different organisms. With this overwhelming amount of data at hand, biologists are now confronted with the challenge of understanding the function of the many different elements of the genome. One of the best places to start gaining insight on the mechanisms by which the genome controls an organism is the study of embryogenesis. There are multiple and inter-related layers of information that must be established in order to understand how the genome controls the formation of an organism. One is cell lineage which describes how patterns of cell division give rise to different parts of an organism. Another is gene expression which describes when and where different genes are turned on. Both of these data types can now be acquired using fluorescent laser-scanning (confocal or 2-photon) microscopy of embryos tagged with fluorescent proteins to generate 3D movies of developing embryos. However, analyzing the wealth of resulting images requires tools capable of interactively visualizing several different types of information as well as being scalable to terabytes of data. This paper describes how the combination of existing large data volume visualization and the new Titan information visualization framework of the Visualization Toolkit (VTK) can be applied to the problem of studying the cell lineage of an organism. In particular, by linking the visualization of spatial and temporal gene expression data with novel ways of visualizing cell lineage data, users can study how the genome regulates different aspects of embryonic development.

Distinct regulation of Snail in two muscle lineages of the ascidian embryo achieves temporal coordination of muscle development.

PubMed

Tokuoka, Miki; Kobayashi, Kenji; Satou, Yutaka

2018-06-06

The transcriptional repressor Snail is required for proper differentiation of the tail muscle of ascidian tadpole larvae. Two muscle lineages (B5.1 and B6.4) contribute to the anterior tail muscle cells, and are consecutively separated from a transcriptionally quiescent germ cell lineage at the 16- and 32-cell stages. Concomitantly, cells of these lineages begin to express Tbx6.b ( Tbx6-r.b ) at the 16- and 32-cell stages, respectively. Meanwhile, Snail expression begins in these two lineages simultaneously at the 32-cell stage. Here, we show that Snail expression is regulated differently between these two lineages. In the B5.1 lineage, Snail was activated through Tbx6.b , which is activated by maternal factors, including Zic-r.a. In the B6.4 lineage, the MAPK pathway was cell-autonomously activated by a constitutively active form of Raf, enabling Zic-r.a to activate Snail independently of Tbx6.b As a result, Snail begins to be expressed at the 32-cell stage simultaneously in these two lineages. Such shortcuts might be required for coordinating developmental programs in embryos in which cells become separated progressively from stem cells, including germline cells. © 2018. Published by The Company of Biologists Ltd.

Transcription factor AP-2γ induces early Cdx2 expression and represses HIPPO signaling to specify the trophectoderm lineage.

PubMed

Cao, Zubing; Carey, Timothy S; Ganguly, Avishek; Wilson, Catherine A; Paul, Soumen; Knott, Jason G

2015-05-01

Cell fate decisions are fundamental to the development of multicellular organisms. In mammals the first cell fate decision involves segregation of the pluripotent inner cell mass and the trophectoderm, a process regulated by cell polarity proteins, HIPPO signaling and lineage-specific transcription factors such as CDX2. However, the regulatory mechanisms that operate upstream to specify the trophectoderm lineage have not been established. Here we report that transcription factor AP-2γ (TFAP2C) functions as a novel upstream regulator of Cdx2 expression and position-dependent HIPPO signaling in mice. Loss- and gain-of-function studies and promoter analysis revealed that TFAP2C binding to an intronic enhancer is required for activation of Cdx2 expression during early development. During the 8-cell to morula transition TFAP2C potentiates cell polarity to suppress HIPPO signaling in the outside blastomeres. TFAP2C depletion triggered downregulation of PARD6B, loss of apical cell polarity, disorganization of F-actin, and activation of HIPPO signaling in the outside blastomeres. Rescue experiments using Pard6b mRNA restored cell polarity but only partially corrected position-dependent HIPPO signaling, suggesting that TFAP2C negatively regulates HIPPO signaling via multiple pathways. Several genes involved in regulation of the actin cytoskeleton (including Rock1, Rock2) were downregulated in TFAP2C-depleted embryos. Inhibition of ROCK1 and ROCK2 activity during the 8-cell to morula transition phenocopied TFAP2C knockdown, triggering a loss of position-dependent HIPPO signaling and decrease in Cdx2 expression. Altogether, these results demonstrate that TFAP2C facilitates trophectoderm lineage specification by functioning as a key regulator of Cdx2 transcription, cell polarity and position-dependent HIPPO signaling. © 2015. Published by The Company of Biologists Ltd.

In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model.

PubMed

Szentpáli-Gavallér, Katalin; Lim, Stephanie M; Dencső, László; Bányai, Krisztián; Koraka, Penelope; Osterhaus, Albert D M E; Martina, Byron E E; Bakonyi, Tamás; Bálint, Ádám

2016-02-19

West Nile virus (WNV) strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe) was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L), NS2A (A30P), NS3 (P249H) NS4B (P38G, C102S, E249G)). The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA.

Lineage-Restricted Mammary Stem Cells Sustain the Development, Homeostasis, and Regeneration of the Estrogen Receptor Positive Lineage.

PubMed

Van Keymeulen, Alexandra; Fioramonti, Marco; Centonze, Alessia; Bouvencourt, Gaëlle; Achouri, Younes; Blanpain, Cédric

2017-08-15

The mammary gland (MG) is composed of different cell lineages, including the basal and the luminal cells (LCs) that are maintained by distinct stem cell (SC) populations. LCs can be subdivided into estrogen receptor (ER) + and ER - cells. LCs act as the cancer cell of origin in different types of mammary tumors. It remains unclear whether the heterogeneity found in luminal-derived mammary tumors arises from a pre-existing heterogeneity within LCs. To investigate LC heterogeneity, we used lineage tracing to assess whether the ER + lineage is maintained by multipotent SCs or by lineage-restricted SCs. To this end, we generated doxycycline-inducible ER-rtTA mice that allowed us to perform genetic lineage tracing of ER + LCs and study their fate and long-term maintenance. Our results show that ER + cells are maintained by lineage-restricted SCs that exclusively contribute to the expansion of the ER + lineage during puberty and their maintenance during adult life. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Macrophages – Key Cells in the Response to Wear Debris from Joint Replacements

PubMed Central

Nich, Christophe; Takakubo, Yuya; Pajarinen, Jukka; Ainola, Mari; Salem, Abdelhakim; Sillat, Tarvo; Rao, Allison J.; Raska, Milan; Tamaki, Yasunobu; Takagi, Michiaki; Konttinen, Yrjö T.; Goodman, Stuart B.; Gallo, Jiri

2013-01-01

The generation of wear debris is an inevitable result of normal usage of joint replacements. Wear debris particles stimulate local and systemic biological reactions resulting in chronic inflammation, periprosthetic bone destruction, and eventually, implant loosening and revision surgery. The latter may be indicated in up to 15% patients in the decade following the arthroplasty using conventional polyethylene. Macrophages play multiple roles in both inflammation and in maintaining tissue homeostasis. As sentinels of the innate immune system, they are central to the initiation of this inflammatory cascade, characterized by the release of pro-inflammatory and pro-osteoclastic factors. Similar to the response to pathogens, wear particles elicit a macrophage response, based on the unique properties of the cells belonging to this lineage, including sensing, chemotaxis, phagocytosis, and adaptive stimulation. The biological processes involved are complex, redundant, both local and systemic, and highly adaptive. Cells of the monocyte/macrophage lineage are implicated in this phenomenon, ultimately resulting in differentiation and activation of bone resorbing osteoclasts. Simultaneously, other distinct macrophage populations inhibit inflammation and protect the bone-implant interface from osteolysis. Here, the current knowledge about the physiology of monocyte/macrophage lineage cells is reviewed. In addition, the pattern and consequences of their interaction with wear debris and the recent developments in this field are presented. PMID:23568608

Increased Expression of PcG Protein YY1 Negatively Regulates B Cell Development while Allowing Accumulation of Myeloid Cells and LT-HSC Cells

PubMed Central

Pan, Xuan; Jones, Morgan; Jiang, Jie; Zaprazna, Kristina; Yu, Duonan; Pear, Warren; Maillard, Ivan; Atchison, Michael L.

2012-01-01

Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs. PMID:22292011

Production of somatic chimera chicks by injection of bone marrow cells into recipient blastoderms.

PubMed

Heo, Young Tae; Lee, Sung Ho; Kim, Teoan; Kim, Nam Hyung; Lee, Hoon Taek

2012-01-01

Several types of cells, including blastoderm cells, primordial germ cells, and embryonic germ cells were injected into early-stage recipient embryos to produce chimera avians and to gain insights into cell development. However, a limited number of studies of avian adult stem cells have also been conducted. This study is, to the best of our knowledge, the first to evaluate chicken bone marrow cells' (chBMC) ability to differentiate into multiple cell lineages and capability to generate chimera chicks. We induced random differentiation of chBMCs in vitro and injected immunologically selected pluripotent cells in chBMCs into the blastoderms of recipient eggs. The multipotency of BMCs from the barred Plymouth rock (BPR) was confirmed via AP staining, RT-PCR, immunocytochemistry, and FACS using specific markers, such as Oct-4 and SSEA-1, 3 and 4. Isolated chBMCs were found to be able to induce in vitro differentiation to multiple cell lineages. Approximately 5,000 chBMCs were injected into the blastoderms of white leghorn (WL) recipients and proved able to contribute to the generation of somatic chimera chicks with a frequency of 2.7% (2 of 73). Confirmation of chimerism in hatched chicks was achieved via PCR analysis using D-loop-specific primers of BPR and WL. Our study demonstrated the successful production of chimera chicks using chBMC. Therefore, we propose that the use of adult chBMCs may constitute a new possible approach to the production of chimera poultry, and may provide helpful studies in avian developmental biology.

Analysis of the genetic phylogeny of multifocal prostate cancer identifies multiple independent clonal expansions in neoplastic and morphologically normal prostate tissue.

PubMed

Cooper, Colin S; Eeles, Rosalind; Wedge, David C; Van Loo, Peter; Gundem, Gunes; Alexandrov, Ludmil B; Kremeyer, Barbara; Butler, Adam; Lynch, Andrew G; Camacho, Niedzica; Massie, Charlie E; Kay, Jonathan; Luxton, Hayley J; Edwards, Sandra; Kote-Jarai, ZSofia; Dennis, Nening; Merson, Sue; Leongamornlert, Daniel; Zamora, Jorge; Corbishley, Cathy; Thomas, Sarah; Nik-Zainal, Serena; O'Meara, Sarah; Matthews, Lucy; Clark, Jeremy; Hurst, Rachel; Mithen, Richard; Bristow, Robert G; Boutros, Paul C; Fraser, Michael; Cooke, Susanna; Raine, Keiran; Jones, David; Menzies, Andrew; Stebbings, Lucy; Hinton, Jon; Teague, Jon; McLaren, Stuart; Mudie, Laura; Hardy, Claire; Anderson, Elizabeth; Joseph, Olivia; Goody, Victoria; Robinson, Ben; Maddison, Mark; Gamble, Stephen; Greenman, Christopher; Berney, Dan; Hazell, Steven; Livni, Naomi; Fisher, Cyril; Ogden, Christopher; Kumar, Pardeep; Thompson, Alan; Woodhouse, Christopher; Nicol, David; Mayer, Erik; Dudderidge, Tim; Shah, Nimish C; Gnanapragasam, Vincent; Voet, Thierry; Campbell, Peter; Futreal, Andrew; Easton, Douglas; Warren, Anne Y; Foster, Christopher S; Stratton, Michael R; Whitaker, Hayley C; McDermott, Ultan; Brewer, Daniel S; Neal, David E

2015-04-01

Genome-wide DNA sequencing was used to decrypt the phylogeny of multiple samples from distinct areas of cancer and morphologically normal tissue taken from the prostates of three men. Mutations were present at high levels in morphologically normal tissue distant from the cancer, reflecting clonal expansions, and the underlying mutational processes at work in morphologically normal tissue were also at work in cancer. Our observations demonstrate the existence of ongoing abnormal mutational processes, consistent with field effects, underlying carcinogenesis. This mechanism gives rise to extensive branching evolution and cancer clone mixing, as exemplified by the coexistence of multiple cancer lineages harboring distinct ERG fusions within a single cancer nodule. Subsets of mutations were shared either by morphologically normal and malignant tissues or between different ERG lineages, indicating earlier or separate clonal cell expansions. Our observations inform on the origin of multifocal disease and have implications for prostate cancer therapy in individual cases.

Thymocyte Maturation Is Regulated by the Activity of the Helix-Loop-Helix Protein, E47

PubMed Central

Bain, Gretchen; Quong, Melanie W.; Soloff, Rachel S.; Hedrick, Stephen M.; Murre, Cornelis

1999-01-01

The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I– and class II–restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I–restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection. PMID:10587351

Genome expansion via lineage splitting and genome reduction in the cicada endosymbiont Hodgkinia.

PubMed

Campbell, Matthew A; Van Leuven, James T; Meister, Russell C; Carey, Kaitlin M; Simon, Chris; McCutcheon, John P

2015-08-18

Comparative genomics from mitochondria, plastids, and mutualistic endosymbiotic bacteria has shown that the stable establishment of a bacterium in a host cell results in genome reduction. Although many highly reduced genomes from endosymbiotic bacteria are stable in gene content and genome structure, organelle genomes are sometimes characterized by dramatic structural diversity. Previous results from Candidatus Hodgkinia cicadicola, an endosymbiont of cicadas, revealed that some lineages of this bacterium had split into two new cytologically distinct yet genetically interdependent species. It was hypothesized that the long life cycle of cicadas in part enabled this unusual lineage-splitting event. Here we test this hypothesis by investigating the structure of the Ca. Hodgkinia genome in one of the longest-lived cicadas, Magicicada tredecim. We show that the Ca. Hodgkinia genome from M. tredecim has fragmented into multiple new chromosomes or genomes, with at least some remaining partitioned into discrete cells. We also show that this lineage-splitting process has resulted in a complex of Ca. Hodgkinia genomes that are 1.1-Mb pairs in length when considered together, an almost 10-fold increase in size from the hypothetical single-genome ancestor. These results parallel some examples of genome fragmentation and expansion in organelles, although the mechanisms that give rise to these extreme genome instabilities are likely different.

A lineage CLOUD for neoblasts.

PubMed

Tran, Thao Anh; Gentile, Luca

2018-05-10

In planarians, pluripotency can be studied in vivo in the adult animal, making these animals a unique model system where pluripotency-based regeneration (PBR)-and its therapeutic potential-can be investigated. This review focuses on recent findings to build a cloud model of fate restriction likelihood for planarian stem and progenitor cells. Recently, a computational approach based on functional and molecular profiling at the single cell level was proposed for human hematopoietic stem cells. Based on data generated both in vivo and ex vivo, we hypothesized that planarian stem cells could acquire multiple direction lineage biases, following a "badlands" landscape. Instead of a discrete tree-like hierarchy, where the potency of stem/progenitor cells reduces stepwise, we propose a Continuum of LOw-primed UnDifferentiated Planarian Stem/Progenitor Cells (CLOUD-PSPCs). Every subclass of neoblast/progenitor cells is a cloud of likelihood, as the single cell transcriptomics data indicate. The CLOUD-HSPCs concept was substantiated by in vitro data from cell culture; therefore, to confirm the CLOUD-PSPCs model, the planarian community needs to develop new tools, like live cell tracking. Future studies will allow a deeper understanding of PBR in planarian, and the possible implications for regenerative therapies in human. Copyright © 2018 Elsevier Ltd. All rights reserved.

Inferring rules of lineage commitment in haematopoiesis.

PubMed

Pina, Cristina; Fugazza, Cristina; Tipping, Alex J; Brown, John; Soneji, Shamit; Teles, Jose; Peterson, Carsten; Enver, Tariq

2012-02-19

How the molecular programs of differentiated cells develop as cells transit from multipotency through lineage commitment remains unexplored. This reflects the inability to access cells undergoing commitment or located in the immediate vicinity of commitment boundaries. It remains unclear whether commitment constitutes a gradual process, or else represents a discrete transition. Analyses of in vitro self-renewing multipotent systems have revealed cellular heterogeneity with individual cells transiently exhibiting distinct biases for lineage commitment. Such systems can be used to molecularly interrogate early stages of lineage affiliation and infer rules of lineage commitment. In haematopoiesis, population-based studies have indicated that lineage choice is governed by global transcriptional noise, with self-renewing multipotent cells reversibly activating transcriptome-wide lineage-affiliated programs. We examine this hypothesis through functional and molecular analysis of individual blood cells captured from self-renewal cultures, during cytokine-driven differentiation and from primary stem and progenitor bone marrow compartments. We show dissociation between self-renewal potential and transcriptome-wide activation of lineage programs, and instead suggest that multipotent cells experience independent activation of individual regulators resulting in a low probability of transition to the committed state.

Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells.

PubMed

Semrau, Stefan; Goldmann, Johanna E; Soumillon, Magali; Mikkelsen, Tarjei S; Jaenisch, Rudolf; van Oudenaarden, Alexander

2017-10-23

Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression dynamics of retinoic acid driven mESC differentiation from pluripotency to lineage commitment, using an unbiased single-cell transcriptomics approach. We find that the exit from pluripotency marks the start of a lineage transition as well as a transient phase of increased susceptibility to lineage specifying signals. Our study reveals several transcriptional signatures of this phase, including a sharp increase of gene expression variability and sequential expression of two classes of transcriptional regulators. In summary, we provide a comprehensive analysis of the exit from pluripotency and lineage commitment at the single cell level, a potential stepping stone to improved lineage manipulation through timing of differentiation cues.

Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.

PubMed Central

Housley, R M; Morris, C F; Boyle, W; Ring, B; Biltz, R; Tarpley, J E; Aukerman, S L; Devine, P L; Whitehead, R H; Pierce, G F

1994-01-01

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut. Images PMID:7962522

Cell Expansion During Directed Differentiation of Stem Cells Toward the Hepatic Lineage.

PubMed

Raju, Ravali; Chau, David; Cho, Dong Seong; Park, Yonsil; Verfaillie, Catherine M; Hu, Wei-Shou

2017-02-15

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 10 9 -10 10 cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications.

Temporal and Embryonic Lineage-Dependent Regulation of Human Vascular SMC Development by NOTCH3

PubMed Central

Granata, Alessandra; Bernard, William G.; Zhao, Ning; Mccafferty, John; Lilly, Brenda

2015-01-01

Vascular smooth muscle cells (SMCs), which arise from multiple embryonic progenitors, have unique lineage-specific properties and this diversity may contribute to spatial patterns of vascular diseases. We developed in vitro methods to generate distinct vascular SMC subtypes from human pluripotent stem cells, allowing us to explore their intrinsic differences and the mechanisms involved in SMC development. Since Notch signaling is thought to be one of the several key regulators of SMC differentiation and function, we profiled the expression of Notch receptors, ligands, and downstream elements during the development of origin-specific SMC subtypes. NOTCH3 expression in our in vitro model varied in a lineage- and developmental stage-specific manner so that the highest expression in mature SMCs was in those derived from paraxial mesoderm (PM). This pattern was consistent with the high expression level of NOTCH3 observed in the 8–9 week human fetal descending aorta, which is populated by SMCs of PM origin. Silencing NOTCH3 in mature SMCs in vitro reduced SMC markers in cells of PM origin preferentially. Conversely, during early development, NOTCH3 was highly expressed in vitro in SMCs of neuroectoderm (NE) origin. Inhibition of NOTCH3 in early development resulted in a significant downregulation of specific SMC markers exclusively in the NE lineage. Corresponding to this prediction, the Notch3-null mouse showed reduced expression of Acta2 in the neural crest-derived SMCs of the aortic arch. Thus, Notch3 signaling emerges as one of the key regulators of vascular SMC differentiation and maturation in vitro and in vivo in a lineage- and temporal-dependent manner. PMID:25539150

Identification and characterization of mouse otic sensory lineage genes

PubMed Central

Hartman, Byron H.; Durruthy-Durruthy, Robert; Laske, Roman D.; Losorelli, Steven; Heller, Stefan

2015-01-01

Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiation potential, giving rise to otic sensory cell populations including hair cells, supporting cells, and ganglion neurons of the auditory and vestibular organs. Here we present a systematic approach to identify transcriptional features that distinguish the otic sensory lineage (from early otic progenitors to otic sensory populations) from other major lineages of vertebrate development. We used a microarray approach to analyze otic sensory lineage populations including microdissected otic vesicles (embryonic day 10.5) as well as isolated neonatal cochlear hair cells and supporting cells at postnatal day 3. Non-otic tissue samples including periotic tissues and whole embryos with otic regions removed were used as reference populations to evaluate otic specificity. Otic populations shared transcriptome-wide correlations in expression profiles that distinguish members of this lineage from non-otic populations. We further analyzed the microarray data using comparative and dimension reduction methods to identify individual genes that are specifically expressed in the otic sensory lineage. This analysis identified and ranked top otic sensory lineage-specific transcripts including Fbxo2, Col9a2, and Oc90, and additional novel otic lineage markers. To validate these results we performed expression analysis on select genes using immunohistochemistry and in situ hybridization. Fbxo2 showed the most striking pattern of specificity to the otic sensory lineage, including robust expression in the early otic vesicle and sustained expression in prosensory progenitors and auditory and vestibular hair cells and supporting cells. PMID:25852475

Pox neuro control of cell lineages that give rise to larval poly-innervated external sensory organs in Drosophila.

PubMed

Jiang, Yanrui; Boll, Werner; Noll, Markus

2015-01-15

The Pox neuro (Poxn) gene of Drosophila plays a crucial role in the development of poly-innervated external sensory (p-es) organs. However, how Poxn exerts this role has remained elusive. In this study, we have analyzed the cell lineages of all larval p-es organs, namely of the kölbchen, papilla 6, and hair 3. Surprisingly, these lineages are distinct from any previously reported cell lineages of sensory organs. Unlike the well-established lineage of mono-innervated external sensory (m-es) organs and a previously proposed model of the p-es lineage, we demonstrate that all wild-type p-es lineages exhibit the following features: the secondary precursor, pIIa, gives rise to all three support cells-socket, shaft, and sheath, whereas the other secondary precursor, pIIb, is neuronal and gives rise to all neurons. We further show that in one of the p-es lineages, that of papilla 6, one cell undergoes apoptosis. By contrast in Poxn null mutants, all p-es lineages have a reduced number of cells and their pattern of cell divisions is changed to that of an m-es organ, with the exception of a lineage in a minority of mutant kölbchen that retains a second bipolar neuron. Indeed, the role of Poxn in p-es lineages is consistent with the specification of the developmental potential of secondary precursors and the regulation of cell division but not apoptosis. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparative Genomic Analysis Reveals Ecological Differentiation in the Genus Carnobacterium.

PubMed

Iskandar, Christelle F; Borges, Frédéric; Taminiau, Bernard; Daube, Georges; Zagorec, Monique; Remenant, Benoît; Leisner, Jørgen J; Hansen, Martin A; Sørensen, Søren J; Mangavel, Cécile; Cailliez-Grimal, Catherine; Revol-Junelles, Anne-Marie

2017-01-01

Lactic acid bacteria (LAB) differ in their ability to colonize food and animal-associated habitats: while some species are specialized and colonize a limited number of habitats, other are generalist and are able to colonize multiple animal-linked habitats. In the current study, Carnobacterium was used as a model genus to elucidate the genetic basis of these colonization differences. Analyses of 16S rRNA gene meta-barcoding data showed that C. maltaromaticum followed by C. divergens are the most prevalent species in foods derived from animals (meat, fish, dairy products), and in the gut. According to phylogenetic analyses, these two animal-adapted species belong to one of two deeply branched lineages. The second lineage contains species isolated from habitats where contact with animal is rare. Genome analyses revealed that members of the animal-adapted lineage harbor a larger secretome than members of the other lineage. The predicted cell-surface proteome is highly diversified in C. maltaromaticum and C. divergens with genes involved in adaptation to the animal milieu such as those encoding biopolymer hydrolytic enzymes, a heme uptake system, and biopolymer-binding adhesins. These species also exhibit genes for gut adaptation and respiration. In contrast, Carnobacterium species belonging to the second lineage encode a poorly diversified cell-surface proteome, lack genes for gut adaptation and are unable to respire. These results shed light on the important genomics traits required for adaptation to animal-linked habitats in generalist Carnobacterium .

Efficient production of erythroid, megakaryocytic and myeloid cells, using single cell-derived iPSC colony differentiation.

PubMed

Hansen, Marten; Varga, Eszter; Aarts, Cathelijn; Wust, Tatjana; Kuijpers, Taco; von Lindern, Marieke; van den Akker, Emile

2018-04-28

Hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) provide opportunities not only for fundamental research and disease modelling/drug testing but also for large-scale production of blood effector cells for future clinical application. Although there are multiple ways to differentiate human iPSCs towards hematopoietic lineages, there is a need to develop reproducible and robust protocols. Here we introduce an efficient way to produce three major blood cell types using a standardized differentiation protocol that starts with a single hematopoietic initiation step. This system is feeder-free, avoids EB-formation, starts with a hematopoietic initiation step based on a novel single cell-derived iPSC colony differentiation and produces multi-potential progenitors within 8-10 days. Followed by lineage-specific growth factor supplementation these cells can be matured into well characterized erythroid, megakaryocytic and myeloid cells with high-purity, without transcription factor overexpression or any kind of pre-purification step. This standardized differentiation system provides a simple platform to produce specific blood cells in a reproducible manner for hematopoietic development studies, disease modelling, drug testing and the potential for future therapeutic applications. Copyright © 2018. Published by Elsevier B.V.

Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model.

PubMed

Fatima, Soghra; Zhou, Sheng; Sorrentino, Brian P

2012-02-01

The side population phenotype is associated with the Hoechst dye efflux activity of the Abcg2 transporter and identifies hematopoietic stem cells (HSCs) in the bone marrow. This association suggests the direct use of Abcg2 expression to identify adult stem cells in various other organs. We have generated a lineage tracing mouse model based on an allele that coexpresses both Abcg2 and a CreERT2 expression cassette. By crossing these mice with lox-STOP-lox reporter lines (LacZ or YFP), cells that express Abcg2 and their progeny were identified following treatment with tamoxifen (Tam). In the liver and kidney, in which mature cells express Abcg2, reporter gene expression verified the expected physiologic expression pattern of the recombinant allele. Long-term marking of HSCs was seen in multiple peripheral blood lineages from adult mice, demonstrating that Abcg2(+) bone marrow HSCs contribute to steady-state hematopoiesis. Stem cell tracing patterns were seen in the small intestine and in seminiferous tubules in the testis 20 months after Tam treatment, proving that stem cells from these organs express Abcg2. Interstitial cells from skeletal and cardiac muscle were labeled, and some cells were costained with endothelial markers, raising the possibility that these cells may function in the repair response to muscle injury. Altogether, these studies prove that Abcg2 is a stem cell marker for blood, small intestine, testicular germ cells, and possibly for injured skeletal and/or cardiac muscle and provide a new model for studying stem cell activity that does not require transplant-based assays. Copyright © 2011 AlphaMed Press.

Investigating the Relatedness of Enteroinvasive Escherichia coli to Other E. coli and Shigella Isolates by Using Comparative Genomics

PubMed Central

Hazen, Tracy H.; Leonard, Susan R.; Lampel, Keith A.; Lacher, David W.

2016-01-01

Enteroinvasive Escherichia coli (EIEC) is a unique pathovar that has a pathogenic mechanism nearly indistinguishable from that of Shigella species. In contrast to isolates of the four Shigella species, which are widespread and can be frequent causes of human illness, EIEC causes far fewer reported illnesses each year. In this study, we analyzed the genome sequences of 20 EIEC isolates, including 14 first described in this study. Phylogenomic analysis of the EIEC genomes demonstrated that 17 of the isolates are present in three distinct lineages that contained only EIEC genomes, compared to reference genomes from each of the E. coli pathovars and Shigella species. Comparative genomic analysis identified genes that were unique to each of the three identified EIEC lineages. While many of the EIEC lineage-specific genes have unknown functions, those with predicted functions included a colicin and putative proteins involved in transcriptional regulation or carbohydrate metabolism. In silico detection of the Shigella virulence plasmid (pINV), which is essential for the invasion of host cells, demonstrated that a form of pINV was present in nearly all EIEC genomes, but the Mxi-Spa-Ipa region of the plasmid that encodes the invasion-associated proteins was absent from several of the EIEC isolates. The comparative genomic findings in this study support the hypothesis that multiple EIEC lineages have evolved independently from multiple distinct lineages of E. coli via the acquisition of the Shigella virulence plasmid and, in some cases, the Shigella pathogenicity islands. PMID:27271741

Investigating the Relatedness of Enteroinvasive Escherichia coli to Other E. coli and Shigella Isolates by Using Comparative Genomics.

PubMed

Hazen, Tracy H; Leonard, Susan R; Lampel, Keith A; Lacher, David W; Maurelli, Anthony T; Rasko, David A

2016-08-01

Enteroinvasive Escherichia coli (EIEC) is a unique pathovar that has a pathogenic mechanism nearly indistinguishable from that of Shigella species. In contrast to isolates of the four Shigella species, which are widespread and can be frequent causes of human illness, EIEC causes far fewer reported illnesses each year. In this study, we analyzed the genome sequences of 20 EIEC isolates, including 14 first described in this study. Phylogenomic analysis of the EIEC genomes demonstrated that 17 of the isolates are present in three distinct lineages that contained only EIEC genomes, compared to reference genomes from each of the E. coli pathovars and Shigella species. Comparative genomic analysis identified genes that were unique to each of the three identified EIEC lineages. While many of the EIEC lineage-specific genes have unknown functions, those with predicted functions included a colicin and putative proteins involved in transcriptional regulation or carbohydrate metabolism. In silico detection of the Shigella virulence plasmid (pINV), which is essential for the invasion of host cells, demonstrated that a form of pINV was present in nearly all EIEC genomes, but the Mxi-Spa-Ipa region of the plasmid that encodes the invasion-associated proteins was absent from several of the EIEC isolates. The comparative genomic findings in this study support the hypothesis that multiple EIEC lineages have evolved independently from multiple distinct lineages of E. coli via the acquisition of the Shigella virulence plasmid and, in some cases, the Shigella pathogenicity islands. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity

PubMed Central

Capoccia, Benjamin J.; Robson, Debra L.; Levac, Krysta D.; Maxwell, Dustin J.; Hohm, Sarah A.; Neelamkavil, Marian J.; Bell, Gillian I.; Xenocostas, Anargyros; Link, Daniel C.; Piwnica-Worms, David; Nolta, Jan A.

2009-01-01

The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDHhi) activity, a progenitor cell function conserved between several lineages. BM ALDHhi cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDHhi cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDHhi cells, mice transplanted with purified ALDHhi cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDHhi cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDHhi cell engraftment stimulated endogenous revascularization. Thus, human BM ALDHhi cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans. PMID:19324906

Marking cell lineages in living tissues.

PubMed

Kurup, Smita; Runions, John; Köhler, Uwe; Laplaze, Laurent; Hodge, Sarah; Haseloff, Jim

2005-05-01

We have generated a novel genetic system to visualize cell lineages in living tissues at high resolution. Heat shock was used to trigger the excision of a specific transposon and activation of a fluorescent marker gene. A histone-YFP marker was used to allow identification of cell lineages and easy counting of cells. Constitutive expression of a green fluorescent membrane protein was used to provide a precise outline of all surrounding cells. Marked lineages can be induced from specific cells within the organism by targeted laser irradiation, and the fate of the marked cells can be followed non-invasively. We have used the system to map cell lineages originating from the initials of primary and lateral roots in Arabidopsis. The lineage marking technique enabled us to measure the differential contribution of primary root pericycle cell files to developing lateral root primordia. The majority of cells in an emerging lateral root primordium derive from the central file of pericycle founder cells while off-centre founder cells contribute only a minor proliferation of tissue near the base of the root. The system shows great promise for the detailed study of cell division during morphogenesis.

Cell lineages of the embryo of the nematode Caenorhabditis elegans.

PubMed

Deppe, U; Schierenberg, E; Cole, T; Krieg, C; Schmitt, D; Yoder, B; von Ehrenstein, G

1978-01-01

Embryogenesis of the free-living soil nematode Caenorhabditis elegans produces a juvenile having about 550 cells at hatching. We have determined the lineages of 182 cells by tracing the divisions of individual cells in living embryos. An invariant pattern of cleavage divisions of the egg generates a set of stem cells. These stem cells are the founders of six stem cell lineages. Each lineage has its own clock--i.e., an autonomous rhythm of synchronous cell divisions. The rhythms are maintained in spite of extensive cellular rearrangement. The rate and the orientation of the cell divisions of the cell lineages are essentially invariant among individuals. Thus, the destiny of cells seems to depend primarily on their lineage history. The anterior position of the site of origin of the stem cells in the egg relates to the rate of the cell cycle clock, suggesting intracellular preprogramming of the uncleaved egg. We used a technique that allows normal embryogenesis, from the fertilized egg to hatching, outside the parent under a cover glass. Embryogenesis was followed microscopically with Nomarski interference optics and high-resolution video recording.

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes

PubMed Central

Baxter, Melissa; Withey, Sarah; Harrison, Sean; Segeritz, Charis-Patricia; Zhang, Fang; Atkinson-Dell, Rebecca; Rowe, Cliff; Gerrard, Dave T.; Sison-Young, Rowena; Jenkins, Roz; Henry, Joanne; Berry, Andrew A.; Mohamet, Lisa; Best, Marie; Fenwick, Stephen W.; Malik, Hassan; Kitteringham, Neil R.; Goldring, Chris E.; Piper Hanley, Karen; Vallier, Ludovic; Hanley, Neil A.

2015-01-01

Background & Aims Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Methods Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. Results HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. Conclusions HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes. PMID:25457200

Cell Expansion During Directed Differentiation of Stem Cells Toward the Hepatic Lineage

PubMed Central

Raju, Ravali; Chau, David; Cho, Dong Seong; Park, Yonsil; Verfaillie, Catherine M.

2017-01-01

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 109–1010 cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications. PMID:27806669

In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model

PubMed Central

Szentpáli-Gavallér, Katalin; Lim, Stephanie M.; Dencső, László; Bányai, Krisztián; Koraka, Penelope; Osterhaus, Albert D.M.E.; Martina, Byron E.E.; Bakonyi, Tamás; Bálint, Ádám

2016-01-01

West Nile virus (WNV) strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe) was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L), NS2A (A30P), NS3 (P249H) NS4B (P38G, C102S, E249G)). The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA. PMID:26907325

Transcription factor interplay in T helper cell differentiation.

PubMed

Evans, Catherine M; Jenner, Richard G

2013-11-01

The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity.

Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets

PubMed Central

Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

2014-01-01

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms. PMID:24903657

Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets.

PubMed

Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

2014-06-06

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms.

Matrix directed adipogenesis and neurogenesis of mesenchymal stem cells derived from adipose tissue and bone marrow.

PubMed

Lee, Junmin; Abdeen, Amr A; Tang, Xin; Saif, Taher A; Kilian, Kristopher A

2016-09-15

Mesenchymal stem cells (MSCs) can differentiate into multiple lineages through guidance from the biophysical and biochemical properties of the extracellular matrix. In this work we conduct a combinatorial study of matrix properties that influence adipogenesis and neurogenesis including: adhesion proteins, stiffness, and cell geometry, for mesenchymal stem cells derived from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs). We uncover distinct differences in integrin expression, the magnitude of traction stress, and lineage specification to adipocytes and neuron-like cells between cell sources. In the absence of media supplements, adipogenesis in AT-MSCs is not significantly influenced by matrix properties, while the converse is true in BM-MSCs. Both cell types show changes in the expression of neurogenesis markers as matrix cues are varied. When cultured on laminin conjugated microislands of the same adhesive area, BM-MSCs display elevated adipogenesis markers, while AT-MSCs display elevated neurogenesis markers; integrin analysis suggests neurogenesis in AT-MSCs is guided by adhesion through integrin αvβ3. Overall, the properties of the extracellular matrix guides MSC adhesion and lineage specification to different degrees and outcomes, in spite of their similarities in general characteristics. This work will help guide the selection of MSCs and matrix components for applications where high fidelity of differentiation outcome is desired. Mesenchymal stem cells (MSCs) are an attractive cell type for stem cell therapies; however, in order for these cells to be useful in medicine, we need to understand how they respond to the physical and chemical environments of tissue. Here, we explore how two promising sources of MSCs-those derived from bone marrow and from adipose tissue-respond to the compliance and composition of tissue using model extracellular matrices. Our results demonstrate a source-specific propensity to undergo adipogenesis and neurogenesis, and uncover a role for adhesion, and the degree of traction force exerted on the substrate in guiding these lineage outcomes. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Discovery of ectosymbiotic Endomicrobium lineages associated with protists in the gut of stolotermitid termites.

PubMed

Izawa, Kazuki; Kuwahara, Hirokazu; Sugaya, Kaito; Lo, Nathan; Ohkuma, Moriya; Hongoh, Yuichi

2017-08-01

The genus Endomicrobium is a dominant bacterial group in the gut of lower termites, and most phylotypes are intracellular symbionts of gut protists. Here we report the discovery of Endomicrobium ectosymbionts of termite gut protists. We found that bristle-like Endomicrobium cells attached to the surface of spirotrichosomid protist cells inhabiting the termite Stolotermes victoriensis. Transmission electron microscopy revealed that a putative Endomicrobium cell likely attached to the protist surface via a protrusion from the tip of the bacterium. A phylotype, sharing 98.9% 16S rRNA sequence identity with the Endomicrobium ectosymbionts of the spirotrichosomid protists, was also found on the cell surface of the protist Trichonympha magna in the gut of the termite Porotermes adamsoni. We propose the novel species 'Candidatus Endomicrobium superficiale' for these bacteria. T. magna simultaneously harboured another Endomicrobium ectosymbiont that shared 93.5-94.2% 16S rRNA sequence identities with 'Ca. Endomicrobium superficiale'. Furthermore, Spirotrichonympha-like protists in P. adamsoni guts were associated with an Endomicrobium phylotype that possibly attached to the host flagella. A phylogenetic analysis suggested that these ectosymbiotic lineages have evolved multiple times from free-living Endomicrobium lineages and are relatively distant from the endosymbionts. Our results provide novel insights into the ecology and evolution of the Endomicrobium. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Reduced Abd-B Hox function during kidney development results in lineage infidelity.

PubMed

Magella, Bliss; Mahoney, Robert; Adam, Mike; Potter, S Steven

2018-06-15

Hox genes can function as key drivers of segment identity, with Hox mutations in Drosophila often resulting in dramatic homeotic transformations. In addition, however, they can serve other essential functions. In mammals, the study of Hox gene roles in development is complicated by the presence of four Hox clusters with a total of 39 genes showing extensive functional overlap. In this study, in order to better understand shared core Hox functions, we examined kidney development in mice with frameshift mutations of multiple Abd-B type Hox genes. The resulting phenotypes included dramatically reduced branching morphogenesis of the ureteric bud, premature depletion of nephron progenitors and abnormal development of the stromal compartment. Most unexpected, however, we also observed a cellular level lineage infidelity in nephron segments. Scattered cells within the proximal tubules, for example, expressed genes normally expressed only in collecting ducts. Multiple combinations of inappropriate nephron segment specific marker expression were found. In some cases, cells within a tubule showed incorrect identity, while in other cases cells showed ambiguous character, with simultaneous expression of genes associated with more than one nephron segment. These results give evidence that Hox genes have an overlapping core function at the cellular level in driving and/or maintaining correct differentiation decisions. Copyright © 2018 Elsevier Inc. All rights reserved.

Lineage Switching in Acute Leukemias: A Consequence of Stem Cell Plasticity?

PubMed Central

Dorantes-Acosta, Elisa; Pelayo, Rosana

2012-01-01

Acute leukemias are the most common cancer in childhood and characterized by the uncontrolled production of hematopoietic precursor cells of the lymphoid or myeloid series within the bone marrow. Even when a relatively high efficiency of therapeutic agents has increased the overall survival rates in the last years, factors such as cell lineage switching and the rise of mixed lineages at relapses often change the prognosis of the illness. During lineage switching, conversions from lymphoblastic leukemia to myeloid leukemia, or vice versa, are recorded. The central mechanisms involved in these phenomena remain undefined, but recent studies suggest that lineage commitment of plastic hematopoietic progenitors may be multidirectional and reversible upon specific signals provided by both intrinsic and environmental cues. In this paper, we focus on the current knowledge about cell heterogeneity and the lineage switch resulting from leukemic cells plasticity. A number of hypothetical mechanisms that may inspire changes in cell fate decisions are highlighted. Understanding the plasticity of leukemia initiating cells might be fundamental to unravel the pathogenesis of lineage switch in acute leukemias and will illuminate the importance of a flexible hematopoietic development. PMID:22852088

Epigenetic restriction of embryonic cell lineage fate by methylation of Elf5

PubMed Central

Ng, Ray Kit; Dean, Wendy; Dawson, Claire; Lucifero, Diana; Madeja, Zofia; Reik, Wolf; Hemberger, Myriam

2008-01-01

Mouse ES cells can differentiate into all three germ layers of the embryo but are generally excluded from the trophoblast lineage. Here we show that ES cells deficient in DNA methylation can differentiate efficiently into trophoblast derivatives. In a genome-wide screen we identify the transcription factor Elf5 as methylated and repressed in ES cells, and hypomethylated and expressed in TS and methylation-deficient ES cells. Elf5 creates a positive feedback loop with TS cell determinants Cdx2 and Eomes that is restricted to the trophoblast lineage by epigenetic regulation of Elf5. Importantly, the late-acting function of Elf5 allows initial plasticity and regulation in the early blastocyst. Thus, Elf5 acts downstream of initial lineage determination as a gatekeeper to reinforce commitment to the trophoblast lineage, or to abort this pathway in epiblast cells. This epigenetic restriction of cell lineage fate provides a molecular mechanism for Waddington’s concept of canalization of developmental pathways. PMID:18836439

A Complete Developmental Sequence of a Drosophila Neuronal Lineage as Revealed by Twin-Spot MARCM

PubMed Central

He, Yisheng; Ding, Peng; Kao, Jui-Chun; Lee, Tzumin

2010-01-01

Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain. PMID:20808769

Genetic and epigenetic variation in the lineage specification of regulatory T cells

PubMed Central

Arvey, Aaron; van der Veeken, Joris; Plitas, George; Rich, Stephen S; Concannon, Patrick; Rudensky, Alexander Y

2015-01-01

Regulatory T (Treg) cells, which suppress autoimmunity and other inflammatory states, are characterized by a distinct set of genetic elements controlling their gene expression. However, the extent of genetic and associated epigenetic variation in the Treg cell lineage and its possible relation to disease states in humans remain unknown. We explored evolutionary conservation of regulatory elements and natural human inter-individual epigenetic variation in Treg cells to identify the core transcriptional control program of lineage specification. Analysis of single nucleotide polymorphisms in core lineage-specific enhancers revealed disease associations, which were further corroborated by high-resolution genotyping to fine map causal polymorphisms in lineage-specific enhancers. Our findings suggest that a small set of regulatory elements specify the Treg lineage and that genetic variation in Treg cell-specific enhancers may alter Treg cell function contributing to polygenic disease. DOI: http://dx.doi.org/10.7554/eLife.07571.001 PMID:26510014

Origins of adult pigmentation: diversity in pigment stem cell lineages and implications for pattern evolution

PubMed Central

Spiewak, Jessica E.

2014-01-01

Summary Teleosts comprise about half of all vertebrate species and exhibit an extraordinary diversity of adult pigment patterns that function in shoaling, camouflage and mate choice and have played important roles in speciation. Here, we review recent studies that have identified several distinct neural crest lineages, with distinct genetic requirements, that give rise to adult pigment cells in fishes. These lineages include post-embryonic, peripheral nerve associated stem cells that generate black melanophores and iridescent iridophores, cells derived directly from embryonic neural crest cells that generate yellow-orange xanthophores, and bipotent stem cells that generate both melanophores and xanthophores. This complexity in adult chromatophore lineages has implications for our understanding of adult traits, melanoma, and the evolutionary diversification of pigment cell lineages and patterns. PMID:25421288

Fibroblast Growth Factor 1 (FGFR1) Modulation Regulates Repair Capacity of Oligodendrocyte Progenitor Cells Following Chronic Demyelination

PubMed Central

Zhou, Yong-Xing; Pannu, Ravinder; Le, Tuan Q.; Armstrong, Regina C.

2011-01-01

The adult mammalian brain contains multiple populations of endogenous progenitor cell types. However, following CNS trauma or disease, the regenerative capacity of progenitor populations is typically insufficient and may actually be limited by non-permissive or inhibitory signals in the damaged parenchyma. Remyelination is the most effective and simplest regenerative process in the adult CNS yet is still insufficient following repeated or chronic demyelination. Our previous in vitro studies demonstrated that fibroblast growth factor receptor 1 (FGFR1) signaling inhibited oligodendrocyte progenitor (OP) differentiation into mature oligodendrocytes. Therefore, we questioned whether FGFR1 signaling may inhibit the capacity of OP cells to generate oligodendrocytes in a demyelinating disease model and whether genetically reducing FGFR1 signaling in oligodendrocyte lineage cells could enhance the capacity for remyelination. FGFR1 was found to be upregulated in the corpus callosum during cuprizone mediated demyelination and expressed on OP cells just prior to remyelination. Plp/CreERT:Fgfr1fl/flmice were administered tamoxifen to induce conditional Fgfr1 deletion in oligodendrocyte lineage cells. Tamoxifen administration during chronic demyelination resulted in reduced FGFR1 expression in OP cells. OP proliferation and population size were not altered one week after tamoxifen treatment. Tamoxifen was then administered during chronic demyelination and mice were given a six week recovery period without cuprizone in the chow. After the recovery period, OP numbers were reduced and the number of mature oligodendrocytes was increased, indicating an effect of FGFR1 reduction on OP differentiation. Importantly, tamoxifen administration in Plp/CreERT:Fgfr1fl/fl mice significantly promoted remyelination and axon integrity. These results demonstrate a direct effect of FGFR1 signaling in oligodendrocyte lineage cells as inhibiting the repair capacity of OP cells following chronic demyelination in the adult CNS. PMID:21854849

Human T-cell leukemia virus type 1 infects multiple lineage hematopoietic cells in vivo

PubMed Central

Sugata, Kenji; Ueno, Takaharu; Koh, Ki-Ryang; Higuchi, Yusuke; Matsuda, Fumihiko; Melamed, Anat; Bangham, Charles R.

2017-01-01

Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo. PMID:29186194

Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution

PubMed Central

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D.; Rainey, Paul B.; de Visser, J. Arjan G. M.; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes–via growth–over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology. PMID:27077662

Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution.

PubMed

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D; Rainey, Paul B; de Visser, J Arjan G M; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes-via growth-over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.

Cell Lineage Analysis of the Mammalian Female Germline

PubMed Central

Elbaz, Judith; Jinich, Adrian; Chapal-Ilani, Noa; Maruvka, Yosef E.; Nevo, Nava; Marx, Zipora; Horovitz, Inna; Wasserstrom, Adam; Mayo, Avi; Shur, Irena; Benayahu, Dafna; Skorecki, Karl; Segal, Eran; Dekel, Nava; Shapiro, Ehud

2012-01-01

Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development. PMID:22383887

The generation of the epicardial lineage from human pluripotent stem cells

PubMed Central

Witty, Alec D.; Mihic, Anton; Tam, Roger Y.; Fisher, Stephanie A.; Mikryukov, Alexander; Shoichet, Molly S.; Li, Ren-Ke; Kattman, Steven J.; Keller, Gordon

2014-01-01

The epicardium supports cardiomyocyte proliferation early in development and provides fibroblasts and vascular smooth muscle cells to the developing heart. The epicardium has been shown to play an important role during tissue remodeling after cardiac injury, making access to this cell lineage necessary for the study of regenerative medicine. Here we describe the generation of epicardial lineage cells from human pluripotent stem cells by stage-specific activation of the BMP and WNT signaling pathways. These cells display morphological characteristics and express markers of the epicardial lineage, including the transcription factors WT1 and TBX18 and the retinoic acid–producing enzyme ALDH1A2. When induced to undergo epicardial-tomesenchymal transition, the cells give rise to populations that display characteristics of the fibroblast and vascular smooth muscle lineages. These findings identify BMP and WNT as key regulators of the epicardial lineage in vitro and provide a model for investigating epicardial function in human development and disease. PMID:25240927

Transcription factor interplay in T helper cell differentiation

PubMed Central

Evans, Catherine M.

2013-01-01

The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity. PMID:23878131

Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios.

PubMed

Hoppe, Philipp S; Schwarzfischer, Michael; Loeffler, Dirk; Kokkaliaris, Konstantinos D; Hilsenbeck, Oliver; Moritz, Nadine; Endele, Max; Filipczyk, Adam; Gambardella, Adriana; Ahmed, Nouraiz; Etzrodt, Martin; Coutu, Daniel L; Rieger, Michael A; Marr, Carsten; Strasser, Michael K; Schauberger, Bernhard; Burtscher, Ingo; Ermakova, Olga; Bürger, Antje; Lickert, Heiko; Nerlov, Claus; Theis, Fabian J; Schroeder, Timm

2016-07-14

The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic-erythroid and granulocytic-monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic-erythroid versus granulocytic-monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.

SONAR: A High-Throughput Pipeline for Inferring Antibody Ontogenies from Longitudinal Sequencing of B Cell Transcripts.

PubMed

Schramm, Chaim A; Sheng, Zizhang; Zhang, Zhenhai; Mascola, John R; Kwong, Peter D; Shapiro, Lawrence

2016-01-01

The rapid advance of massively parallel or next-generation sequencing technologies has made possible the characterization of B cell receptor repertoires in ever greater detail, and these developments have triggered a proliferation of software tools for processing and annotating these data. Of especial interest, however, is the capability to track the development of specific antibody lineages across time, which remains beyond the scope of most current programs. We have previously reported on the use of techniques such as inter- and intradonor analysis and CDR3 tracing to identify transcripts related to an antibody of interest. Here, we present Software for the Ontogenic aNalysis of Antibody Repertoires (SONAR), capable of automating both general repertoire analysis and specialized techniques for investigating specific lineages. SONAR annotates next-generation sequencing data, identifies transcripts in a lineage of interest, and tracks lineage development across multiple time points. SONAR also generates figures, such as identity-divergence plots and longitudinal phylogenetic "birthday" trees, and provides interfaces to other programs such as DNAML and BEAST. SONAR can be downloaded as a ready-to-run Docker image or manually installed on a local machine. In the latter case, it can also be configured to take advantage of a high-performance computing cluster for the most computationally intensive steps, if available. In summary, this software provides a useful new tool for the processing of large next-generation sequencing datasets and the ontogenic analysis of neutralizing antibody lineages. SONAR can be found at https://github.com/scharch/SONAR, and the Docker image can be obtained from https://hub.docker.com/r/scharch/sonar/.

Investigational Antibody-Drug Conjugates for Treatment of B-lineage Malignancies.

PubMed

Herrera, Alex F; Molina, Arturo

2018-05-10

Antibody-drug conjugates (ADCs) are tripartite molecules consisting of a monoclonal antibody, a covalent linker, and a cytotoxic payload. ADC development has aimed to target the specificity inherent in antigen-antibody interactions to deliver potent cytotoxins preferentially to tumor cells and maximize antitumor activity and simultaneously minimize off-target toxicity. The earliest ADCs provided disappointing results in the clinic; however, the lessons learned regarding the need for human or humanized antibodies, more stable linkers, and greater potency payloads led to improved ADCs. Three ADCs, gemtuzumab ozogamicin, brentuximab vedotin (BV), and inotuzumab ozogamicin, have been approved for hematologic malignancies. Site-specific conjugation methods have now resulted in a new generation of more uniform, molecularly defined ADCs. These are expected to display improved in vivo properties and have recently entered the clinic. We reviewed investigational ADCs currently in clinical testing for the treatment of B-cell lineage malignancies, including leukemias, lymphomas, and multiple myeloma. The rationales for antigen targeting, data reported to date, current trial status, and preclinical results for several newer ADCs expected to enter first-in-human studies are presented. Owing to the large number of ongoing and reported BV clinical studies, only the studies of BV for diffuse large B-cell lymphoma and those combining BV with checkpoint inhibitors in B-lineage malignancies have been reviewed. With > 40 ongoing clinical trials and 7 investigational ADCs already having advanced to phase II studies, the role of ADCs in the armamentarium for the treatment of B-lineage malignancies continues to be elucidated. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells.

PubMed

Macaulay, Iain C; Svensson, Valentine; Labalette, Charlotte; Ferreira, Lauren; Hamey, Fiona; Voet, Thierry; Teichmann, Sarah A; Cvejic, Ana

2016-02-02

The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells

PubMed Central

Sojka, Dorothy K; Plougastel-Douglas, Beatrice; Yang, Liping; Pak-Wittel, Melissa A; Artyomov, Maxim N; Ivanova, Yulia; Zhong, Chao; Chase, Julie M; Rothman, Paul B; Yu, Jenny; Riley, Joan K; Zhu, Jinfang; Tian, Zhigang; Yokoyama, Wayne M

2014-01-01

Natural killer (NK) cells belong to the innate immune system; they can control virus infections and developing tumors by cytotoxicity and producing inflammatory cytokines. Most studies of mouse NK cells, however, have focused on conventional NK (cNK) cells in the spleen. Recently, we described two populations of liver NK cells, tissue-resident NK (trNK) cells and those resembling splenic cNK cells. However, their lineage relationship was unclear; trNK cells could be developing cNK cells, related to thymic NK cells, or a lineage distinct from both cNK and thymic NK cells. Herein we used detailed transcriptomic, flow cytometric, and functional analysis and transcription factor-deficient mice to determine that liver trNK cells form a distinct lineage from cNK and thymic NK cells. Taken together with analysis of trNK cells in other tissues, there are at least four distinct lineages of NK cells: cNK, thymic, liver (and skin) trNK, and uterine trNK cells. DOI: http://dx.doi.org/10.7554/eLife.01659.001 PMID:24714492

A Simplified and Systematic Method to Isolate, Culture, and Characterize Multiple Types of Human Dental Stem Cells from a Single Tooth.

PubMed

Bakkar, Mohammed; Liu, Younan; Fang, Dongdong; Stegen, Camille; Su, Xinyun; Ramamoorthi, Murali; Lin, Li-Chieh; Kawasaki, Takako; Makhoul, Nicholas; Pham, Huan; Sumita, Yoshinori; Tran, Simon D

2017-01-01

This chapter describes a simplified method that allows the systematic isolation of multiple types of dental stem cells such as dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC), and stem cells of the apical papilla (SCAP) from a single tooth. Of specific interest is the modified laboratory approach to harvest/retrieve the dental pulp tissue by minimizing trauma to DPSC by continuous irrigation, reduction of frictional heat from the bur rotation, and reduction of the bur contact time with the dentin. Also, the use of a chisel and a mallet will maximize the number of live DPSC for culture. Steps demonstrating the potential for multiple cell differentiation lineages of each type of dental stem cell into either osteocytes, adipocytes, or chondrocytes are described. Flow cytometry, with a detailed strategy for cell gating and analysis, is described to verify characteristic markers of human mesenchymal multipotent stromal cells (MSC) from DPSC, PDLSC, or SCAP for subsequent experiments in cell therapy and in tissue engineering. Overall, this method can be adapted to any laboratory with a general setup for cell culture experiments.

Fibroblast growth factor signaling in oligodendrocyte-lineage cells facilitates recovery of chronically demyelinated lesions but is redundant in acute lesions

PubMed Central

Furusho, M; Roulois, A; Franklin, RJM; Bansal, R

2015-01-01

Remyelination is a potent regenerative process in demyelinating diseases, such as multiple sclerosis, the effective therapeutic promotion of which will fill an unmet clinical need. The development of pro-regenerative therapies requires the identification of key regulatory targets that are likely to be involved in the integration of multiple signaling mechanisms. Fibroblast growth factor (FGF) signaling system, which comprises multiple ligands and receptors, potentially provides one such target. Since the FGF/FGF receptor (FGFR) interactions are complex and regulate multiple diverse functions of oligodendrocyte lineage cells, it is difficult to predict their overall therapeutic potential in the regeneration of oligodendrocytes and myelin. Therefore, to assess the integrated effects of FGFR signaling on this process, we simultaneously inactivated both FGFR1 and FGFR2 in oligodendrocytes and their precursors using two Cre-driver mouse lines. Acute and chronic cuprizone-induced or lysolecithin-induced demyelination was established in Fgfr1/Fgfr2 double knockout mice (dKO). We found that in the acute cuprizone model, there was normal differentiation of oligodendrocytes and recovery of myelin in the corpus callosum of both control and dKO mice. Similarly, in the spinal cord, lysolecithin-induced demyelinated lesions regenerated similarly in the dKO and control mice. In contrast, in the chronic cuprizone model, fewer differentiated oligodendrocytes and less efficient myelin recovery were observed in the dKO compared to control mice. These data suggest that while cell-autonomous FGF signaling is redundant during recovery of acute demyelinated lesions, it facilitates regenerative processes in chronic demyelination. Thus, FGF-based therapies have potential value in stimulating oligodendrocyte and myelin regeneration in late-stage disease. PMID:25913734

High resolution fate map of the zebrafish diencephalon.

PubMed

Russek-Blum, Niva; Nabel-Rosen, Helit; Levkowitz, Gil

2009-07-01

The diencephalon acts as an interactive site between the sensory, central, and endocrine systems and is one of the most elaborate structures in the vertebrate brain. To better understand the embryonic development and morphogenesis of the diencephalon, we developed an improved photoactivation (uncaging)-based lineage tracing strategy. To determine the exact position of a given diencephalic progenitor domain, we used a transgenic line driving green fluorescent protein (GFP) in cells expressing the proneural protein, Neurogenin1 (Neurog1), which was used as a visible neural plate landmark. This approach facilitated precise labeling of defined groups of cells in the prospective diencephalon of the zebrafish neural plate. In this manner, we labeled multiple overlapping areas of the diencephalon, thereby ensuring both accuracy and reproducibility of our lineage tracing regardless of the dynamic changes of the developing neural plate. We present a fate map of the zebrafish diencephalon at a higher spatial resolution than previously described. (c) 2009 Wiley-Liss, Inc.

Using Genotype Abundance to Improve Phylogenetic Inference

PubMed Central

Mesin, Luka; Victora, Gabriel D; Minin, Vladimir N; Matsen, Frederick A

2018-01-01

Abstract Modern biological techniques enable very dense genetic sampling of unfolding evolutionary histories, and thus frequently sample some genotypes multiple times. This motivates strategies to incorporate genotype abundance information in phylogenetic inference. In this article, we synthesize a stochastic process model with standard sequence-based phylogenetic optimality, and show that tree estimation is substantially improved by doing so. Our method is validated with extensive simulations and an experimental single-cell lineage tracing study of germinal center B cell receptor affinity maturation. PMID:29474671

Accuracy of Answers to Cell Lineage Questions Depends on Single-Cell Genomics Data Quality and Quantity.

PubMed

Spiro, Adam; Shapiro, Ehud

2016-06-01

Advances in single-cell (SC) genomics enable commensurate improvements in methods for uncovering lineage relations among individual cells, as determined by phylogenetic analysis of the somatic mutations harbored by each cell. Theoretically, complete and accurate knowledge of the genome of each cell of an individual can produce an extremely accurate cell lineage tree of that individual. However, the reality of SC genomics is that such complete and accurate knowledge would be wanting, in quality and in quantity, for the foreseeable future. In this paper we offer a framework for systematically exploring the feasibility of answering cell lineage questions based on SC somatic mutational analysis, as a function of SC genomics data quality and quantity. We take into consideration the current limitations of SC genomics in terms of mutation data quality, most notably amplification bias and allele dropouts (ADO), as well as cost, which puts practical limits on mutation data quantity obtained from each cell as well as on cell sample density. We do so by generating in silico cell lineage trees using a dedicated formal language, eSTG, and show how the ability to answer correctly a cell lineage question depends on the quality and quantity of the SC mutation data. The presented framework can serve as a baseline for the potential of current SC genomics to unravel cell lineage dynamics, as well as the potential contributions of future advancement, both biochemical and computational, for the task.

ILK modulates epithelial polarity and matrix formation in hair follicles.

PubMed

Rudkouskaya, Alena; Welch, Ian; Dagnino, Lina

2014-03-01

Hair follicle morphogenesis requires coordination of multiple signals and communication between its epithelial and mesenchymal constituents. Cell adhesion protein platforms, which include integrins and integrin-linked kinase (ILK), are critical for hair follicle formation. However, their precise contribution to this process is poorly understood. We show that in the absence of ILK, the hair follicle matrix lineage fails to develop, likely due to abnormalities in development of apical-basal cell polarity, as well as in laminin-511 and basement membrane assembly at the tip of the hair bud. These defects also result in impaired specification of hair matrix and absence of precortex and inner sheath root cell lineages. The molecular pathways affected in ILK-deficient follicles are similar to those in the absence of epidermal integrin β1 and include Wnt, but not sonic hedgehog, signaling. ILK-deficient hair buds also show abnormalities in the dermal papilla. Addition of exogenous laminin-511 restores morphological and molecular markers associated with hair matrix formation, indicating that ILK regulates hair bud cell polarity and functions upstream from laminin-511 assembly to regulate the developmental progression of hair follicles beyond the germ stage.

Selective Roles of Normal and Mutant Huntingtin in Neural Induction and Early Neurogenesis

PubMed Central

Nguyen, Giang D.; Gokhan, Solen; Molero, Aldrin E.; Mehler, Mark F.

2013-01-01

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal polyglutamine expansion in the amino-terminal end of the huntingtin protein (Htt) and characterized by progressive striatal and cortical pathology. Previous reports have shown that Htt is essential for embryogenesis, and a recent study by our group revealed that the pathogenic form of Htt (mHtt) causes impairments in multiple stages of striatal development. In this study, we have examined whether HD-associated striatal developmental deficits are reflective of earlier maturational alterations occurring at the time of neurulation by assessing differential roles of Htt and mHtt during neural induction and early neurogenesis using an in vitro mouse embryonic stem cell (ESC) clonal assay system. We demonstrated that the loss of Htt in ESCs (KO ESCs) severely disrupts the specification of primitive and definitive neural stem cells (pNSCs, dNSCs, respectively) during the process of neural induction. In addition, clonally derived KO pNSCs and dNSCs displayed impaired proliferative potential, enhanced cell death and altered multi-lineage potential. Conversely, as observed in HD knock-in ESCs (Q111 ESCs), mHtt enhanced the number and size of pNSC clones, which exhibited enhanced proliferative potential and precocious neuronal differentiation. The transition from Q111 pNSCs to fibroblast growth factor 2 (FGF2)-responsive dNSCs was marked by potentiation in the number of dNSCs and altered proliferative potential. The multi-lineage potential of Q111 dNSCs was also enhanced with precocious neurogenesis and oligodendrocyte progenitor elaboration. The generation of Q111 epidermal growth factor (EGF)-responsive dNSCs was also compromised, whereas their multi-lineage potential was unaltered. These abnormalities in neural induction were associated with differential alterations in the expression profiles of Notch, Hes1 and Hes5. These cumulative observations indicate that Htt is required for multiple stages of neural induction, whereas mHtt enhances this process and promotes precocious neurogenesis and oligodendrocyte progenitor cell elaboration. PMID:23691206

Theory and Practice of Lineage Tracing.

PubMed

Hsu, Ya-Chieh

2015-11-01

Lineage tracing is a method that delineates all progeny produced by a single cell or a group of cells. The possibility of performing lineage tracing initiated the field of Developmental Biology and continues to revolutionize Stem Cell Biology. Here, I introduce the principles behind a successful lineage-tracing experiment. In addition, I summarize and compare different methods for conducting lineage tracing and provide examples of how these strategies can be implemented to answer fundamental questions in development and regeneration. The advantages and limitations of each method are also discussed. © 2015 AlphaMed Press.

T-cell stimuli independently sum to regulate an inherited clonal division fate

PubMed Central

Marchingo, J. M.; Prevedello, G.; Kan, A.; Heinzel, S.; Hodgkin, P. D.; Duffy, K. R.

2016-01-01

In the presence of antigen and costimulation, T cells undergo a characteristic response of expansion, cessation and contraction. Previous studies have revealed that population-level reproducibility is a consequence of multiple clones exhibiting considerable disparity in burst size, highlighting the requirement for single-cell information in understanding T-cell fate regulation. Here we show that individual T-cell clones resulting from controlled stimulation in vitro are strongly lineage imprinted with highly correlated expansion fates. Progeny from clonal families cease dividing in the same or adjacent generations, with inter-clonal variation producing burst-size diversity. The effects of costimulatory signals on individual clones sum together with stochastic independence; therefore, the net effect across multiple clones produces consistent, but heterogeneous population responses. These data demonstrate that substantial clonal heterogeneity arises through differences in experience of clonal progenitors, either through stochastic antigen interaction or by differences in initial receptor sensitivities. PMID:27869196

Enforcement of γδ-lineage commitment by the pre-T-cell receptor in precursors with weak γδ-TCR signals.

PubMed

Zarin, Payam; Wong, Gladys W; Mohtashami, Mahmood; Wiest, David L; Zúñiga-Pflücker, Juan Carlos

2014-04-15

Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-β and γδ-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.

Bone Morphogenetic Protein (BMP) signaling in animal reproductive system development and function.

PubMed

Lochab, Amaneet K; Extavour, Cassandra G

2017-07-15

In multicellular organisms, the specification, maintenance, and transmission of the germ cell lineage to subsequent generations are critical processes that ensure species survival. A number of studies suggest that the Bone Morphogenetic Protein (BMP) pathway plays multiple roles in this cell lineage. We wished to use a comparative framework to examine the role of BMP signaling in regulating these processes, to determine if patterns would emerge that might shed light on the evolution of molecular mechanisms that may play germ cell-specific or other reproductive roles across species. To this end, here we review evidence to date from the literature supporting a role for BMP signaling in reproductive processes across Metazoa. We focus on germ line-specific processes, and separately consider somatic reproductive processes. We find that from primordial germ cell (PGC) induction to maintenance of PGC identity and gametogenesis, BMP signaling regulates these processes throughout embryonic development and adult life in multiple deuterostome and protostome clades. In well-studied model organisms, functional genetic evidence suggests that BMP signaling is required in the germ line across all life stages, with the exception of PGC specification in species that do not use inductive signaling to induce germ cell formation. The current evidence is consistent with the hypothesis that BMP signaling is ancestral in bilaterian inductive PGC specification. While BMP4 appears to be the most broadly employed ligand for the reproductive processes considered herein, we also noted evidence for sex-specific usage of different BMP ligands. In gametogenesis, BMP6 and BMP15 seem to have roles restricted to oogenesis, while BMP8 is restricted to spermatogenesis. We hypothesize that a BMP-based mechanism may have been recruited early in metazoan evolution to specify the germ line, and was subsequently co-opted for use in other germ line-specific and somatic reproductive processes. We suggest that if future studies assessing the function of the BMP pathway across extant species were to include a reproductive focus, that we would be likely to find continued evidence in favor of an ancient association between BMP signaling and the reproductive cell lineage in animals. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation of enteroendocrine cell diversity in midgut stem cell lineages

PubMed Central

Beehler-Evans, Ryan; Micchelli, Craig A.

2015-01-01

The endocrine system mediates long-range peptide hormone signaling to broadcast changes in metabolic status to distant target tissues via the circulatory system. In many animals, the diffuse endocrine system of the gut is the largest endocrine tissue, with the full spectrum of endocrine cell subtypes not yet fully characterized. Here, we combine molecular mapping, lineage tracing and genetic analysis in the adult fruit fly to gain new insight into the cellular and molecular mechanisms governing enteroendocrine cell diversity. Neuropeptide hormone distribution was used as a basis to generate a high-resolution cellular map of the diffuse endocrine system. Our studies show that cell diversity is seen at two distinct levels: regional and local. We find that class I and class II enteroendocrine cells can be distinguished locally by combinatorial expression of secreted neuropeptide hormones. Cell lineage tracing studies demonstrate that class I and class II cells arise from a common stem cell lineage and that peptide profiles are a stable feature of enteroendocrine cell identity during homeostasis and following challenge with the enteric pathogen Pseudomonas entomophila. Genetic analysis shows that Notch signaling controls the establishment of class II cells in the lineage, but is insufficient to reprogram extant class I cells into class II enteroendocrine cells. Thus, one mechanism by which secretory cell diversity is achieved in the diffuse endocrine system is through cell-cell signaling interactions within individual adult stem cell lineages. PMID:25670792

Regulation and function of mTOR signalling in T cell fate decision

PubMed Central

Chi, Hongbo

2012-01-01

The evolutionary conserved kinase mTOR couples cell growth and metabolism to environmental inputs in eukaryotes. T cells depend on mTOR signalling to integrate immune signals and metabolic cues for their proper maintenance and activation. Under steady-state conditions, mTOR is actively controlled by multiple inhibitory mechanisms, and this enforces normal T cell homeostasis. Antigen recognition by naïve CD4+ and CD8+ T cells triggers mTOR activation, which in turn programs their differentiation into functionally distinct lineages. This Review focuses on the signalling mechanisms of mTOR in T cell homeostatic and functional fates and therapeutic implications of targeting mTOR in T cells. PMID:22517423

Bioprinting for stem cell research

PubMed Central

Tasoglu, Savas; Demirci, Utkan

2012-01-01

Recently, there has been a growing interest to apply bioprinting techniques to stem cell research. Several bioprinting methods have been developed utilizing acoustics, piezoelectricity, and lasers to deposit living cells onto receiving substrates. Using these technologies, spatially defined gradients of immobilized proteins can be engineered to direct stem cell differentiation into multiple subpopulations of different lineages. Stem cells can also be patterned in a high-throughput manner onto flexible implementation patches for tissue regeneration or onto substrates with the goal of accessing encapsulated stem cell of interest for genomic analysis. Here, we review recent achievements with bioprinting technologies in stem cell research, and identify future challenges and potential applications including tissue engineering and regenerative medicine, wound healing, and genomics. PMID:23260439

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ying, E-mail: ying.chen@hc.msu.edu; Wang, Kai; Chandramouli, Gadisetti V.R.

Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomesmore » a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.« less

Retinoic Acid Is Essential for Th1 Cell Lineage Stability and Prevents Transition to a Th17 Cell Program

PubMed Central

Brown, Chrysothemis C.; Esterhazy, Daria; Sarde, Aurelien; London, Mariya; Pullabhatla, Venu; Osma-Garcia, Ines; al-Bader, Raya; Ortiz, Carla; Elgueta, Raul; Arno, Matthew; de Rinaldis, Emanuele; Mucida, Daniel; Lord, Graham M.; Noelle, Randolph J.

2015-01-01

Summary CD4+ T cells differentiate into phenotypically distinct T helper cells upon antigenic stimulation. Regulation of plasticity between these CD4+ T-cell lineages is critical for immune homeostasis and prevention of autoimmune disease. However, the factors that regulate lineage stability are largely unknown. Here we investigate a role for retinoic acid (RA) in the regulation of lineage stability using T helper 1 (Th1) cells, traditionally considered the most phenotypically stable Th subset. We found that RA, through its receptor RARα, sustains stable expression of Th1 lineage specifying genes, as well as repressing genes that instruct Th17-cell fate. RA signaling is essential for limiting Th1-cell conversion into Th17 effectors and for preventing pathogenic Th17 responses in vivo. Our study identifies RA-RARα as a key component of the regulatory network governing maintenance and plasticity of Th1-cell fate and defines an additional pathway for the development of Th17 cells. PMID:25769610

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.

PubMed

Baxter, Melissa; Withey, Sarah; Harrison, Sean; Segeritz, Charis-Patricia; Zhang, Fang; Atkinson-Dell, Rebecca; Rowe, Cliff; Gerrard, Dave T; Sison-Young, Rowena; Jenkins, Roz; Henry, Joanne; Berry, Andrew A; Mohamet, Lisa; Best, Marie; Fenwick, Stephen W; Malik, Hassan; Kitteringham, Neil R; Goldring, Chris E; Piper Hanley, Karen; Vallier, Ludovic; Hanley, Neil A

2015-03-01

Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Origin of Matrix-Producing Cells That Contribute to Aortic Fibrosis in Hypertension.

PubMed

Wu, Jing; Montaniel, Kim Ramil C; Saleh, Mohamed A; Xiao, Liang; Chen, Wei; Owens, Gary K; Humphrey, Jay D; Majesky, Mark W; Paik, David T; Hatzopoulos, Antonis K; Madhur, Meena S; Harrison, David G

2016-02-01

Various hypertensive stimuli lead to exuberant adventitial collagen deposition in large arteries, exacerbating blood pressure elevation and end-organ damage. Collagen production is generally attributed to resident fibroblasts; however, other cells, including resident and bone marrow-derived stem cell antigen positive (Sca-1(+)) cells and endothelial and vascular smooth muscle cells, can produce collagen and contribute to vascular stiffening. Using flow cytometry and immunofluorescence, we found that adventitial Sca-1(+) progenitor cells begin to produce collagen and acquire a fibroblast-like phenotype in hypertension. We also found that bone marrow-derived cells represent more than half of the matrix-producing cells in hypertension, and that one-third of these are Sca-1(+). Cell sorting and lineage-tracing studies showed that cells of endothelial origin contribute to no more than one fourth of adventitial collagen I(+) cells, whereas those of vascular smooth muscle lineage do not contribute. Our findings indicate that Sca-1(+) progenitor cells and bone marrow-derived infiltrating fibrocytes are major sources of arterial fibrosis in hypertension. Endothelial to mesenchymal transition likely also contributes, albeit to a lesser extent and pre-existing resident fibroblasts represent a minority of aortic collagen-producing cells in hypertension. This study shows that vascular stiffening represents a complex process involving recruitment and transformation of multiple cells types that ultimately elaborate adventitial extracellular matrix. © 2015 American Heart Association, Inc.

Neural stem cells induce the formation of their physical niche during organogenesis

PubMed Central

Riebesehl, Bea F; Ambrosio, Elizabeth M; Stolper, Julian S; Lischik, Colin Q; Dross, Nicolas

2017-01-01

Most organs rely on stem cells to maintain homeostasis during post-embryonic life. Typically, stem cells of independent lineages work coordinately within mature organs to ensure proper ratios of cell types. Little is known, however, on how these different stem cells locate to forming organs during development. Here we show that neuromasts of the posterior lateral line in medaka are composed of two independent life-long lineages with different embryonic origins. Clonal analysis and 4D imaging revealed a hierarchical organisation with instructing and responding roles: an inner, neural lineage induces the formation of an outer, border cell lineage (nBC) from the skin epithelium. Our results demonstrate that the neural lineage is necessary and sufficient to generate nBCs highlighting self-organisation principles at the level of the entire embryo. We hypothesise that induction of surrounding tissues plays a major role during the establishment of vertebrate stem cell niches. PMID:28950935

Constraint of gene expression by the chromatin remodelling protein CHD4 facilitates lineage specification

PubMed Central

O'Shaughnessy-Kirwan, Aoife; Signolet, Jason; Costello, Ita; Gharbi, Sarah; Hendrich, Brian

2015-01-01

Chromatin remodelling proteins are essential for different aspects of metazoan biology, yet functional details of why these proteins are important are lacking. Although it is possible to describe the biochemistry of how they remodel chromatin, their chromatin-binding profiles in cell lines, and gene expression changes upon loss of a given protein, in very few cases can this easily translate into an understanding of how the function of that protein actually influences a developmental process. Here, we investigate how the chromatin remodelling protein CHD4 facilitates the first lineage decision in mammalian embryogenesis. Embryos lacking CHD4 can form a morphologically normal early blastocyst, but are unable to successfully complete the first lineage decision and form functional trophectoderm (TE). In the absence of a functional TE, Chd4 mutant blastocysts do not implant and are hence not viable. By measuring transcript levels in single cells from early embryos, we show that CHD4 influences the frequency at which unspecified cells in preimplantation stage embryos express lineage markers prior to the execution of this first lineage decision. In the absence of CHD4, this frequency is increased in 16-cell embryos, and by the blastocyst stage cells fail to properly adopt a TE gene expression programme. We propose that CHD4 allows cells to undertake lineage commitment in vivo by modulating the frequency with which lineage-specification genes are expressed. This provides novel insight into both how lineage decisions are made in mammalian cells, and how a chromatin remodelling protein functions to facilitate lineage commitment. PMID:26116663

Molecular dissection of prethymic progenitor entry into the T lymphocyte developmental pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fung, Elizabeth-sharon

2008-01-01

Notch signaling activates T lineage differentiation from hemopoietic progenitors, but relatively few regulators that initiate this program have been identified, e.g., GATA3 and T cell factor-I (TCF-1) (gene name Tcli). To identify additional regulators of T cell specification, a cDNA libnlrY from mouse Pro-T cells was screened for genes that are specifically up-regulated in intrathymic T cell precursors as compared with myeloid progenitors. Over 90 genes of interest were identified, and 35 of 44 tested were confirmed to be more highly expressed in T lineage precursors relative to precursors of B and/or myeloid lineage. To a remarkable extent, however, expressionmore » of these T lineage-enriched genes, including zinc finger transcription factor, helicase, and signaling adaptor genes, was also shared by stem cells (Lin{sup -}Sca-1{sup +}Kit{sup +}CD27{sup -}) and multipotent progenitors (Lin{sup -}Sca-l{sup +}Kit{sup +}CD27{sup +}), although down-regulated in other lineages. Thus, a major fraction of these early T lineage genes are a regulatory legacy from stem cells. The few genes sharply up-regulated between multipotent progenitors and Pro-T cell stages included those encoding transcription factors Bclllb, TCF-I (Tcli), and HEBalt, Notch target Deltexl, Deltex3L, Fkbp5, Eval, and Tmem13l. Like GATA3 and Deltexl, Bclllb, Fkbp5, and Eval were dependent on Notch/Delta signaling for induction in fetal liver precursors, but only BcIlI band HEBalt were up-regulated between the first two stages of intrathymic T cell development (double negative I and double negative 2) corresponding to T lineage specification. Bclllb was uniquely T lineage restricted and induced by NotchlDelta signaling specifically upon entry into the T lineage differentiation pathway.« less

Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities.

PubMed

Casey, Alison E; Sinha, Ankit; Singhania, Rajat; Livingstone, Julie; Waterhouse, Paul; Tharmapalan, Pirashaanthy; Cruickshank, Jennifer; Shehata, Mona; Drysdale, Erik; Fang, Hui; Kim, Hyeyeon; Isserlin, Ruth; Bailey, Swneke; Medina, Tiago; Deblois, Genevieve; Shiah, Yu-Jia; Barsyte-Lovejoy, Dalia; Hofer, Stefan; Bader, Gary; Lupien, Mathieu; Arrowsmith, Cheryl; Knapp, Stefan; De Carvalho, Daniel; Berman, Hal; Boutros, Paul C; Kislinger, Thomas; Khokha, Rama

2018-06-19

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology. © 2018 Casey et al.

Microenvironment Determines Lineage Fate in a Human Model of MLL-AF9 Leukemia

PubMed Central

Wei, Junping; Wunderlich, Mark; Fox, Catherine; Alvarez, Sara; Cigudosa, Juan C.; Wilhelm, Jamie S.; Zheng, Yi; Cancelas, Jose A.; Gu, Yi; Jansen, Michael; DiMartino, Jorge F.; Mulloy, James C.

2008-01-01

Summary Faithful modeling of mixed lineage leukemia in murine cells has been difficult to achieve. We show that expression of MLL-AF9 in human CD34+ cells induces acute myeloid, lymphoid or mixed lineage leukemia in immunodeficient mice. Some leukemia stem cells (LSC) were multipotent and could be lineage directed by altering either the growth factors or the recipient strain of mouse, highlighting the importance of microenviromental cues. Other LSC were strictly lineage committed, demonstrating the heterogeneity of the stem cell compartment in MLL disease. Targeting the Rac signaling pathway by pharmacologic or genetic means resulted in rapid and specific apoptosis of MLL-AF9 cells, suggesting that the Rac signaling pathway may be a valid therapeutic target in MLL-rearranged AML. PMID:18538732

Human stem cell neuronal differentiation on silk-carbon nanotube composite

NASA Astrophysics Data System (ADS)

Chen, Chi-Shuo; Soni, Sushant; Le, Catherine; Biasca, Matthew; Farr, Erik; Chen, Eric Y.-T.; Chin, Wei-Chun

2012-02-01

Human embryonic stem cells [hESCs] are able to differentiate into specific lineages corresponding to regulated spatial and temporal signals. This unique attribute holds great promise for regenerative medicine and cell-based therapy for many human diseases such as spinal cord injury [SCI] and multiple sclerosis [MS]. Carbon nanotubes [CNTs] have been successfully used to promote neuronal differentiation, and silk has been widely applied in tissue engineering. This study aims to build silk-CNT composite scaffolds for improved neuron differentiation efficiency from hESCs. Two neuronal markers (β-III tubulin and nestin) were utilized to determine the hESC neuronal lineage differentiation. In addition, axonal lengths were measured to evaluate the progress of neuronal development. The results demonstrated that cells on silk-CNT scaffolds have a higher β-III tubulin and nestin expression, suggesting augmented neuronal differentiation. In addition, longer axons with higher density were found to associate with silk-CNT scaffolds. Our silk-CNT-based composite scaffolds can promote neuronal differentiation of hESCs. The silk-CNT composite scaffolds developed here can serve as efficient supporting matrices for stem cell-derived neuronal transplants, offering a promising opportunity for nerve repair treatments for SCI and MS patients.

FOG-2, a Heart- and Brain-Enriched Cofactor for GATA Transcription Factors

PubMed Central

Lu, Jian-rong; McKinsey, Timothy A.; Xu, Hongtao; Wang, Da-zhi; Richardson, James A.; Olson, Eric N.

1999-01-01

Members of the GATA family of zinc finger transcription factors have been shown to play important roles in the control of gene expression in a variety of cell types. GATA-1, -2, and -3 are expressed primarily in hematopoietic cell lineages and are required for proliferation and differentiation of multiple hematopoietic cell types, whereas GATA-4, -5, and -6 are expressed in the heart, where they activate cardiac muscle structural genes. Friend of GATA-1 (FOG) is a multitype zinc finger protein that interacts with GATA-1 and serves as a cofactor for GATA-1-mediated transcription. FOG is coexpressed with GATA-1 in developing erythroid and megakaryocyte cell lineages and cooperates with GATA-1 to control erythropoiesis. We describe a novel FOG-related factor, FOG-2, that is expressed predominantly in the developing and adult heart, brain, and testis. FOG-2 interacts with GATA factors, and interaction of GATA-4 and FOG-2 results in either synergistic activation or repression of GATA-dependent cardiac promoters, depending on the specific promoter and the cell type in which they are tested. The properties of FOG-2 suggest its involvement in the control of cardiac and neural gene expression by GATA transcription factors. PMID:10330188

Progenitor cell dose determines the pace and completeness of engraftment in a xenograft model for cord blood transplantation

PubMed Central

Liu, Congxiao; Chen, Benny J.; DeOliveira, Divinomar; Sempowski, Gregory D.; Chao, Nelson J.

2010-01-01

Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma–null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34+ human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks), human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses, as documented by the presence of CD4+ CD8+ T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation, human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses, the chimerism was weak and the human hematopoietic lineage development was frequently incomplete. PMID:20833978

Brachyury expression in tailless Molgulid ascidian embryos.

PubMed

Takada, Norio; York, Jonathan; Davis, J Muse; Schumpert, Brenda; Yasuo, Hitoyoshi; Satoh, Nori; Swalla, Billie J

2002-01-01

The T-box transcription factor gene Brachyury is important for the differentiation of notochord in all chordates, including the ascidians Halocynthia roretzi and Ciona intestinalis. We isolated Brachyury from molgulid ascidians, which have evolved tailless larvae multiple times independently, and found the genes appear functional by cDNA sequence analyses. We then compared the expression of Mocu-Bra in tailed Molgula oculata embryos to two tailless species, Molgula occulta (Mocc-Bra) and Molgula tectiformis (Mt-Bra). Here we show that both tailless species express Brachyury in the notochord lineage during embryogenesis. Initial expression of Mocu-Bra is normal in tailed M. oculata embryos; 10 precursor notochord cells divide twice to result in 40 notochord cells that converge and extend to make a notochord down the center of the tail. In contrast, in tailless Molgula occulta, Mocc-Bra expression disappears prematurely, and there is only one round of division, resulting in 20 cells in the final notochord lineage that never converge or extend. In M. occulta x M. oculata hybrid embryos, expression of Mocu-Bra is prolonged, and the embryos form a tail with 20 notochord cells that converge and extend normally. However, in Molgula tectiformis, a different tailless ascidian, Mt-Bra was expressed only in the 10 notochord precursor cells, which never divide, converge, or extend. In summary, neither Brachyury function nor the early establishment of the notochord lineage appears to be impaired in tailless embryos. In light of these results, we are continuing to investigate how and why notochord development is lost in tailless molgulid ascidian embryos.

Mesenchymal stem cells as a vector for the inflammatory prostate microenvironment

PubMed Central

Brennen, W Nathaniel; Denmeade, Samuel R; Isaacs, John T

2014-01-01

Mesenchymal stem cells (MSCs) have an inherent tropism for sites of inflammation, which are frequently present in sites of cancer, including prostatic lesions. MSCs have been defined as CD73/CD90/CD105 triple-positive cells in the absence of hematopoietic lineage markers with the ability to differentiate into multiple mesodermal lineages, including osteoblasts, adipocytes, and chondrocytes. Our group has previously demonstrated that MSCs represent between 0.01 and 1.1% of the total cells present in human prostatectomy tissue. In addition to their multi-lineage differentiation potential, MSCs are immunoprivileged in nature and have a range of immunomodulatory effects on both the innate and adaptive arms of the immune system. MSCs have been detected in an increasing array of tissues, and evidence suggests that they are likely present in perivascular niches throughout the body. These observations suggest that MSCs represent critical mediators of the overall immune response during physiological homeostasis and likely contribute to pathophysiological conditions as well. Chronic inflammation has been suggested as an initiating event and progression factor in prostate carcinogenesis, a process in which the immunosuppressive properties of MSCs may play a role. MSCs have also been shown to influence malignant progression through a variety of other mechanisms, including effects on tumor proliferation, angiogenesis, survival, and metastasis. Additionally, human bone marrow-derived MSCs have been shown to traffic to human prostate cancer xenografts in immunocompromised murine hosts. The trafficking properties and immunoprivileged status of MSCs suggest that they can be exploited as an allogeneic cell-based vector to deliver cytotoxic or diagnostic agents for therapy. PMID:23975882

Progenitors of Secondary Crest Myofibroblasts are Developmentally Committed in Early Lung Mesoderm

PubMed Central

Li, Changgong; Li, Min; Li, Sha; Xing, Yiming; Yang, Chang-Yo; Li, Aimin; Borok, Zea; De Langhe, Stijn; Minoo, Parviz

2015-01-01

Development of the mammalian lung is predicated on cross-communications between two highly interactive tissues, the endodermally-derived epithelium and the mesodermally-derived pulmonary mesenchyme. While much attention has been paid the lung epithelium, the pulmonary mesenchyme, partly due to lack of specific tractable markers remains under-investigated. The lung mesenchyme is derived from the lateral plate mesoderm and is the principal recipient of Hedgehog (Hh) signaling, a morphogenetic network that regulates multiple aspects of embryonic development. Using the Hh-responsive Gli1-creERT2 mouse line, we identified the mesodermal targets of Hh signaling at various time points during embryonic and postnatal lung development. Cell lineage analysis showed these cells serve as progenitors to contribute to multiple lineages of mesodermally-derived differentiated cell types that include parenchymal or interstitial myofibroblasts, parabronchial and perivascular smooth muscle as well as rare populations of cells within the mesothelium. Most importantly, Gli1-creERT2 identified the progenitors of secondary crest myofibroblasts, a hitherto intractable cell type that plays a key role in alveolar formation, a vital process about which little is currently known. Transcriptome analysis of Hh-targeted progenitor cells transitioning from the pseudoglandular to the saccular phase of lung development revealed important modulations of key signaling pathways. Amongst these, there was significant down-regulation of canonical WNT signaling. Ectopic stabilization of β-Catenin via inactivation of Apc by Gli1-creERT2 expanded the Hh-targeted progenitor pools, which caused the formation of fibroblastic masses within the lung parenchyma. The Gli1-creERT2 mouse line represents a novel tool in the analysis of mesenchymal cell biology and alveolar formation during lung development. PMID:25448080

Epigenetic modulation by TFII-I during embryonic stem cell differentiation.

PubMed

Bayarsaihan, Dashzeveg; Makeyev, Aleksandr V; Enkhmandakh, Badam

2012-10-01

TFII-I transcription factors play an essential role during early vertebrate embryogenesis. Genome-wide mapping studies by ChIP-seq and ChIP-chip revealed that TFII-I primes multiple genomic loci in mouse embryonic stem cells and embryonic tissues. Moreover, many TFII-I-bound regions co-localize with H3K4me3/K27me3 bivalent chromatin within the promoters of lineage-specific genes. This minireview provides a summary of current knowledge regarding the function of TFII-I in epigenetic control of stem cell differentiation. Copyright © 2012 Wiley Periodicals, Inc.

Pan-cancer genome and transcriptome analyses of 1,699 paediatric leukaemias and solid tumours | Office of Cancer Genomics

Cancer.gov

Analysis of molecular aberrations across multiple cancer types, known as pan-cancer analysis, identifies commonalities and differences in key biological processes that are dysregulated in cancer cells from diverse lineages. Pan-cancer analyses have been performed for adult1–4 but not paediatric cancers, which commonly occur in developing mesodermic rather than adult epithelial tissues5.

The impact of Pleistocene climate change on an ancient arctic–alpine plant: multiple lineages of disparate history in Oxyria digyna

PubMed Central

Allen, Geraldine A; Marr, Kendrick L; McCormick, Laurie J; Hebda, Richard J

2012-01-01

The ranges of arctic–alpine species have shifted extensively with Pleistocene climate changes and glaciations. Using sequence data from the trnH-psbA and trnT-trnL chloroplast DNA spacer regions, we investigated the phylogeography of the widespread, ancient (>3 million years) arctic–alpine plant Oxyria digyna (Polygonaceae). We identified 45 haplotypes and six highly divergent major lineages; estimated ages of these lineages (time to most recent common ancestor, TMRCA) ranged from ∼0.5 to 2.5 million years. One lineage is widespread in the arctic, a second is restricted to the southern Rocky Mountains of the western United States, and a third was found only in the Himalayan and Altai regions of Asia. Three other lineages are widespread in western North America, where they overlap extensively. The high genetic diversity and the presence of divergent major cpDNA lineages within Oxyria digyna reflect its age and suggest that it was widespread during much of its history. The distributions of individual lineages indicate repeated spread of Oxyria digyna through North America over multiple glacial cycles. During the Last Glacial Maximum it persisted in multiple refugia in western North America, including Beringia, south of the continental ice, and within the northern limits of the Cordilleran ice sheet. Our data contribute to a growing body of evidence that arctic–alpine species have migrated from different source regions over multiple glacial cycles and that cryptic refugia contributed to persistence through the Last Glacial Maximum. PMID:22822441

Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes

PubMed Central

Liu, Qiaozhen; Yang, Rui; Huang, Xiuzhen; Zhang, Hui; He, Lingjuan; Zhang, Libo; Tian, Xueying; Nie, Yu; Hu, Shengshou; Yan, Yan; Zhang, Li; Qiao, Zengyong; Wang, Qing-Dong; Lui, Kathy O; Zhou, Bin

2016-01-01

Cardiac cells marked by c-Kit or Kit, dubbed cardiac stem cells (CSCs), are in clinical trials to investigate their ability to stimulate cardiac regeneration and repair. These studies were initially motivated by the purported cardiogenic activity of these cells. Recent lineage tracing studies using Kit promoter to drive expression of the inducible Cre recombinase showed that these CSCs had highly limited cardiogenic activity, inadequate to support efficient cardiac repair. Here we reassess the lineage tracing data by investigating the identity of cells immediately after Cre labeling. Our instant lineage tracing approach identifies Kit-expressing cardiomyocytes, which are labeled immediately after tamoxifen induction. In combination with long-term lineage tracing experiments, these data reveal that the large majority of long-term labeled cardiomyocytes are pre-existing Kit-expressing cardiomyocytes rather than cardiomyocytes formed de novo from CSCs. This study presents a new interpretation for the contribution of Kit+ cells to cardiomyocytes and shows that Kit genetic lineage tracing over-estimates the cardiogenic activity of Kit+ CSCs. PMID:26634606

Genetically distant American Canine distemper virus lineages have recently caused epizootics with somewhat different characteristics in raccoons living around a large suburban zoo in the USA

PubMed Central

Lednicky, John A; Dubach, Jean; Kinsel, Michael J; Meehan, Thomas P; Bocchetta, Maurizio; Hungerford, Laura L; Sarich, Nicolene A; Witecki, Kelley E; Braid, Michael D; Pedrak, Casandra; Houde, Christiane M

2004-01-01

Background Mortality rates have differed during distemper outbreaks among free-ranging raccoons (Procyon lotor) living around a large Chicago-area zoo, and appeared higher in year 2001 than in 1998 and 2000. We hypothesized that a more lethal variant of the local Canine distemper virus (CDV) lineage had emerged in 2001, and sought the genetic basis that led to increased virulence. However, a more complex model surfaced during preliminary analyses of CDV genomic sequences in infected tissues and of virus isolated in vitro from the raccoons. Results Phylogenetic analyses of subgenomic CDV fusion (F) -, phosphoprotein (P) -, and complete hemagglutinin (H) – gene sequences indicated that distinct American CDV lineages caused the distemper epizootics. The 1998 outbreak was caused by viruses that are likely from an old CDV lineage that includes CDV Snyder Hill and Lederle, which are CDV strains from the early 1950's. The 2000 and 2001 viruses appear to stem from the lineage of CDV A75/17, which was isolated in the mid 1970's. Only the 2001 viruses formed large syncytia in brain and/or lung tissue, and during primary isolation in-vitro in Vero cells, demonstrating at least one phenotypic property by which they differed from the other viruses. Conclusions Two different American CDV lineages caused the raccoon distemper outbreaks. The 1998 viruses are genetically distant to the 2000/2001 viruses. Since CDV does not cause persistent infections, the cycling of different CDV lineages within the same locale suggests multiple reintroductions of the virus to area raccoons. Our findings establish a precedent for determining whether the perceived differences in mortality rates are actual and attributable in part to inherent differences between CDV strains arising from different CDV lineages. PMID:15507154

Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells.

PubMed

Trylinski, Mateusz; Mazouni, Khalil; Schweisguth, François

2017-08-07

Notch receptors regulate cell fate decisions during embryogenesis and throughout adult life. In many cell lineages, binary fate decisions are mediated by directional Notch signaling between the two sister cells produced by cell division. How Notch signaling is restricted to sister cells after division to regulate intra-lineage decision is poorly understood. More generally, where ligand-dependent activation of Notch occurs at the cell surface is not known, as methods to detect receptor activation in vivo are lacking. In Drosophila pupae, Notch signals during cytokinesis to regulate the intra-lineage pIIa/pIIb decision in the sensory organ lineage. Here, we identify two pools of Notch along the pIIa-pIIb interface, apical and basal to the midbody. Analysis of the dynamics of Notch, Delta, and Neuralized distribution in living pupae suggests that ligand endocytosis and receptor activation occur basal to the midbody. Using selective photo-bleaching of GFP-tagged Notch and photo-tracking of photo-convertible Notch, we show that nuclear Notch is indeed produced by receptors located basal to the midbody. Thus, only a specific subset of receptors, located basal to the midbody, contributes to signaling in pIIa. This is the first in vivo characterization of the pool of Notch contributing to signaling. We propose a simple mechanism of cell fate decision based on intra-lineage signaling: ligands and receptors localize during cytokinesis to the new cell-cell interface, thereby ensuring signaling between sister cells, hence intra-lineage fate decision. Copyright © 2017 Elsevier Ltd. All rights reserved.

Joint morphogenetic cells in the adult mammalian synovium

PubMed Central

Roelofs, Anke J.; Zupan, Janja; Riemen, Anna H. K.; Kania, Karolina; Ansboro, Sharon; White, Nathan; Clark, Susan M.; De Bari, Cosimo

2017-01-01

The stem cells that safeguard synovial joints in adulthood are undefined. Studies on mesenchymal stromal/stem cells (MSCs) have mainly focused on bone marrow. Here we show that lineage tracing of Gdf5-expressing joint interzone cells identifies in adult mouse synovium an MSC population largely negative for the skeletal stem cell markers Nestin-GFP, Leptin receptor and Gremlin1. Following cartilage injury, Gdf5-lineage cells underpin synovial hyperplasia through proliferation, are recruited to a Nestin-GFPhigh perivascular population, and contribute to cartilage repair. The transcriptional co-factor Yap is upregulated after injury, and its conditional ablation in Gdf5-lineage cells prevents synovial lining hyperplasia and decreases contribution of Gdf5-lineage cells to cartilage repair. Cultured Gdf5-lineage cells exhibit progenitor activity for stable chondrocytes and are able to self-organize three-dimensionally to form a synovial lining-like layer. Finally, human synovial MSCs transduced with Bmp7 display morphogenetic properties by patterning a joint-like organ in vivo. Our findings further the understanding of the skeletal stem/progenitor cells in adult life. PMID:28508891

Spatially Correlated Gene Expression in Bacterial Groups: The Role of Lineage History, Spatial Gradients, and Cell-Cell Interactions.

PubMed

van Vliet, Simon; Dal Co, Alma; Winkler, Annina R; Spriewald, Stefanie; Stecher, Bärbel; Ackermann, Martin

2018-04-25

Gene expression levels in clonal bacterial groups have been found to be spatially correlated. These correlations can partly be explained by the shared lineage history of nearby cells, although they could also arise from local cell-cell interactions. Here, we present a quantitative framework that allows us to disentangle the contributions of lineage history, long-range spatial gradients, and local cell-cell interactions to spatial correlations in gene expression. We study pathways involved in toxin production, SOS stress response, and metabolism in Escherichia coli microcolonies and find for all pathways that shared lineage history is the main cause of spatial correlations in gene expression levels. However, long-range spatial gradients and local cell-cell interactions also contributed to spatial correlations in SOS response, amino acid biosynthesis, and overall metabolic activity. Together, our data show that the phenotype of a cell is influenced by its lineage history and population context, raising the question of whether bacteria can arrange their activities in space to perform functions they cannot achieve alone. Copyright © 2018 Elsevier Inc. All rights reserved.

Cell lineage and cell cycling analyses of the 4d micromere using live imaging in the marine annelid Platynereis dumerilii

PubMed Central

Handberg-Thorsager, Mette; Vervoort, Michel

2017-01-01

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos. PMID:29231816

SONAR: A High-Throughput Pipeline for Inferring Antibody Ontogenies from Longitudinal Sequencing of B Cell Transcripts

PubMed Central

Schramm, Chaim A.; Sheng, Zizhang; Zhang, Zhenhai; Mascola, John R.; Kwong, Peter D.; Shapiro, Lawrence

2016-01-01

The rapid advance of massively parallel or next-generation sequencing technologies has made possible the characterization of B cell receptor repertoires in ever greater detail, and these developments have triggered a proliferation of software tools for processing and annotating these data. Of especial interest, however, is the capability to track the development of specific antibody lineages across time, which remains beyond the scope of most current programs. We have previously reported on the use of techniques such as inter- and intradonor analysis and CDR3 tracing to identify transcripts related to an antibody of interest. Here, we present Software for the Ontogenic aNalysis of Antibody Repertoires (SONAR), capable of automating both general repertoire analysis and specialized techniques for investigating specific lineages. SONAR annotates next-generation sequencing data, identifies transcripts in a lineage of interest, and tracks lineage development across multiple time points. SONAR also generates figures, such as identity–divergence plots and longitudinal phylogenetic “birthday” trees, and provides interfaces to other programs such as DNAML and BEAST. SONAR can be downloaded as a ready-to-run Docker image or manually installed on a local machine. In the latter case, it can also be configured to take advantage of a high-performance computing cluster for the most computationally intensive steps, if available. In summary, this software provides a useful new tool for the processing of large next-generation sequencing datasets and the ontogenic analysis of neutralizing antibody lineages. SONAR can be found at https://github.com/scharch/SONAR, and the Docker image can be obtained from https://hub.docker.com/r/scharch/sonar/. PMID:27708645

A heterogeneous lineage origin underlies the phenotypic and molecular differences of white and beige adipocytes

PubMed Central

Liu, Weiyi; Shan, Tizhong; Yang, Xin; Liang, Sandra; Zhang, Pengpeng; Liu, Yaqin; Liu, Xiaoqi; Kuang, Shihuan

2013-01-01

Summary A worldwide epidemic of obesity and its associated metabolic disorders raise the significance of adipocytes, their origins and characteristics. Our previous study has demonstrated that interscapular brown adipose tissue (BAT), but not intramuscular adipose, is derived from the Pax3-expressing cell lineage. Here, we show that various depots of subcutaneous (SAT) and visceral adipose tissue (VAT) are highly heterogeneous in the Pax3 lineage origin. Interestingly, the relative abundance of Pax3 lineage cells in SAT depots is inversely correlated to expression of BAT signature genes including Prdm16, Pgc1a (Ppargc1a) and Ucp1. FACS analysis further demonstrates that adipocytes differentiated from non-Pax3 lineage preadipocytes express higher levels of BAT and beige adipocyte signature genes compared with the Pax3 lineage adipocytes within the same depots. Although both Pax3 and non-Pax3 lineage preadipocytes can give rise to beige adipocytes, the latter contributes more significantly. Consistently, genetic ablation of Pax3 lineage cells in SAT leads to increased expression of beige cell markers. Finally, non-Pax3 lineage beige adipocytes are more responsive to cAMP-agonist-induced Ucp1 expression. Taken together, these results demonstrate widespread heterogeneity in Pax3 lineage origin, and its inverse association with BAT gene expression within and among subcutaneous adipose depots. PMID:23781029

EED and KDM6B Coordinate the First Mammalian Cell Lineage Commitment To Ensure Embryo Implantation

PubMed Central

Saha, Biswarup; Home, Pratik; Ray, Soma; Larson, Melissa; Paul, Arindam; Rajendran, Ganeshkumar; Behr, Barry

2013-01-01

The first mammalian cell lineage commitment is the formation of the trophectoderm (TE) and the inner cell mass (ICM) lineages during preimplantation development. Proper development of the TE and ICM lineages is dependent upon establishment of specific transcriptional programs. However, the epigenetic mechanisms that functionally contribute to establish TE- and ICM-specific transcriptional programs are poorly understood. Here, we show that proper development of the TE and ICM lineages is coordinated via combinatorial regulation of embryonic ectoderm development (EED) and lysine-specific demethylase 6B (KDM6B). During blastocyst formation, the relative levels of EED and KDM6B expression determine altered polycomb repressor 2 (PRC2) complex recruitment and incorporation of the repressive histone H3 lysine 27 trimethylation (H3K27Me3) mark at the chromatin domains of TE-specific master regulators CDX2 and GATA3, leading to their activation in the TE lineage and repression in the ICM lineage. Furthermore, ectopic gain of EED along with depletion of KDM6B in preimplantation mouse embryos abrogates CDX2 and GATA3 expression in the nascent TE lineage. The loss of CDX2 and GATA3 in the nascent TE lineage results in improper TE development, leading to failure in embryo implantation to the uterus. Our study delineates a novel epigenetic mechanism that orchestrates proper development of the first mammalian cell lineages. PMID:23671187

Epstein-Barr virus-positive nodal T/NK-cell lymphoma: an analysis of 15 cases with distinct clinicopathological features.

PubMed

Jeon, Yoon Kyung; Kim, Jo-Heon; Sung, Ji-Youn; Han, Jae Ho; Ko, Young-Hyeh

2015-07-01

Nodal peripheral T-cell lymphoma, not otherwise specified, is a heterogeneous entity with variable biologic behavior. We analyze the clinicopathological features of 15 patients with Epstein-Barr virus-positive (EBV+) nodal T/NK-cell lymphoma, including 9 males and 6 females, with a median age of 64 years. All patients presented with multiple lymphadenopathy with common B symptoms (80%, 12/15) at an advanced Ann Arbor stage (III, IV) (87%, 13/15). The International Prognostic Index was high or high/intermediate in 87% (13/15) of patients, and the prognostic index for peripheral T-cell lymphoma was group 3 or 4 in 73% (11/15). Spleen and liver involvement was observed in 73% (11/15) and 60% (9/15) of patients, respectively. In contrast, extranodal involvement was infrequent, with no more than 1 site in 71% (10/15) of patients. Moreover, none had nasal lesions, and only 1 had mucocutaneous involvement. The cell lineage of EBV+ tumor cells was determined to be T cell in all except 1 patient, who was NK-cell lineage. Cytotoxic molecules were expressed in all cases, and 64% (9/14) of patients expressed the αβT-cell receptor. Moreover, most patients (67%, 10/15) showed CD8 positivity, with 2 of them being CD4CD8 double positive; the others were CD4 positive (n = 2) or CD4CD8 double negative (n = 3). The clinical course was very aggressive, with a median survival time of 3.5 months, and 10 patients died within 6 months of diagnosis. Taken together, our data demonstrate that EBV+ nodal T/NK-cell lymphoma is a distinct clinicopathological entity characterized by cytotoxic molecule expression, a frequent CD8-positive αβT-cell lineage, and a very aggressive clinical behavior. Copyright © 2015 Elsevier Inc. All rights reserved.

Developmental heterogeneity of cardiac fibroblasts does not predict pathological proliferation and activation.

PubMed

Ali, Shah R; Ranjbarvaziri, Sara; Talkhabi, Mahmood; Zhao, Peng; Subat, Ali; Hojjat, Armin; Kamran, Paniz; Müller, Antonia M S; Volz, Katharina S; Tang, Zhaoyi; Red-Horse, Kristy; Ardehali, Reza

2014-09-12

Fibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown. We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury. We showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles. The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response. © 2014 American Heart Association, Inc.

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells.

PubMed

Yu, Qing Cissy; Song, Wenqian; Lai, Dengwen; Zeng, Yi Arial

2017-08-03

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr + VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

Lineage-specific effects of Notch/Numb signaling in post-embryonic development of the Drosophila brain.

PubMed

Lin, Suewei; Lai, Sen-Lin; Yu, Huang-Hsiang; Chihara, Takahiro; Luo, Liqun; Lee, Tzumin

2010-01-01

Numb can antagonize Notch signaling to diversify the fates of sister cells. We report here that paired sister cells acquire different fates in all three Drosophila neuronal lineages that make diverse types of antennal lobe projection neurons (PNs). Only one in each pair of postmitotic neurons survives into the adult stage in both anterodorsal (ad) and ventral (v) PN lineages. Notably, Notch signaling specifies the PN fate in the vPN lineage but promotes programmed cell death in the missing siblings in the adPN lineage. In addition, Notch/Numb-mediated binary sibling fates underlie the production of PNs and local interneurons from common precursors in the lAL lineage. Furthermore, Numb is needed in the lateral but not adPN or vPN lineages to prevent the appearance of ectopic neuroblasts and to ensure proper self-renewal of neural progenitors. These lineage-specific outputs of Notch/Numb signaling show that a universal mechanism of binary fate decision can be utilized to govern diverse neural sibling differentiations.

The Diversity of Nanos Expression in Echinoderm Embryos Supports Different Mechanisms in Germ Cell Specification

PubMed Central

Fresques, Tara; Swartz, S. Zachary; Juliano, Celina; Morino, Yoshiaki; Kikuchi, Mani; Akasaka, Koji; Wada, Hiroshi; Yajima, Mamiko; Wessel, Gary M.

2016-01-01

Specification of the germ cell lineage is required for sexual reproduction in all animals. However, the timing and mechanisms of germ cell specification is remarkably diverse in animal development. Echinoderms, such as sea urchins and sea stars, are excellent model systems to study the molecular and cellular mechanisms that contribute to germ cell specification. In several echinoderm embryos tested, the germ cell factor Vasa accumulates broadly during early development and is restricted after gastrulation to cells that contribute to the germ cell lineage. In the sea urchin, however, the germ cell factor Vasa is restricted to a specific lineage by the 32-cell stage. We therefore hypothesized that the germ cell specification program in the sea urchin/Euechinoid lineage has evolved to an earlier developmental time point. To test this hypothesis we determined the expression pattern of a second germ cell factor, Nanos, in four out of five extant echinoderm clades. Here we find that Nanos mRNA does not accumulate until the blastula stage or later during the development of all other echinoderm embryos except those that belong to the Echinoid lineage. Instead, Nanos is expressed in a restricted domain at the 32–128 cell stage in Echinoid embryos. Our results support the model that the germ cell specification program underwent a heterochronic shift in the Echinoid lineage. A comparison of Echinoid and non-Echinoid germ cell specification mechanisms will contribute to our understanding of how these mechanisms have changed during animal evolution. PMID:27402572

Braveheart, a long non-coding RNA required for cardiovascular lineage commitment

PubMed Central

Klattenhoff, Carla; Scheuermann, Johanna C.; Surface, Lauren E.; Bradley, Robert K.; Fields, Paul A.; Steinhauser, Matthew L.; Ding, Huiming; Butty, Vincent L.; Torrey, Lillian; Haas, Simon; Abo, Ryan; Tabebordbar, Mohammadsharif; Lee, Richard T.; Burge, Christopher B.; Boyer, Laurie A.

2013-01-01

Summary Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm towards a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of MesP1 (mesoderm posterior 1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of Polycomb Repressive Complex 2 (PRC2), during cardiomyocyte differentiation suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development. PMID:23352431

Repression by PRDM13 is critical for generating precision in neuronal identity

PubMed Central

Kollipara, Rahul K; Ma, Zhenzhong; Borromeo, Mark D; Chang, Joshua C

2017-01-01

The mechanisms that activate some genes while silencing others are critical to ensure precision in lineage specification as multipotent progenitors become restricted in cell fate. During neurodevelopment, these mechanisms are required to generate the diversity of neuronal subtypes found in the nervous system. Here we report interactions between basic helix-loop-helix (bHLH) transcriptional activators and the transcriptional repressor PRDM13 that are critical for specifying dorsal spinal cord neurons. PRDM13 inhibits gene expression programs for excitatory neuronal lineages in the dorsal neural tube. Strikingly, PRDM13 also ensures a battery of ventral neural tube specification genes such as Olig1, Olig2 and Prdm12 are excluded dorsally. PRDM13 does this via recruitment to chromatin by multiple neural bHLH factors to restrict gene expression in specific neuronal lineages. Together these findings highlight the function of PRDM13 in repressing the activity of bHLH transcriptional activators that together are required to achieve precise neuronal specification during mouse development. PMID:28850031

Discovering sparse transcription factor codes for cell states and state transitions during development

PubMed Central

Furchtgott, Leon A; Melton, Samuel; Menon, Vilas; Ramanathan, Sharad

2017-01-01

Computational analysis of gene expression to determine both the sequence of lineage choices made by multipotent cells and to identify the genes influencing these decisions is challenging. Here we discover a pattern in the expression levels of a sparse subset of genes among cell types in B- and T-cell developmental lineages that correlates with developmental topologies. We develop a statistical framework using this pattern to simultaneously infer lineage transitions and the genes that determine these relationships. We use this technique to reconstruct the early hematopoietic and intestinal developmental trees. We extend this framework to analyze single-cell RNA-seq data from early human cortical development, inferring a neocortical-hindbrain split in early progenitor cells and the key genes that could control this lineage decision. Our work allows us to simultaneously infer both the identity and lineage of cell types as well as a small set of key genes whose expression patterns reflect these relationships. DOI: http://dx.doi.org/10.7554/eLife.20488.001 PMID:28296636

Clonal tracing of Sox9+ liver progenitors in oval cell injury

PubMed Central

Tarlow, Branden D.; Finegold, Milton J.; Grompe, Markus

2014-01-01

Proliferating ducts, termed “oval cells”, have long thought to be bipotential, i.e. produce both biliary ducts and hepatocytes during chronic liver injury. The precursor to oval cells is considered to be a facultative liver stem cell (LSC). Recent lineage tracing experiments indicated that the LSC is Sox9+ and can replace the bulk of hepatocyte mass in several settings. However, no clonal relationship between Sox9+ cells and the two epithelial liver lineages was established. We labeled Sox9+ mouse liver cells at low density with a multicolor fluorescent confetti reporter. Organoid formation validated the progenitor activity of the labeled population. Sox9+ cells were traced in multiple oval cell injury models using both histology and FACS. Surprisingly, only rare clones containing both hepatocytes and oval cells were found in any experiment. Quantitative analysis showed that Sox9+ cells contributed only minimally (<1%) to the hepatocyte pool, even in classic oval cell injury models. In contrast, clonally marked mature hepatocytes demonstrated the ability to self-renew in all classic mouse oval cell activation injuries. A hepatocyte chimera model to trace hepatocytes and non-parenchymal cells also demonstrated the prevalence of hepatocyte-driven regeneration in mouse oval cell injury models. Conclusion Sox9+ ductal progenitor cells give rise to clonal oval cell proliferation and bipotential organoids but rarely produce hepatocytes in vivo. Hepatocytes themselves are the predominant source of new parenchyma cells in prototypical mouse models of oval cell activation. PMID:24700457

IL-4/IL-13 Signaling Inhibits the Potential of Early Thymic Progenitors To Commit to the T Cell Lineage.

PubMed

Barik, Subhasis; Miller, Mindy M; Cattin-Roy, Alexis N; Ukah, Tobechukwu K; Chen, Weirong; Zaghouani, Habib

2017-10-15

Early thymic progenitors (ETPs) are endowed with diverse potencies and can give rise to myeloid and lymphoid lineage progenitors. How the thymic environment guides ETP commitment and maturation toward a specific lineage remains obscure. We have previously shown that ETPs expressing the heteroreceptor (HR) comprising IL-4Rα and IL-13Rα1 give rise to myeloid cells but not T cells. In this article, we show that signaling through the HR inhibits ETP maturation to the T cell lineage but enacts commitment toward the myeloid cells. Indeed, HR + ETPs, but not HR - ETPs, exhibit activated STAT6 transcription factor, which parallels with downregulation of Notch1, a critical factor for T cell development. Meanwhile, the myeloid-specific transcription factor C/EBPα, usually under the control of Notch1, is upregulated. Furthermore, in vivo inhibition of STAT6 phosphorylation restores Notch1 expression in HR + ETPs, which regain T lineage potential. In addition, upon stimulation with IL-4 or IL-13, HR - ETPs expressing virally transduced HR also exhibit STAT6 phosphorylation and downregulation of Notch1, leading to inhibition of lymphoid, but not myeloid, lineage potential. These observations indicate that environmental cytokines play a role in conditioning ETP lineage choice, which would impact T cell development. Copyright © 2017 by The American Association of Immunologists, Inc.

Evolutionary origins of germline segregation in Metazoa: evidence for a germ stem cell lineage in the coral Orbicella faveolata (Cnidaria, Anthozoa).

PubMed

Barfield, Sarah; Aglyamova, Galina V; Matz, Mikhail V

2016-01-13

The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions. © 2016 The Author(s).

Evolutionary origins of germline segregation in Metazoa: evidence for a germ stem cell lineage in the coral Orbicella faveolata (Cnidaria, Anthozoa)

PubMed Central

Barfield, Sarah; Aglyamova, Galina V.; Matz, Mikhail V.

2016-01-01

The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions. PMID:26763699

Live cell imaging reveals marked variability in myoblast proliferation and fate

PubMed Central

2013-01-01

Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

The Innate Lymphoid Cell Precursor.

PubMed

Ishizuka, Isabel E; Constantinides, Michael G; Gudjonson, Herman; Bendelac, Albert

2016-05-20

The discovery of tissue-resident innate lymphoid cell populations effecting different forms of type 1, 2, and 3 immunity; tissue repair; and immune regulation has transformed our understanding of mucosal immunity and allergy. The emerging complexity of these populations along with compounding issues of redundancy and plasticity raise intriguing questions about their precise lineage relationship. Here we review advances in mapping the emergence of these lineages from early lymphoid precursors. We discuss the identification of a common innate lymphoid cell precursor characterized by transient expression of the transcription factor PLZF, and the lineage relationships of innate lymphoid cells with conventional natural killer cells and lymphoid tissue inducer cells. We also review the rapidly growing understanding of the network of transcription factors that direct the development of these lineages.

Unique Features of Fish Immune Repertoires: Particularities of Adaptive Immunity Within the Largest Group of Vertebrates

PubMed Central

Sunyer, Oriol J.

2016-01-01

Fishes (i.e., teleost fishes) are the largest group of vertebrates. Although their immune system is based on the fundamental receptors, pathways, and cell types found in all groups of vertebrates, fishes show a diversity of particular features that challenge some classical concepts of immunology. In this chapter, we discuss the particularities of fish immune repertoires from a comparative perspective. We examine how allelic exclusion can be achieved when multiple Ig loci are present, how isotypic diversity and functional specificity impact clonal complexity, how loss of the MHC class II molecules affects the cooperation between T and B cells, and how deep sequencing technologies bring new insights about somatic hypermutation in the absence of germinal centers. The unique coexistence of two distinct B-cell lineages respectively specialized in systemic and mucosal responses is also discussed. Finally, we try to show that the diverse adaptations of immune repertoires in teleosts can help in understanding how somatic adaptive mechanisms of immunity evolved in parallel in different lineages across vertebrates. PMID:26537384

Stochasticity and stereotypy in the Ciona notochord.

PubMed

Carlson, Maia; Reeves, Wendy; Veeman, Michael

2015-01-15

Fate mapping with single cell resolution has typically been confined to embryos with completely stereotyped development. The lineages giving rise to the 40 cells of the Ciona notochord are invariant, but the intercalation of those cells into a single-file column is not. Here we use genetic labeling methods to fate map the Ciona notochord with both high resolution and large sample sizes. We find that the ordering of notochord cells into a single column is not random, but instead shows a distinctive signature characteristic of mediolaterally-biased intercalation. We find that patterns of cell intercalation in the notochord are somewhat stochastic but far more stereotyped than previously believed. Cell behaviors vary by lineage, with the secondary notochord lineage being much more constrained than the primary lineage. Within the primary lineage, patterns of intercalation reflect the geometry of the intercalating tissue. We identify the latest point at which notochord morphogenesis is largely stereotyped, which is shortly before the onset of mediolateral intercalation and immediately after the final cell divisions in the primary lineage. These divisions are consistently oriented along the AP axis. Our results indicate that the interplay between stereotyped and stochastic cell behaviors in morphogenesis can only be assessed by fate mapping experiments that have both cellular resolution and large sample sizes. Copyright © 2014 Elsevier Inc. All rights reserved.

Stochasticity and Stereotypy in the Ciona Notochord

PubMed Central

Carlson, Maia; Reeves, Wendy; Veeman, Michael

2015-01-01

Fate mapping with single cell resolution has typically been confined to embryos with completely stereotyped development. The lineages giving rise to the 40 cells of the Ciona notochord are invariant, but the intercalation of those cells into a single-file column is not. Here we use genetic labeling methods to fate map the Ciona notochord with both high resolution and large sample sizes. We find that the ordering of notochord cells into a single column is not random, but instead shows a distinctive signature characteristic of mediolaterally-biased intercalation. We find that patterns of cell intercalation in the notochord are somewhat stochastic but far more stereotyped than previously believed. Cell behaviors vary by lineage, with the secondary notochord lineage being much more constrained than the primary lineage. Within the primary lineage, patterns of intercalation reflect the geometry of the intercalating tissue. We identify the latest point at which notochord morphogenesis is largely stereotyped, which is shortly before the onset of mediolateral intercalation and immediately after the final cell divisions in the primary lineage. These divisions are consistently oriented along the AP axis. Our results indicate that the interplay between stereotyped and stochastic cell behaviors in morphogenesis can only be assessed by fate mapping experiments that have both cellular resolution and large sample sizes. PMID:25459659

Neuroinflammation and physical exercise as modulators of adult hippocampal neural precursor cell behavior.

PubMed

Pérez-Domínguez, Martha; Tovar-Y-Romo, Luis B; Zepeda, Angélica

2018-01-26

The dentate gyrus of the hippocampus is a plastic structure where adult neurogenesis constitutively occurs. Cell components of the neurogenic niche are source of paracrine as well as membrane-bound factors such as Notch, Bone Morphogenetic Proteins, Wnts, Sonic Hedgehog, cytokines, and growth factors that regulate adult hippocampal neurogenesis and cell fate decision. The integration and coordinated action of multiple extrinsic and intrinsic cues drive a continuous decision process: if adult neural stem cells remain quiescent or proliferate, if they take a neuronal or a glial lineage, and if new cells proliferate, undergo apoptotic death, or survive. The proper balance in the molecular milieu of this neurogenic niche leads to the production of neurons in a higher rate as that of astrocytes. But this rate changes in face of microenvironment modifications as those driven by physical exercise or with neuroinflammation. In this work, we first review the cellular and molecular components of the subgranular zone, focusing on the molecules, active signaling pathways and genetic programs that maintain quiescence, induce proliferation, or promote differentiation. We then summarize the evidence regarding the role of neuroinflammation and physical exercise in the modulation of adult hippocampal neurogenesis with emphasis on the activation of progression from adult neural stem cells to lineage-committed progenitors to their progeny mainly in murine models.

Phylogeographic separation and formation of sexually discrete lineages in a global population of Yersinia pseudotuberculosis

PubMed Central

Seecharran, Tristan; Kalin-Manttari, Laura; Koskela, Katja; Nikkari, Simo; Dickins, Benjamin; Corander, Jukka; Skurnik, Mikael

2017-01-01

Yersinia pseudotuberculosis is a Gram-negative intestinal pathogen of humans and has been responsible for several nationwide gastrointestinal outbreaks. Large-scale population genomic studies have been performed on the other human pathogenic species of the genus Yersinia, Yersinia pestis and Yersinia enterocolitica allowing a high-resolution understanding of the ecology, evolution and dissemination of these pathogens. However, to date no purpose-designed large-scale global population genomic analysis of Y. pseudotuberculosis has been performed. Here we present analyses of the genomes of 134 strains of Y. pseudotuberculosis isolated from around the world, from multiple ecosystems since the 1960s. Our data display a phylogeographic split within the population, with an Asian ancestry and subsequent dispersal of successful clonal lineages into Europe and the rest of the world. These lineages can be differentiated by CRISPR cluster arrays, and we show that the lineages are limited with respect to inter-lineage genetic exchange. This restriction of genetic exchange maintains the discrete lineage structure in the population despite co-existence of lineages for thousands of years in multiple countries. Our data highlights how CRISPR can be informative of the evolutionary trajectory of bacterial lineages, and merits further study across bacteria. PMID:29177091

Production of imidazole alkaloids in cell cultures of jaborandi as affected by the medium pH.

PubMed

Andreazza, N L; Abreu, I N; Sawaya, A C H F; Eberlin, M N; Mazzafera, P

2009-04-01

The effect of pH (from 4.8 to 9.8) on the production of pilosine and pilocarpine and on their partition between cell and medium was studied in two lineages (P and PP) of Pilocarpus microphyllus cell suspension cultures. Highest mass accumulation was observed at high pHs and both lineages produced pilocarpine while only lineage PP produced pilosine. Both alkaloids were released in the medium but higher accumulation occurred in the cells. The highest production of pilocarpine was at pH 8.8-9.8 in both cell lineages. Other imidazole alkaloids were also identified in both lineages. At all pHs tested, the pH in the media cultures tended to stabilize around 6 after 10-15 days of cultivation. NO3(-) and NH4+ variation in the media might partially explain the pH stabilization.

IFN Regulatory Factor 8 Represses GM-CSF Expression in T cells to Affect Myeloid Cell Lineage Differentiation

PubMed Central

Paschall, Amy V.; Zhang, Ruihua; Qi, Chen-Feng; Bardhan, Kankana; Peng, Liang; Lu, Geming; Yang, Jianjun; Merad, Miriam; McGaha, Tracy; Zhou, Gang; Mellor, Andrew; Abrams, Scott I.; Morse, Herbert C.; Ozato, Keiko; Xiong, Huabao; Liu, Kebin

2015-01-01

During hematopoiesis, hematopoietic stem cells constantly differentiate into granulocytes and macrophages via a distinct differentiation program that is tightly controlled by myeloid lineage-specific transcription factors. Mice with a null mutation of IFN Regulatory Factor 8 (IRF8) accumulate CD11b+Gr1+ myeloid cells that phenotypically and functionally resemble tumor-induced myeloid-derived suppressor cells (MDSCs), indicating an essential role of IRF8 in myeloid cell lineage differentiation. However, IRF8 is expressed in various types of immune cells and whether IRF8 functions intrinsically or extrinsically in regulation of myeloid cell lineage differentiation is not fully understood. Here we report an intriguing finding that although IRF8-deficient mice exhibit deregulated myeloid cell differentiation and resultant accumulation of CD11b+Gr1+ MDSCs, surprisingly, mice with IRF8 deficiency only in myeloid cells exhibit no abnormal myeloid cell lineage differentiation. Instead, mice with IRF8 deficiency only in T cells exhibited deregulated myeloid cell differentiation and MDSC accumulation. We further demonstrated that IRF8-deficient T cells exhibit elevated GM-CSF expression and secretion. Treatment of mice with GM-CSF increased MDSC accumulation, and adoptive transfer of IRF8-deficient T cells, but not GM-CSF-deficient T cells, increased MDSC accumulation in the recipient chimeric mice. Moreover, overexpression of IRF8 decreased GM-CSF expression in T cells. Our data determine that in addition to its intrinsic function as an apoptosis regulator in myeloid cells, IRF8 also acts extrinsically to represses GM-CSF expression in T cells to control myeloid cell lineage differentiation, revealing a novel mechanism that the adaptive immune component of the immune system regulates the innate immune cell myelopoiesis in vivo. PMID:25646302

Single cell gene expression profiling of cortical osteoblast lineage cells.

PubMed

Flynn, James M; Spusta, Steven C; Rosen, Clifford J; Melov, Simon

2013-03-01

In tissues with complex architectures such as bone, it is often difficult to purify and characterize specific cell types via molecular profiling. Single cell gene expression profiling is an emerging technology useful for characterizing transcriptional profiles of individual cells isolated from heterogeneous populations. In this study we describe a novel procedure for the isolation and characterization of gene expression profiles of single osteoblast lineage cells derived from cortical bone. Mixed populations of different cell types were isolated from adult long bones of C57BL/6J mice by enzymatic digestion, and subsequently subjected to FACS to purify and characterize osteoblast lineage cells via a selection strategy using antibodies against CD31, CD45, and alkaline phosphatase (AP), specific for mature osteoblasts. The purified individual osteoblast lineage cells were then profiled at the single cell level via nanofluidic PCR. This method permits robust gene expression profiling on single osteoblast lineage cells derived from mature bone, potentially from anatomically distinct sites. In conjunction with this technique, we have also shown that it is possible to carry out single cell profiling on cells purified from fixed and frozen bone samples without compromising the gene expression signal. The latter finding means the technique can be extended to biopsies of bone from diseased individuals. Our approach for single cell expression profiling provides a new dimension to the transcriptional profile of the primary osteoblast lineage population in vivo, and has the capacity to greatly expand our understanding of how these cells may function in vivo under normal and diseased states. Copyright © 2012 Elsevier Inc. All rights reserved.

ILK modulates epithelial polarity and matrix formation in hair follicles

PubMed Central

Rudkouskaya, Alena; Welch, Ian; Dagnino, Lina

2014-01-01

Hair follicle morphogenesis requires coordination of multiple signals and communication between its epithelial and mesenchymal constituents. Cell adhesion protein platforms, which include integrins and integrin-linked kinase (ILK), are critical for hair follicle formation. However, their precise contribution to this process is poorly understood. We show that in the absence of ILK, the hair follicle matrix lineage fails to develop, likely due to abnormalities in development of apical–basal cell polarity, as well as in laminin-511 and basement membrane assembly at the tip of the hair bud. These defects also result in impaired specification of hair matrix and absence of precortex and inner sheath root cell lineages. The molecular pathways affected in ILK-deficient follicles are similar to those in the absence of epidermal integrin β1 and include Wnt, but not sonic hedgehog, signaling. ILK-deficient hair buds also show abnormalities in the dermal papilla. Addition of exogenous laminin-511 restores morphological and molecular markers associated with hair matrix formation, indicating that ILK regulates hair bud cell polarity and functions upstream from laminin-511 assembly to regulate the developmental progression of hair follicles beyond the germ stage. PMID:24371086

Frequent mechanical stress suppresses proliferation of mesenchymal stem cells from human bone marrow without loss of multipotency

NASA Astrophysics Data System (ADS)

Frank, Viktoria; Kaufmann, Stefan; Wright, Rebecca; Horn, Patrick; Yoshikawa, Hiroshi Y.; Wuchter, Patrick; Madsen, Jeppe; Lewis, Andrew L.; Armes, Steven P.; Ho, Anthony D.; Tanaka, Motomu

2016-04-01

Mounting evidence indicated that human mesenchymal stem cells (hMSCs) are responsive not only to biochemical but also to physical cues, such as substrate topography and stiffness. To simulate the dynamic structures of extracellular environments of the marrow in vivo, we designed a novel surrogate substrate for marrow derived hMSCs based on physically cross-linked hydrogels whose elasticity can be adopted dynamically by chemical stimuli. Under frequent mechanical stress, hMSCs grown on our hydrogel substrates maintain the expression of STRO-1 over 20 d, irrespective of the substrate elasticity. On exposure to the corresponding induction media, these cultured hMSCs can undergo adipogenesis and osteogenesis without requiring cell transfer onto other substrates. Moreover, we demonstrated that our surrogate substrate suppresses the proliferation of hMSCs by up to 90% without any loss of multiple lineage potential by changing the substrate elasticity every 2nd days. Such “dynamic in vitro niche” can be used not only for a better understanding of the role of dynamic mechanical stresses on the fate of hMSCs but also for the synchronized differentiation of adult stem cells to a specific lineage.

Identification of cancer genes that are independent of dominant proliferation and lineage programs

PubMed Central

Selfors, Laura M.; Stover, Daniel G.; Harris, Isaac S.; Brugge, Joan S.; Coloff, Jonathan L.

2017-01-01

Large, multidimensional cancer datasets provide a resource that can be mined to identify candidate therapeutic targets for specific subgroups of tumors. Here, we analyzed human breast cancer data to identify transcriptional programs associated with tumors bearing specific genetic driver alterations. Using an unbiased approach, we identified thousands of genes whose expression was enriched in tumors with specific genetic alterations. However, expression of the vast majority of these genes was not enriched if associations were analyzed within individual breast tumor molecular subtypes, across multiple tumor types, or after gene expression was normalized to account for differences in proliferation or tumor lineage. Together with linear modeling results, these findings suggest that most transcriptional programs associated with specific genetic alterations in oncogenes and tumor suppressors are highly context-dependent and are predominantly linked to differences in proliferation programs between distinct breast cancer subtypes. We demonstrate that such proliferation-dependent gene expression dominates tumor transcriptional programs relative to matched normal tissues. However, we also identified a relatively small group of cancer-associated genes that are both proliferation- and lineage-independent. A subset of these genes are attractive candidate targets for combination therapy because they are essential in breast cancer cell lines, druggable, enriched in stem-like breast cancer cells, and resistant to chemotherapy-induced down-regulation. PMID:29229826

HLS7, a hemopoietic lineage switch gene homologous to the leukemia-inducing gene MLF1.

PubMed Central

Williams, J H; Daly, L N; Ingley, E; Beaumont, J G; Tilbrook, P A; Lalonde, J P; Stillitano, J P; Klinken, S P

1999-01-01

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid-specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin- or chemically-induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin-responsive colonies in semi-solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells. PMID:10523300

HLS7, a hemopoietic lineage switch gene homologous to the leukemia-inducing gene MLF1.

PubMed

Williams, J H; Daly, L N; Ingley, E; Beaumont, J G; Tilbrook, P A; Lalonde, J P; Stillitano, J P; Klinken, S P

1999-10-15

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid-specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin- or chemically-induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin-responsive colonies in semi-solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells.

Derivation of lung mesenchymal lineages from the fetal mesothelium requires hedgehog signaling for mesothelial cell entry

PubMed Central

Dixit, Radhika; Ai, Xingbin; Fine, Alan

2013-01-01

Recent studies have shown that mesothelial progenitors contribute to mesenchymal lineages of developing organs. To what extent the overlying mesothelium contributes to lung development remains unknown. To rigorously address this question, we employed Wt1CreERT2/+ mice for high-fidelity lineage tracing after confirming that Cre recombinase was mesothelial specific and faithfully recapitulated endogenous Wilms’ tumor 1 (Wt1) gene expression. We visualized WT1+ mesothelial cell entry into the lung by live imaging and identified their progenies in subpopulations of bronchial smooth muscle cells, vascular smooth muscle cells and desmin+ fibroblasts by lineage tagging. Derivation of these lineages was only observed with Cre recombinase activation during early lung development. Using loss-of-function assays in organ cultures, and targeted mesothelial-restricted hedgehog loss-of-function mice, we demonstrated that mesothelial cell movement into the lung requires the direct action of hedgehog signaling. By contrast, hedgehog signaling was not required for fetal mesothelial heart entry. These findings further support a paradigm wherein the mesothelium is a source of progenitors for mesenchymal lineages during organogenesis and indicate that signals controlling mesothelial cell entry are organ specific. PMID:24130328

Derivation of lung mesenchymal lineages from the fetal mesothelium requires hedgehog signaling for mesothelial cell entry.

PubMed

Dixit, Radhika; Ai, Xingbin; Fine, Alan

2013-11-01

Recent studies have shown that mesothelial progenitors contribute to mesenchymal lineages of developing organs. To what extent the overlying mesothelium contributes to lung development remains unknown. To rigorously address this question, we employed Wt1(CreERT2/+) mice for high-fidelity lineage tracing after confirming that Cre recombinase was mesothelial specific and faithfully recapitulated endogenous Wilms' tumor 1 (Wt1) gene expression. We visualized WT1(+) mesothelial cell entry into the lung by live imaging and identified their progenies in subpopulations of bronchial smooth muscle cells, vascular smooth muscle cells and desmin(+) fibroblasts by lineage tagging. Derivation of these lineages was only observed with Cre recombinase activation during early lung development. Using loss-of-function assays in organ cultures, and targeted mesothelial-restricted hedgehog loss-of-function mice, we demonstrated that mesothelial cell movement into the lung requires the direct action of hedgehog signaling. By contrast, hedgehog signaling was not required for fetal mesothelial heart entry. These findings further support a paradigm wherein the mesothelium is a source of progenitors for mesenchymal lineages during organogenesis and indicate that signals controlling mesothelial cell entry are organ specific.

Stochastic Cell Fate Progression in Embryonic Stem Cells

NASA Astrophysics Data System (ADS)

Zou, Ling-Nan; Doyle, Adele; Jang, Sumin; Ramanathan, Sharad

2013-03-01

Studies on the directed differentiation of embryonic stem (ES) cells suggest that some early developmental decisions may be stochastic in nature. To identify the sources of this stochasticity, we analyzed the heterogeneous expression of key transcription factors in single ES cells as they adopt distinct germ layer fates. We find that under sufficiently stringent signaling conditions, the choice of lineage is unambiguous. ES cells flow into differentiated fates via diverging paths, defined by sequences of transitional states that exhibit characteristic co-expression of multiple transcription factors. These transitional states have distinct responses to morphogenic stimuli; by sequential exposure to multiple signaling conditions, ES cells are steered towards specific fates. However, the rate at which cells travel down a developmental path is stochastic: cells exposed to the same signaling condition for the same amount of time can populate different states along the same path. The heterogeneity of cell states seen in our experiments therefore does not reflect the stochastic selection of germ layer fates, but the stochastic rate of progression along a chosen developmental path. Supported in part by the Jane Coffin Childs Fund

Mbd3/NuRD controls lymphoid cell fate and inhibits tumorigenesis by repressing a B cell transcriptional program

PubMed Central

Hamey, Fiona K.; Errami, Youssef

2017-01-01

Differentiation of lineage-committed cells from multipotent progenitors requires the establishment of accessible chromatin at lineage-specific transcriptional enhancers and promoters, which is mediated by pioneer transcription factors that recruit activating chromatin remodeling complexes. Here we show that the Mbd3/nucleosome remodeling and deacetylation (NuRD) chromatin remodeling complex opposes this transcriptional pioneering during B cell programming of multipotent lymphoid progenitors by restricting chromatin accessibility at B cell enhancers and promoters. Mbd3/NuRD-deficient lymphoid progenitors therefore prematurely activate a B cell transcriptional program and are biased toward overproduction of pro–B cells at the expense of T cell progenitors. The striking reduction in early thymic T cell progenitors results in compensatory hyperproliferation of immature thymocytes and development of T cell lymphoma. Our results reveal that Mbd3/NuRD can regulate multilineage differentiation by constraining the activation of dormant lineage-specific enhancers and promoters. In this way, Mbd3/NuRD protects the multipotency of lymphoid progenitors, preventing B cell–programming transcription factors from prematurely enacting lineage commitment. Mbd3/NuRD therefore controls the fate of lymphoid progenitors, ensuring appropriate production of lineage-committed progeny and suppressing tumor formation. PMID:28899870

The diversity of nanos expression in echinoderm embryos supports different mechanisms in germ cell specification.

PubMed

Fresques, Tara; Swartz, Steven Zachary; Juliano, Celina; Morino, Yoshiaki; Kikuchi, Mani; Akasaka, Koji; Wada, Hiroshi; Yajima, Mamiko; Wessel, Gary M

2016-07-01

Specification of the germ cell lineage is required for sexual reproduction in all animals. However, the timing and mechanisms of germ cell specification is remarkably diverse in animal development. Echinoderms, such as sea urchins and sea stars, are excellent model systems to study the molecular and cellular mechanisms that contribute to germ cell specification. In several echinoderm embryos tested, the germ cell factor Vasa accumulates broadly during early development and is restricted after gastrulation to cells that contribute to the germ cell lineage. In the sea urchin, however, the germ cell factor Vasa is restricted to a specific lineage by the 32-cell stage. We therefore hypothesized that the germ cell specification program in the sea urchin/Euechinoid lineage has evolved to an earlier developmental time point. To test this hypothesis we determined the expression pattern of a second germ cell factor, Nanos, in four out of five extant echinoderm clades. Here we find that Nanos mRNA does not accumulate until the blastula stage or later during the development of all other echinoderm embryos except those that belong to the Echinoid lineage. Instead, Nanos is expressed in a restricted domain at the 32-128 cell stage in Echinoid embryos. Our results support the model that the germ cell specification program underwent a heterochronic shift in the Echinoid lineage. A comparison of Echinoid and non-Echinoid germ cell specification mechanisms will contribute to our understanding of how these mechanisms have changed during animal evolution. © 2016 Wiley Periodicals, Inc.

The origin of pre-neoplastic metaplasia in the stomach: Chief cells emerge from the Mist

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goldenring, James R., E-mail: jim.goldenring@vanderbilt.edu; Departments of Surgery and Cell and Developmental Biology, Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN; Nam, Ki Taek

2011-11-15

The digestive-enzyme secreting, gastric epithelial chief (zymogenic) cell is remarkable and underappreciated. Here, we discuss how all available evidence suggests that mature chief cells in the adult, mammalian stomach are postmitotic, slowly turning over cells that arise via a relatively long-lived progenitor, the mucous neck cell, The differentiation of chief cells from neck cells does not involve cell division, and the neck cell has its own distinct pattern of gene expression and putative physiological function. Thus, the ontogeny of the normal chief cell lineage exemplifies transdifferentiation. Furthermore, under pathophysiogical loss of acid-secreting parietal cell, the chief cell lineage can itselfmore » trasndifferentiate into a mucous cell metaplasia designated Spasmolytic Polypeptide Expressing Metaplasia (SPEM). Especially in the presence of inflammation, this metaplastic lineage can regain proliferative capacity and, in humans may also further differentiate into intestinal metaplasia. The results indicate that gastric fundic lineages display remarkable plasticity in both physiological ontogeny and pathophysiological pre-neoplastic metaplasia.« less

Identification of aberrant gene expression associated with aberrant promoter methylation in primordial germ cells between E13 and E16 rat F3 generation vinclozolin lineage.

PubMed

Taguchi, Y-h

2015-01-01

Transgenerational epigenetics (TGE) are currently considered important in disease, but the mechanisms involved are not yet fully understood. TGE abnormalities expected to cause disease are likely to be initiated during development and to be mediated by aberrant gene expression associated with aberrant promoter methylation that is heritable between generations. However, because methylation is removed and then re-established during development, it is not easy to identify promoter methylation abnormalities by comparing normal lineages with those expected to exhibit TGE abnormalities. This study applied the recently proposed principal component analysis (PCA)-based unsupervised feature extraction to previously reported and publically available gene expression/promoter methylation profiles of rat primordial germ cells, between E13 and E16 of the F3 generation vinclozolin lineage that are expected to exhibit TGE abnormalities, to identify multiple genes that exhibited aberrant gene expression/promoter methylation during development. The biological feasibility of the identified genes were tested via enrichment analyses of various biological concepts including pathway analysis, gene ontology terms and protein-protein interactions. All validations suggested superiority of the proposed method over three conventional and popular supervised methods that employed t test, limma and significance analysis of microarrays, respectively. The identified genes were globally related to tumors, the prostate, kidney, testis and the immune system and were previously reported to be related to various diseases caused by TGE. Among the genes reported by PCA-based unsupervised feature extraction, we propose that chemokine signaling pathways and leucine rich repeat proteins are key factors that initiate transgenerational epigenetic-mediated diseases, because multiple genes included in these two categories were identified in this study.

Identification of aberrant gene expression associated with aberrant promoter methylation in primordial germ cells between E13 and E16 rat F3 generation vinclozolin lineage

PubMed Central

2015-01-01

Background Transgenerational epigenetics (TGE) are currently considered important in disease, but the mechanisms involved are not yet fully understood. TGE abnormalities expected to cause disease are likely to be initiated during development and to be mediated by aberrant gene expression associated with aberrant promoter methylation that is heritable between generations. However, because methylation is removed and then re-established during development, it is not easy to identify promoter methylation abnormalities by comparing normal lineages with those expected to exhibit TGE abnormalities. Methods This study applied the recently proposed principal component analysis (PCA)-based unsupervised feature extraction to previously reported and publically available gene expression/promoter methylation profiles of rat primordial germ cells, between E13 and E16 of the F3 generation vinclozolin lineage that are expected to exhibit TGE abnormalities, to identify multiple genes that exhibited aberrant gene expression/promoter methylation during development. Results The biological feasibility of the identified genes were tested via enrichment analyses of various biological concepts including pathway analysis, gene ontology terms and protein-protein interactions. All validations suggested superiority of the proposed method over three conventional and popular supervised methods that employed t test, limma and significance analysis of microarrays, respectively. The identified genes were globally related to tumors, the prostate, kidney, testis and the immune system and were previously reported to be related to various diseases caused by TGE. Conclusions Among the genes reported by PCA-based unsupervised feature extraction, we propose that chemokine signaling pathways and leucine rich repeat proteins are key factors that initiate transgenerational epigenetic-mediated diseases, because multiple genes included in these two categories were identified in this study. PMID:26677731

Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

PubMed

Drent, Esther; Groen, Richard W J; Noort, Willy A; Themeli, Maria; Lammerts van Bueren, Jeroen J; Parren, Paul W H I; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W C J; Martens, Anton C M; Lokhorst, Henk M; Mutis, Tuna

2016-05-01

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. Copyright© Ferrata Storti Foundation.

Genome-wide chromatin state transitions associated with developmental and environmental cues.

PubMed

Zhu, Jiang; Adli, Mazhar; Zou, James Y; Verstappen, Griet; Coyne, Michael; Zhang, Xiaolan; Durham, Timothy; Miri, Mohammad; Deshpande, Vikram; De Jager, Philip L; Bennett, David A; Houmard, Joseph A; Muoio, Deborah M; Onder, Tamer T; Camahort, Ray; Cowan, Chad A; Meissner, Alexander; Epstein, Charles B; Shoresh, Noam; Bernstein, Bradley E

2013-01-31

Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background, yet the landscapes of in vivo tissues remain largely uncharted. Here, we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages, lineages, and cellular environments. They also reveal global features of the epigenome, related to nuclear architecture, that also vary across cellular phenotypes. Specifically, developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture, and discuss their implications for lineage fidelity, cellular senescence, and reprogramming. Copyright © 2013 Elsevier Inc. All rights reserved.

Lineage-specific effects of Notch/Numb signaling in post-embryonic development of the Drosophila brain

PubMed Central

Lin, Suewei; Lai, Sen-Lin; Yu, Huang-Hsiang; Chihara, Takahiro; Luo, Liqun; Lee, Tzumin

2010-01-01

Numb can antagonize Notch signaling to diversify the fates of sister cells. We report here that paired sister cells acquire different fates in all three Drosophila neuronal lineages that make diverse types of antennal lobe projection neurons (PNs). Only one in each pair of postmitotic neurons survives into the adult stage in both anterodorsal (ad) and ventral (v) PN lineages. Notably, Notch signaling specifies the PN fate in the vPN lineage but promotes programmed cell death in the missing siblings in the adPN lineage. In addition, Notch/Numb-mediated binary sibling fates underlie the production of PNs and local interneurons from common precursors in the lAL lineage. Furthermore, Numb is needed in the lateral but not adPN or vPN lineages to prevent the appearance of ectopic neuroblasts and to ensure proper self-renewal of neural progenitors. These lineage-specific outputs of Notch/Numb signaling show that a universal mechanism of binary fate decision can be utilized to govern diverse neural sibling differentiations. PMID:20023159

Roles of CONSTITUTIVE PHOTOMORPHOGENIC 10 in Arabidopsis stomata development

PubMed Central

Delgado, Dolores; Ballesteros, Isabel; Mena, Montaña; Fenoll, Carmen

2012-01-01

Stomata are epidermal bi-celled structures that differentiate within special cell lineages initiated by a subset of protodermal cells. Recently, we showed that the Arabidopsis photomorphogenic repressor COP10 controls specific cell-lineage and cell-signaling developmental mechanisms in stomatal lineages. Loss-of-function cop10-1 mutant cotyledons and leaves produced (in the light and in the dark) abundant stomatal clusters, but nonlineage epidermal cells were not affected. Here we examine COP10 role in hypocotyls, cylindrical organs displaying a distinct epidermal organization with alternate files of protruding and non-protruding cells, with the latter producing a limited number of stomata. COP10 prevents stomatal clusters and restricts stomata production in hypocotyls; these roles are specific to lineage cells as in cotyledons, since COP10 loss of function does not elicit stomatal fate in nonlineage cells; COP10 also sustains the directional cell expansion of all hypocotyl epidermal cell types, and seems necessary for the differentiation between protruding and non-protruding cell files. PMID:22836493

Disruption of Insulin Signaling in Myf5-Expressing Progenitors Leads to Marked Paucity of Brown Fat but Normal Muscle Development

PubMed Central

Lynes, Matthew D.; Schulz, Tim J.; Pan, Andrew J.

2015-01-01

Insulin exerts pleiotropic effects on cell growth, survival, and metabolism, and its role in multiple tissues has been dissected using conditional knockout mice; however, its role in development has not been studied. Lineage tracing experiments have demonstrated that interscapular brown adipose tissue (BAT) arises from a Myf5-positive lineage shared with skeletal muscle and distinct from the majority of white adipose tissue (WAT) precursors. In this study, we sought to investigate the effects of impaired insulin signaling in the Myf5-expressing precursor cells by deleting the insulin receptor gene. Mice lacking insulin receptor in the Myf5 lineage (Myf5IRKO) have a decrease of interscapular BAT mass; however, muscle development appeared normal. Histologically, the residual BAT had decreased cell size but appeared mature and potentially functional. Expression of adipogenic inhibitors preadipocyte factor-1, Necdin, and wingless-type MMTV integration site member 10a in the residual BAT tissue was nonetheless increased compared with controls, and there was an enrichment of progenitor cells with impaired adipogenic differentiation capacity, suggesting a suppression of adipogenesis in BAT. Surprisingly, when cold challenged, Myf5IRKO mice did not show impaired thermogenesis. This resistance to cold could be attributed to an increased presence of uncoupling protein 1-positive brown adipocytes in sc WAT as well as increased expression of lipolytic activity in BAT. These data suggest a critical role of insulin signaling in the development of interscapular BAT from Myf5-positive progenitor cells, but it appears to be dispensable for muscle development. They also underscore the importance of compensatory browning of sc WAT in the absence of BAT for thermoregulation. PMID:25625589

Osteoblastic/Cementoblastic and Neural Differentiation of Dental Stem Cells and Their Applications to Tissue Engineering and Regenerative Medicine

PubMed Central

Kim, Byung-Chul; Bae, Hojae; Kwon, Il-Keun; Lee, Eun-Jun; Park, Jae-Hong

2012-01-01

Recently, dental stem and progenitor cells have been harvested from periodontal tissues such as dental pulp, periodontal ligament, follicle, and papilla. These cells have received extensive attention in the field of tissue engineering and regenerative medicine due to their accessibility and multilineage differentiation capacity. These dental stem and progenitor cells are known to be derived from ectomesenchymal origin formed during tooth development. A great deal of research has been accomplished for directing osteoblastic/cementoblastic differentiation and neural differentiation from dental stem cells. To differentiate dental stem cells for use in tissue engineering and regenerative medicine, there needs to be efficient in vitro differentiation toward the osteoblastic/cementoblastic and neural lineage with well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source. This review focuses on the multilineage differentiation capacity, especially into osteoblastic/cementoblastic lineage and neural lineages, of dental stem cells such as dental pulp stem cells (DPSC), dental follicle stem cells (DFSC), periodontal ligament stem cells (PDLSC), and dental papilla stem cells (DPPSC). It also covers various experimental strategies that could be used to direct lineage-specific differentiation, and their potential applications in tissue engineering and regenerative medicine. PMID:22224548

Osteoblastic/cementoblastic and neural differentiation of dental stem cells and their applications to tissue engineering and regenerative medicine.

PubMed

Kim, Byung-Chul; Bae, Hojae; Kwon, Il-Keun; Lee, Eun-Jun; Park, Jae-Hong; Khademhosseini, Ali; Hwang, Yu-Shik

2012-06-01

Recently, dental stem and progenitor cells have been harvested from periodontal tissues such as dental pulp, periodontal ligament, follicle, and papilla. These cells have received extensive attention in the field of tissue engineering and regenerative medicine due to their accessibility and multilineage differentiation capacity. These dental stem and progenitor cells are known to be derived from ectomesenchymal origin formed during tooth development. A great deal of research has been accomplished for directing osteoblastic/cementoblastic differentiation and neural differentiation from dental stem cells. To differentiate dental stem cells for use in tissue engineering and regenerative medicine, there needs to be efficient in vitro differentiation toward the osteoblastic/cementoblastic and neural lineage with well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source. This review focuses on the multilineage differentiation capacity, especially into osteoblastic/cementoblastic lineage and neural lineages, of dental stem cells such as dental pulp stem cells (DPSC), dental follicle stem cells (DFSC), periodontal ligament stem cells (PDLSC), and dental papilla stem cells (DPPSC). It also covers various experimental strategies that could be used to direct lineage-specific differentiation, and their potential applications in tissue engineering and regenerative medicine.

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system

PubMed Central

Kutleša, Snježana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B.; Jurecic, Roland

2011-01-01

Objective Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. Materials and Methods EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. Results The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Tα, RAG-1, and T-cell receptor – Vβ genes; and 3) produced interferon-γ in response to T-cell receptor stimulation. Conclusions These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation. PMID:19447159

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.

PubMed

Kutlesa, Snjezana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B; Jurecic, Roland

2009-08-01

Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.

Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential.

PubMed

Bolton, Helen; Graham, Sarah J L; Van der Aa, Niels; Kumar, Parveen; Theunis, Koen; Fernandez Gallardo, Elia; Voet, Thierry; Zernicka-Goetz, Magdalena

2016-03-29

Most human pre-implantation embryos are mosaics of euploid and aneuploid cells. To determine the fate of aneuploid cells and the developmental potential of mosaic embryos, here we generate a mouse model of chromosome mosaicism. By treating embryos with a spindle assembly checkpoint inhibitor during the four- to eight-cell division, we efficiently generate aneuploid cells, resulting in embryo death during peri-implantation development. Live-embryo imaging and single-cell tracking in chimeric embryos, containing aneuploid and euploid cells, reveal that the fate of aneuploid cells depends on lineage: aneuploid cells in the fetal lineage are eliminated by apoptosis, whereas those in the placental lineage show severe proliferative defects. Overall, the proportion of aneuploid cells is progressively depleted from the blastocyst stage onwards. Finally, we show that mosaic embryos have full developmental potential, provided they contain sufficient euploid cells, a finding of significance for the assessment of embryo vitality in the clinic.

Lineage specific expression of Polycomb Group Proteins in human embryonic stem cells in vitro.

PubMed

Pethe, Prasad; Pursani, Varsha; Bhartiya, Deepa

2015-05-01

Human embryonic (hES) stem cells are an excellent model to study lineage specification and differentiation into various cell types. Differentiation necessitates repression of specific genes not required for a particular lineage. Polycomb Group (PcG) proteins are key histone modifiers, whose primary function is gene repression. PcG proteins form complexes called Polycomb Repressive Complexes (PRCs), which catalyze histone modifications such as H2AK119ub1, H3K27me3, and H3K9me3. PcG proteins play a crucial role during differentiation of stem cells. The expression of PcG transcripts during differentiation of hES cells into endoderm, mesoderm, and ectoderm lineage is yet to be shown. In-house derived hES cell line KIND1 was differentiated into endoderm, mesoderm, and ectoderm lineages; followed by characterization using RT-PCR for HNF4A, CDX2, MEF2C, TBX5, SOX1, and MAP2. qRT-PCR and western blotting was performed to compare expression of PcG transcripts and proteins across all the three lineages. We observed that cells differentiated into endoderm showed upregulation of RING1B, BMI1, EZH2, and EED transcripts. Mesoderm differentiation was characterized by significant downregulation of all PcG transcripts during later stages. BMI1 and RING1B were upregulated while EZH2, SUZ12, and EED remained low during ectoderm differentiation. Western blotting also showed distinct expression of BMI1 and EZH2 during differentiation into three germ layers. Our study shows that hES cells differentiating into endoderm, mesoderm, and ectoderm lineages show distinct PcG expression profile at transcript and protein level. © 2015 International Federation for Cell Biology.

Adult DRG Stem/Progenitor Cells Generate Pericytes in the Presence of Central Nervous System (CNS) Developmental Cues, and Schwann Cells in Response to CNS Demyelination.

PubMed

Vidal, Marie; Maniglier, Madlyne; Deboux, Cyrille; Bachelin, Corinne; Zujovic, Violetta; Baron-Van Evercooren, Anne

2015-06-01

It has been proposed that the adult dorsal root ganglia (DRG) harbor neural stem/progenitor cells (NPCs) derived from the neural crest. However, the thorough characterization of their stemness and differentiation plasticity was not addressed. In this study, we investigated adult DRG-NPC stem cell properties overtime, and their fate when ectopically grafted in the central nervous system. We compared them in vitro and in vivo to the well-characterized adult spinal cord-NPCs derived from the same donors. Using micro-dissection and neurosphere cultures, we demonstrate that adult DRG-NPCs have quasi unlimited self-expansion capacities without compromising their tissue specific molecular signature. Moreover, they differentiate into multiple peripheral lineages in vitro. After transplantation, adult DRG-NPCs generate pericytes in the developing forebrain but remyelinating Schwann cells in response to spinal cord demyelination. In addition, we show that axonal and endothelial/astrocytic factors as well astrocytes regulate the fate of adult DRG-NPCs in culture. Although the adult DRG-NPC multipotency is restricted to the neural crest lineage, their dual responsiveness to developmental and lesion cues highlights their impressive adaptive and repair potentials making them valuable targets for regenerative medicine. © 2015 AlphaMed Press.

Cell reprogramming: Therapeutic potential and the promise of rejuvenation for the aging brain.

PubMed

López-León, Micaela; Outeiro, Tiago F; Goya, Rodolfo G

2017-11-01

Aging is associated with a progressive increase in the incidence of neurodegenerative diseases, with Alzheimer's (AD) and Parkinson's (PD) disease being the most conspicuous examples. Within this context, the absence of efficacious therapies for most age-related brain pathologies has increased the interest in regenerative medicine. In particular, cell reprogramming technologies have ushered in the era of personalized therapies that not only show a significant potential for the treatment of neurodegenerative diseases but also promise to make biological rejuvenation feasible. We will first review recent evidence supporting the emerging view that aging is a reversible epigenetic phenomenon. Next, we will describe novel reprogramming approaches that overcome some of the intrinsic limitations of conventional induced-pluripotent-stem-cell technology. One of the alternative approaches, lineage reprogramming, consists of the direct conversion of one adult cell type into another by transgenic expression of multiple lineage-specific transcription factors (TF). Another strategy, termed pluripotency factor-mediated direct reprogramming, uses universal TF to generate epigenetically unstable intermediates able to differentiate into somatic cell types in response to specific differentiation factors. In the third part we will review studies showing the potential relevance of the above approaches for the treatment of AD and PD. Copyright © 2017 Elsevier B.V. All rights reserved.

TBX1 Represses Vegfr2 Gene Expression and Enhances the Cardiac Fate of VEGFR2+ Cells

PubMed Central

Lania, Gabriella; Ferrentino, Rosa; Baldini, Antonio

2015-01-01

The T-box transcription factor TBX1 has critical roles in maintaining proliferation and inhibiting differentiation of cardiac progenitor cells of the second heart field (SHF). Haploinsufficiency of the gene that encodes it is a cause of congenital heart disease. Here, we developed an embryonic stem (ES) cell-based model in which Tbx1 expression can be modulated by tetracycline. Using this model, we found that TBX1 down regulates the expression of VEGFR2, and we confirmed this finding in vivo during embryonic development. In addition, we found a Vegfr2 domain of expression, not previously described, in the posterior SHF and this expression is extended by loss of Tbx1. VEGFR2 has been previously described as a marker of a subpopulation of cardiac progenitors. Clonal analysis of ES-derived VEGFR2+ cells indicated that 12.5% of clones expressed three markers of cardiac lineage (cardiomyocyte, smooth muscle and endothelium). However, a pulse of Tbx1 expression was sufficient to increase the percentage to 20.8%. In addition, the percentage of clones expressing markers of multiple cardiac lineages increased from 41.6% to 79.1% after Tbx1 pulse. These results suggest that TBX1 plays a role in maintaining a progenitor state in VEGFR2+ cells. PMID:26382615

Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2.

PubMed

Priddle, Helen; Allegrucci, Cinzia; Burridge, Paul; Munoz, Maria; Smith, Nigel M; Devlin, Lyndsey; Sjoblom, Cecilia; Chamberlain, Sarah; Watson, Sue; Young, Lorraine E; Denning, Chris

2010-04-01

The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

Inflammatory Cytokines Induce a Unique Mineralizing Phenotype in Mesenchymal Stem Cells Derived from Human Bone Marrow*

PubMed Central

Ferreira, Elisabeth; Porter, Ryan M.; Wehling, Nathalie; O'Sullivan, Regina P.; Liu, Fangjun; Boskey, Adele; Estok, Daniel M.; Harris, Mitchell B.; Vrahas, Mark S.; Evans, Christopher H.; Wells, James W.

2013-01-01

Bone marrow contains mesenchymal stem cells (MSCs) that can differentiate along multiple mesenchymal lineages. In this capacity they are thought to be important in the intrinsic turnover and repair of connective tissues while also serving as a basis for tissue engineering and regenerative medicine. However, little is known of the biological responses of human MSCs to inflammatory conditions. When cultured with IL-1β, marrow-derived MSCs from 8 of 10 human subjects deposited copious hydroxyapatite, in which authenticity was confirmed by Fourier transform infrared spectroscopy. Transmission electron microscopy revealed the production of fine needles of hydroxyapatite in conjunction with matrix vesicles. Alkaline phosphatase activity did not increase in response to inflammatory mediators, but PPi production fell, reflecting lower ectonucleotide pyrophosphatase activity in cells and matrix vesicles. Because PPi is the major physiological inhibitor of mineralization, its decline generated permissive conditions for hydroxyapatite formation. This is in contrast to MSCs treated with dexamethasone, where PPi levels did not fall and mineralization was fuelled by a large and rapid increase in alkaline phosphatase activity. Bone sialoprotein was the only osteoblast marker strongly induced by IL-1β; thus these cells do not become osteoblasts despite depositing abundant mineral. RT-PCR did not detect transcripts indicative of alternative mesenchymal lineages, including chondrocytes, myoblasts, adipocytes, ligament, tendon, or vascular smooth muscle cells. IL-1β phosphorylated multiple MAPKs and activated nuclear factor-κB (NF-κB). Certain inhibitors of MAPK and PI3K, but not NF-κB, prevented mineralization. The findings are of importance to soft tissue mineralization, tissue engineering, and regenerative medicine. PMID:23970554

Strength of signal: a fundamental mechanism for cell fate specification.

PubMed

Hayes, Sandra M; Love, Paul E

2006-02-01

How equipotent cells develop into complex tissues containing many diverse cell types is still a mystery. However, evidence is accumulating from different tissue systems in multiple organisms that many of the specific receptor families known to regulate cell fate decisions target conserved signaling pathways. A mechanism for preserving specificity in the cellular response that has emerged from these studies is one in which quantitative differences in receptor signaling regulate the cell fate decision. A signal strength model has recently gained support as a means to explain alphabeta/gammadelta lineage commitment. In this review, we compare the alphabeta/gammadelta fate decision with other cell fate decisions that occur outside of the lymphoid system to attain a better picture of the quantitative signaling mechanism for cell fate specification.

Historical biogeography and diversification of truffles in the Tuberaceae and their newly identified Southern hemisphere sister lineage

Treesearch

Gregory Bonito; Matthew E. Smith; Michael Nowak; Rosanne A. Healy; Gonzalo Guevara; Efren Cazares; Akihiko Kinoshita; Eduardo R. Nouhra; Laura S. Dominguez; Leho Tedersoo; Claude Murat; Yun Wang; Baldomero Arroyo Moreno; Donald H. Pfister; Kazuhide Nara; Alessandra Zambonelli; James M. Trappe; Rytas Vilgalys

2013-01-01

In this study we reassessed the biogeography and origin of the Tuberaceae and their relatives using multiple loci and a global sampling of taxa. Multiple independent transitions from an aboveground to a belowground truffie fruiting body form have occurred in the Tuberaceae and in its newly recognized sister lineage...

Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction.

PubMed

Poleshko, Andrey; Shah, Parisha P; Gupta, Mudit; Babu, Apoorva; Morley, Michael P; Manderfield, Lauren J; Ifkovits, Jamie L; Calderon, Damelys; Aghajanian, Haig; Sierra-Pagán, Javier E; Sun, Zheng; Wang, Qiaohong; Li, Li; Dubois, Nicole C; Morrisey, Edward E; Lazar, Mitchell A; Smith, Cheryl L; Epstein, Jonathan A; Jain, Rajan

2017-10-19

Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin, and independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery. Copyright © 2017 Elsevier Inc. All rights reserved.

Single-Cell Resolution of Temporal Gene Expression during Heart Development.

PubMed

DeLaughter, Daniel M; Bick, Alexander G; Wakimoto, Hiroko; McKean, David; Gorham, Joshua M; Kathiriya, Irfan S; Hinson, John T; Homsy, Jason; Gray, Jesse; Pu, William; Bruneau, Benoit G; Seidman, J G; Seidman, Christine E

2016-11-21

Activation of complex molecular programs in specific cell lineages governs mammalian heart development, from a primordial linear tube to a four-chamber organ. To characterize lineage-specific, spatiotemporal developmental programs, we performed single-cell RNA sequencing of >1,200 murine cells isolated at seven time points spanning embryonic day 9.5 (primordial heart tube) to postnatal day 21 (mature heart). Using unbiased transcriptional data, we classified cardiomyocytes, endothelial cells, and fibroblast-enriched cells, thus identifying markers for temporal and chamber-specific developmental programs. By harnessing these datasets, we defined developmental ages of human and mouse pluripotent stem-cell-derived cardiomyocytes and characterized lineage-specific maturation defects in hearts of mice with heterozygous mutations in Nkx2.5 that cause human heart malformations. This spatiotemporal transcriptome analysis of heart development reveals lineage-specific gene programs underlying normal cardiac development and congenital heart disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of whole bone marrow, whole bone marrow cultured cells, and mesenchymal stem cells in chronic wound healing.

PubMed

Rodriguez-Menocal, Luis; Shareef, Shahjahan; Salgado, Marcela; Shabbir, Arsalan; Van Badiavas, Evangelos

2015-03-13

Recent evidence has shown that bone marrow cells play critical roles during the inflammatory, proliferative and remodeling phases of cutaneous wound healing. Among the bone marrow cells delivered to wounds are stem cells, which can differentiate into multiple tissue-forming cell lineages to effect, healing. Gaining insight into which lineages are most important in accelerating wound healing would be quite valuable in designing therapeutic approaches for difficult to heal wounds. In this report we compared the effect of different bone marrow preparations on established in vitro wound healing assays. The preparations examined were whole bone marrow (WBM), whole bone marrow (long term initiating/hematopoietic based) cultured cells (BMC), and bone marrow derived mesenchymal stem cells (BM-MSC). We also applied these bone marrow preparations in two murine models of radiation induced delayed wound healing to determine which had a greater effect on healing. Angiogenesis assays demonstrated that tube formation was stimulated by both WBM and BMC, with WBM having the greatest effect. Scratch wound assays showed higher fibroblast migration at 24, 48, and 72 hours in presence of WBM as compared to BM-MSC. WBM also appeared to stimulate a greater healing response than BMC and BM-MSC in a radiation induced delayed wound healing animal model. These studies promise to help elucidate the role of stem cells during repair of chronic wounds and reveal which cells present in bone marrow might contribute most to the wound healing process.

Src Homology 2–containing 5-Inositol Phosphatase (SHIP) Suppresses an Early Stage of Lymphoid Cell Development through Elevated Interleukin-6 Production by Myeloid Cells in Bone Marrow

PubMed Central

Nakamura, Koji; Kouro, Taku; Kincade, Paul W.; Malykhin, Alexander; Maeda, Kazuhiko; Coggeshall, K. Mark

2004-01-01

The Src homology (SH)2–containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP−/− animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP−/− mice. Lineage-negative marrow (Lin−) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP−/− mice. Furthermore, culture supernatants of SHIP−/− Lin− cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP−/− Lin− cells contained more IL-6 transcripts than wild-type Lin− cells, and neutralizing anti–IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP−/− Lin− cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin− bone marrow cells reproduced the phenotype of SHIP−/− bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow. PMID:14718513

What happens in the thymus does not stay in the thymus: how T cells recycle the CD4+-CD8+ lineage commitment transcriptional circuitry to control their function

PubMed Central

Vacchio, Melanie S.; Bosselut, Rémy

2016-01-01

MHC-restricted CD4+ and CD8+ T cell are at the core of most adaptive immune responses. Although these cells carry distinct functions, they arise from a common precursor during thymic differentiation, in a developmental sequence that matches CD4 and CD8 expression and functional potential with MHC restriction. While the transcriptional control of CD4+-CD8+ lineage choice in the thymus is now better understood, less was known about what maintains the CD4+- and CD8+-lineage integrity of mature T cells. In this review, we discuss the mechanisms that establish in the thymus, and maintain in post-thymic cells, the separation of these lineages. We focus on recent studies that address the mechanisms of epigenetic control of Cd4 expression and emphasize how maintaining a transcriptional circuitry nucleated around Thpok and Runx proteins, the key architects of CD4+-CD8+ lineage commitment in the thymus, is critical for CD4+ T cell helper functions. PMID:27260768

Early cell lineage specification in a marsupial: a case for diverse mechanisms among mammals.

PubMed

Frankenberg, Stephen; Shaw, Geoff; Freyer, Claudia; Pask, Andrew J; Renfree, Marilyn B

2013-03-01

Early cell lineage specification in eutherian mammals results in the formation of a pluripotent inner cell mass (ICM) and trophoblast. By contrast, marsupials have no ICM. Here, we present the first molecular analysis of mechanisms of early cell lineage specification in a marsupial, the tammar wallaby. There was no overt differential localisation of key lineage-specific transcription factors in cleavage and early unilaminar blastocyst stages. Pluriblast cells (equivalent to the ICM) became distinguishable from trophoblast cells by differential expression of POU5F1 and, to a greater extent, POU2, a paralogue of POU5F1. Unlike in the mouse, pluriblast-trophoblast differentiation coincided with a global nuclear-to-cytoplasmic transition of CDX2 localisation. Also unlike in the mouse, Hippo pathway factors YAP and WWTR1 showed mutually distinct localisation patterns that suggest non-redundant roles. NANOG and GATA6 were conserved as markers of epiblast and hypoblast, respectively, but some differences to the mouse were found in their mode of differentiation. Our results suggest that there is considerable evolutionary plasticity in the mechanisms regulating early lineage specification in mammals.

An organizing activity is required for head patterning and cell fate specification in the polychaete annelid Capitella teleta: new insights into cell-cell signaling in Lophotrochozoa.

PubMed

Amiel, Aldine R; Henry, Jonathan Q; Seaver, Elaine C

2013-07-01

Many lophotrochozoans (i.e., molluscs, annelids, nemerteans, and polyclad flatworms) display a well-conserved early developmental program called spiral cleavage that contrasts with the high diversity of adult body forms present in this group. Due to this stereotypical development, each cell can be uniquely identified and its lineage history known following intracellular injection of lineage tracers. Cell deletion experiments performed mainly in molluscs have demonstrated that one or two cells associated with the endomesodermal lineage represent an embryonic organizer of subsequent development and are causally involved in cell fate and body patterning. Utilizing the published fate map of the spiral-cleaving annelid Capitella teleta, we used infrared laser cell deletions to dissect the role of individual cells on the patterning of the larval body. Thirteen uniquely identifiable individual blastomeres and two double cell combination deletions were studied to assess larval phenotypes by scoring multiple morphological structures and cell type-specific molecular markers differentially expressed along the antero-posterior and dorso-ventral axes. Surprisingly, our results show that in C. teleta, the cellular identity of the "organizing cell" and the timing of the organizing activity are different from that of other spiralians. retain-->In C. teleta, the ectodermal primary somatoblast, 2d, is the key cell responsible for organizing activity during early embryonic development, and is necessary for bilateral symmetry and dorso-ventral axis organization of the head as well as neural, foregut and mesoderm tissue formation. Furthermore, we show that the ERK/MAPK signaling pathway does not appear to be involved in organizing activity in retain-->C. teleta. This contrasts with data from molluscs and the molecular mechanism suggested for another polychaete, Hydroides elegans, highlighting likely molecular level variation among spiralian embryos. These results reinforce the idea that an embryonic organizing activity is present across spiralians. Our data also emphasize the developmental variation within lophotrochozoans, and may ultimately provide insight into the role of developmental processes in the evolution of diverse body forms in metazoans. Copyright © 2013 Elsevier Inc. All rights reserved.

Immunocytochemical markers in acute leukaemias diagnosis.

PubMed

Gluzman, D F; Nadgornaya, V A; Sklyarenko, L M; Ivanovskaya, T S; Poludnenko, L Yu; Ukrainskaya, N I

2010-09-01

The study included 1742 patients with acute myeloblastic leukaemias (AML) and acute lymphoblastic leukaemias (ALL), Kyiv city residents and patients from 20 regions of Ukraine. Bone marrow and blood smears were sent at diagnosis to Reference Center. The analysis was based on May-Grünvald-Giemza (MGG) stain and cytochemical reactions (MPO, acNSE, CAE, AP, PAS). Immunocytochemical techniques (APAAP, LSAB) and broad panel of monoclonal antibodies (MoAbs) against lineage specific and differentiation antigens of leukocytes were employed for immunophenotyping of leukemic blast cells directly in blood and bone marrow smears. Different types of AML were defined by the expression of the cell surface and cytoplasmic antigens. Immunocytochemical study was required especially in diagnosing of AML with minimal differentiation, acute megakaryoblastic leukaemia, acute erythroid leukaemia and acute leukaemias of ambiguous lineage. Acute lymphoblastic leukaemias was broadly classified into B-lineage and T-lineage ALL. According to the degree of B-lymphoid differentiation of the blast cells four subtypes of B-lineage ALL were established. T-lineage ALL observed in patients were also divided into four subtypes. Immunocytochemical examination was required to diagnose AL of ambiguous lineage with no clear evidence of lineage differentiation (acute undifferentiated leukaemia) or those with blasts that express markers of more than one lineage (mixed phenotype acute leukaemias).

A CD133-expressing murine liver oval cell population with bilineage potential.

PubMed

Rountree, C Bart; Barsky, Lora; Ge, Shundi; Zhu, Judy; Senadheera, Shantha; Crooks, Gay M

2007-10-01

Although oval cells are postulated to be adult liver stem cells, a well-defined phenotype of a bipotent liver stem cell remains elusive. The heterogeneity of cells within the oval cell fraction has hindered lineage potential studies. Our goal was to identify an enriched population of bipotent oval cells using a combination of flow cytometry and single cell gene expression in conjunction with lineage-specific liver injury models. Expression of cell surface markers on nonparenchymal, nonhematopoietic (CD45-) cells were characterized. Cell populations were isolated by flow cytometry for gene expression studies. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine toxic injury induced cell cycling and expansion specifically in the subpopulation of oval cells in the periportal zone that express CD133. CD133+CD45- cells expressed hepatoblast and stem cell-associated genes, and single cells coexpressed both hepatocyte and cholangiocyte-associated genes, indicating bilineage potential. CD133+CD45- cells proliferated in response to liver injury. Following toxic hepatocyte damage, CD133+CD45- cells demonstrated upregulated expression of the hepatocyte gene Albumin. In contrast, toxic cholangiocyte injury resulted in upregulation of the cholangiocyte gene Ck19. After 21-28 days in culture, CD133+CD45- cells continued to generate cells of both hepatocyte and cholangiocyte lineages. Thus, CD133 expression identifies a population of oval cells in adult murine liver with the gene expression profile and function of primitive, bipotent liver stem cells. In response to lineage-specific injury, these cells demonstrate a lineage-appropriate genetic response. Disclosure of potential conflicts of interest is found at the end of this article.

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

PubMed

Vicinanza, Carla; Aquila, Iolanda; Scalise, Mariangela; Cristiano, Francesca; Marino, Fabiola; Cianflone, Eleonora; Mancuso, Teresa; Marotta, Pina; Sacco, Walter; Lewis, Fiona C; Couch, Liam; Shone, Victoria; Gritti, Giulia; Torella, Annalaura; Smith, Andrew J; Terracciano, Cesare Mn; Britti, Domenico; Veltri, Pierangelo; Indolfi, Ciro; Nadal-Ginard, Bernardo; Ellison-Hughes, Georgina M; Torella, Daniele

2017-12-01

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kit pos ) cells. The adult heart indeed contains a heterogeneous mixture of c-kit pos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit pos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kit pos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit pos sorting. The blood/endothelial lineage-committed (Lineage pos ) CD45 pos c-kit pos cardiac cells were compared to CD45 neg (Lineage neg /Lin neg ) c-kit pos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit pos cardiac cells are blood/endothelial lineage-committed CD45 pos CD31 pos c-kit pos cells. In contrast, the Lin neg CD45 neg c-kit pos cardiac cell cohort, which represents ⩽10% of the total c-kit pos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit neg and the blood/endothelial lineage-committed c-kit pos cardiac cells. Single Lin neg c-kit pos cell-derived clones, which represent only 1-2% of total c-kit pos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Lin neg c-kit pos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kit pos cardiac cells were injected. Thus, among the cardiac c-kit pos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

Adaptive and Pathogenic Responses to Stress by Stem Cells during Development.

PubMed

Mansouri, Ladan; Xie, Yufen; Rappolee, Daniel A

2012-12-10

Cellular stress is the basis of a dose-dependent continuum of responses leading to adaptive health or pathogenesis. For all cells, stress leads to reduction in macromolecular synthesis by shared pathways and tissue and stress-specific homeostatic mechanisms. For stem cells during embryonic, fetal, and placental development, higher exposures of stress lead to decreased anabolism, macromolecular synthesis and cell proliferation. Coupled with diminished stem cell proliferation is a stress-induced differentiation which generates minimal necessary function by producing more differentiated product/cell. This compensatory differentiation is accompanied by a second strategy to insure organismal survival as multipotent and pluripotent stem cells differentiate into the lineages in their repertoire. During stressed differentiation, the first lineage in the repertoire is increased and later lineages are suppressed, thus prioritized differentiation occurs. Compensatory and prioritized differentiation is regulated by at least two types of stress enzymes. AMP-activated protein kinase (AMPK) which mediates loss of nuclear potency factors and stress-activated protein kinase (SAPK) that does not. SAPK mediates an increase in the first essential lineage and decreases in later lineages in placental stem cells. The clinical significance of compensatory and prioritized differentiation is that stem cell pools are depleted and imbalanced differentiation leads to gestational diseases and long term postnatal pathologies.

Adaptive and Pathogenic Responses to Stress by Stem Cells during Development

PubMed Central

Mansouri, Ladan; Xie, Yufen; Rappolee, Daniel A

2012-01-01

Cellular stress is the basis of a dose-dependent continuum of responses leading to adaptive health or pathogenesis. For all cells, stress leads to reduction in macromolecular synthesis by shared pathways and tissue and stress-specific homeostatic mechanisms. For stem cells during embryonic, fetal, and placental development, higher exposures of stress lead to decreased anabolism, macromolecular synthesis and cell proliferation. Coupled with diminished stem cell proliferation is a stress-induced differentiation which generates minimal necessary function by producing more differentiated product/cell. This compensatory differentiation is accompanied by a second strategy to insure organismal survival as multipotent and pluripotent stem cells differentiate into the lineages in their repertoire. During stressed differentiation, the first lineage in the repertoire is increased and later lineages are suppressed, thus prioritized differentiation occurs. Compensatory and prioritized differentiation is regulated by at least two types of stress enzymes. AMP-activated protein kinase (AMPK) which mediates loss of nuclear potency factors and stress-activated protein kinase (SAPK) that does not. SAPK mediates an increase in the first essential lineage and decreases in later lineages in placental stem cells. The clinical significance of compensatory and prioritized differentiation is that stem cell pools are depleted and imbalanced differentiation leads to gestational diseases and long term postnatal pathologies. PMID:24710551

Template DNA-strand co-segregation and asymmetric cell division in skeletal muscle stem cells.

PubMed

Shinin, Vasily; Gayraud-Morel, Barbara; Tajbakhsh, Shahragim

2009-01-01

Stem cells are present in all tissues and organs, and are crucial for normal regulated growth. How the pool size of stem cells and their progeny is regulated to establish the tissue prenatally, then maintain it throughout life, is a key question in biology and medicine. The ability to precisely locate stem and progenitors requires defining lineage progression from stem to differentiated cells, assessing the mode of cell expansion and self-renewal and identifying markers to assess the different cell states within the lineage. We have shown that during lineage progression from a quiescent adult muscle satellite cell to a differentiated myofibre, both symmetric and asymmetric divisions take place. Furthermore, we provide evidence that a sub-population of label retaining satellite cells co-segregate template DNA strands to one daughter cell. These findings provide a means of identifying presumed stem and progenitor cells within the lineage. In addition, asymmetric segregation of template DNA and the cytoplasmic protein Numb provides a landmark to define cell behaviour as self-renewal and differentiation decisions are being executed.

Directed differentiation of embryonic stem cells using a bead-based combinatorial screening method.

PubMed

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.

Dental pulp stem cells express tendon markers under mechanical loading and are a potential cell source for tissue engineering of tendon-like tissue.

PubMed

Chen, Yu-Ying; He, Sheng-Teng; Yan, Fu-Hua; Zhou, Peng-Fei; Luo, Kai; Zhang, Yan-Ding; Xiao, Yin; Lin, Min-Kui

2016-12-16

Postnatal mesenchymal stem cells have the capacity to differentiate into multiple cell lineages. This study explored the possibility of dental pulp stem cells (DPSCs) for potential application in tendon tissue engineering. The expression of tendon-related markers such as scleraxis, tenascin-C, tenomodulin, eye absent homologue 2, collagens I and VI was detected in dental pulp tissue. Interestingly, under mechanical stimulation, these tendon-related markers were significantly enhanced when DPSCs were seeded in aligned polyglycolic acid (PGA) fibre scaffolds. Furthermore, mature tendon-like tissue was formed after transplantation of DPSC-PGA constructs under mechanical loading conditions in a mouse model. This study demonstrates that DPSCs could be a potential stem cell source for tissue engineering of tendon-like tissue.

Multiple lineages of Avian malaria parasites (Plasmodium) in the Galapagos Islands and evidence for arrival via migratory birds.

PubMed

Levin, I I; Zwiers, P; Deem, S L; Geest, E A; Higashiguchi, J M; Iezhova, T A; Jiménez-Uzcátegui, G; Kim, D H; Morton, J P; Perlut, N G; Renfrew, R B; Sari, E H R; Valkiunas, G; Parker, P G

2013-12-01

Haemosporidian parasites in the genus Plasmodium were recently detected through molecular screening in the Galapagos Penguin (Spheniscus mendiculus). We summarized results of an archipelago-wide screen of 3726 endemic birds representing 22 species for Plasmodium spp. through a combination of molecular and microscopy techniques. Three additional Plasmodium lineages were present in Galapagos. Lineage A-infected penguins, Yellow Warblers (Setophaga petechia aureola), and one Medium Ground Finch (Geospiza fortis) and was detected at multiple sites in multiple years [corrected]. The other 3 lineages were each detected at one site and at one time; apparently, they were transient infections of parasites not established on the archipelago. No gametocytes were found in blood smears of infected individuals; thus, endemic Galapagos birds may be dead-end hosts for these Plasmodium lineages. Determining when and how parasites and pathogens arrive in Galapagos is key to developing conservation strategies to prevent and mitigate the effects of introduced diseases. To assess the potential for Plasmodium parasites to arrive via migratory birds, we analyzed blood samples from 438 North American breeding Bobolinks (Dolichonyx oryzivorus), the only songbird that regularly migrates through Galapagos. Two of the ephemeral Plasmodium lineages (B and C) found in Galapagos birds matched parasite sequences from Bobolinks. Although this is not confirmation that Bobolinks are responsible for introducing these lineages, evidence points to higher potential arrival rates of avian pathogens than previously thought. Linajes Múltiples de Parásitos de Malaria Aviar (Plasmodium) en las Islas Galápagos y Evidencia de su Arribo por Medio de Aves Migratorias. © 2013 Society for Conservation Biology.

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers

PubMed Central

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-01-01

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks. PMID:26563448

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers.

PubMed

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-11-13

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Endele, Max; Etzrodt, Martin; Schroeder, Timm, E-mail: timm.schroeder@bsse.ethz.ch

Hematopoiesis is the cumulative consequence of finely tuned signaling pathways activated through extrinsic factors, such as local niche signals and systemic hematopoietic cytokines. Whether extrinsic factors actively instruct the lineage choice of hematopoietic stem and progenitor cells or are only selectively allowing survival and proliferation of already intrinsically lineage-committed cells has been debated over decades. Recent results demonstrated that cytokines can instruct lineage choice. However, the precise function of individual cytokine-triggered signaling molecules in inducing cellular events like proliferation, lineage choice, and differentiation remains largely elusive. Signal transduction pathways activated by different cytokine receptors are highly overlapping, but support themore » production of distinct hematopoietic lineages. Cellular context, signaling dynamics, and the crosstalk of different signaling pathways determine the cellular response of a given extrinsic signal. New tools to manipulate and continuously quantify signaling events at the single cell level are therefore required to thoroughly interrogate how dynamic signaling networks yield a specific cellular response. - Highlights: • Recent studies provided definite proof for lineage-instructive action of cytokines. • Signaling pathways involved in hematopoietic lineage instruction remain elusive. • New tools are emerging to quantitatively study dynamic signaling networks over time.« less

Humoral Activity of Cord Blood-Derived Stem/Progenitor Cells: Implications for Stem Cell-Based Adjuvant Therapy of Neurodegenerative Disorders

PubMed Central

Paczkowska, Edyta; Kaczyńska, Katarzyna; Pius-Sadowska, Ewa; Rogińska, Dorota; Kawa, Miłosz; Ustianowski, Przemysław; Safranow, Krzysztof; Celewicz, Zbigniew; Machaliński, Bogusław

2013-01-01

Background Stem/progenitor cells (SPCs) demonstrate neuro-regenerative potential that is dependent upon their humoral activity by producing various trophic factors regulating cell migration, growth, and differentiation. Herein, we compared the expression of neurotrophins (NTs) and their receptors in specific umbilical cord blood (UCB) SPC populations, including lineage-negative, CD34+, and CD133+ cells, with that in unsorted, nucleated cells (NCs). Methods and Results The expression of NTs and their receptors was detected by QRT-PCR, western blotting, and immunofluorescent staining in UCB-derived SPC populations (i.e., NCs vs. lineage-negative, CD34+, and CD133+ cells). To better characterize, global gene expression profiles of SPCs were determined using genome-wide RNA microarray technology. Furthermore, the intracellular production of crucial neuro-regenerative NTs (i.e., BDNF and NT-3) was assessed in NCs and lineage-negative cells after incubation for 24, 48, and 72 h in both serum and serum-free conditions. We discovered significantly higher expression of NTs and NT receptors at both the mRNA and protein level in lineage-negative, CD34+, and CD133+ cells than in NCs. Global gene expression analysis revealed considerably higher expression of genes associated with the production and secretion of proteins, migration, proliferation, and differentiation in lineage-negative cells than in CD34+ or CD133+ cell populations. Notably, after short-term incubation under serum-free conditions, lineage-negative cells and NCs produced significantly higher amounts of BDNF and NT-3 than under steady-state conditions. Finally, conditioned medium (CM) from lineage-negative SPCs exerted a beneficial impact on neural cell survival and proliferation. Conclusions Collectively, our findings demonstrate that UCB-derived SPCs highly express NTs and their relevant receptors under steady-state conditions, NT expression is greater under stress-related conditions and that CM from SPCs favorable influence neural cell proliferation and survival. Understanding the mechanisms governing the characterization and humoral activity of subsets of SPCs may yield new therapeutic strategies that might be more effective in treating neurodegenerative disorders. PMID:24391835

Humoral activity of cord blood-derived stem/progenitor cells: implications for stem cell-based adjuvant therapy of neurodegenerative disorders.

PubMed

Paczkowska, Edyta; Kaczyńska, Katarzyna; Pius-Sadowska, Ewa; Rogińska, Dorota; Kawa, Miłosz; Ustianowski, Przemysław; Safranow, Krzysztof; Celewicz, Zbigniew; Machaliński, Bogusław

2013-01-01

Stem/progenitor cells (SPCs) demonstrate neuro-regenerative potential that is dependent upon their humoral activity by producing various trophic factors regulating cell migration, growth, and differentiation. Herein, we compared the expression of neurotrophins (NTs) and their receptors in specific umbilical cord blood (UCB) SPC populations, including lineage-negative, CD34(+), and CD133(+) cells, with that in unsorted, nucleated cells (NCs). The expression of NTs and their receptors was detected by QRT-PCR, western blotting, and immunofluorescent staining in UCB-derived SPC populations (i.e., NCs vs. lineage-negative, CD34(+), and CD133(+) cells). To better characterize, global gene expression profiles of SPCs were determined using genome-wide RNA microarray technology. Furthermore, the intracellular production of crucial neuro-regenerative NTs (i.e., BDNF and NT-3) was assessed in NCs and lineage-negative cells after incubation for 24, 48, and 72 h in both serum and serum-free conditions. We discovered significantly higher expression of NTs and NT receptors at both the mRNA and protein level in lineage-negative, CD34(+), and CD133(+) cells than in NCs. Global gene expression analysis revealed considerably higher expression of genes associated with the production and secretion of proteins, migration, proliferation, and differentiation in lineage-negative cells than in CD34(+) or CD133(+) cell populations. Notably, after short-term incubation under serum-free conditions, lineage-negative cells and NCs produced significantly higher amounts of BDNF and NT-3 than under steady-state conditions. Finally, conditioned medium (CM) from lineage-negative SPCs exerted a beneficial impact on neural cell survival and proliferation. Collectively, our findings demonstrate that UCB-derived SPCs highly express NTs and their relevant receptors under steady-state conditions, NT expression is greater under stress-related conditions and that CM from SPCs favorable influence neural cell proliferation and survival. Understanding the mechanisms governing the characterization and humoral activity of subsets of SPCs may yield new therapeutic strategies that might be more effective in treating neurodegenerative disorders.

Colon Stem Cell and Crypt Dynamics Exposed by Cell Lineage Reconstruction

PubMed Central

Itzkovitz, Shalev; Elbaz, Judith; Maruvka, Yosef E.; Segev, Elad; Shlush, Liran I.; Dekel, Nava; Shapiro, Ehud

2011-01-01

Stem cell dynamics in vivo are often being studied by lineage tracing methods. Our laboratory has previously developed a retrospective method for reconstructing cell lineage trees from somatic mutations accumulated in microsatellites. This method was applied here to explore different aspects of stem cell dynamics in the mouse colon without the use of stem cell markers. We first demonstrated the reliability of our method for the study of stem cells by confirming previously established facts, and then we addressed open questions. Our findings confirmed that colon crypts are monoclonal and that, throughout adulthood, the process of monoclonal conversion plays a major role in the maintenance of crypts. The absence of immortal strand mechanism in crypts stem cells was validated by the age-dependent accumulation of microsatellite mutations. In addition, we confirmed the positive correlation between physical and lineage proximity of crypts, by showing that the colon is separated into small domains that share a common ancestor. We gained new data demonstrating that colon epithelium is clustered separately from hematopoietic and other cell types, indicating that the colon is constituted of few progenitors and ruling out significant renewal of colonic epithelium from hematopoietic cells during adulthood. Overall, our study demonstrates the reliability of cell lineage reconstruction for the study of stem cell dynamics, and it further addresses open questions in colon stem cells. In addition, this method can be applied to study stem cell dynamics in other systems. PMID:21829376

Gata4-Dependent Differentiation of c-Kit+ Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the Heart.

PubMed

Maliken, Bryan D; Kanisicak, Onur; Karch, Jason; Khalil, Hadi; Fu, Xing; Boyer, Justin G; Prasad, Vikram; Zheng, Yi; Molkentin, Jeffery D

2018-04-17

Background -While c-Kit + adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells (CPCs). Methods - Kit allele-dependent lineage tracing and fusion analysis was performed in mice following simultaneous Gata4 and Gata6 cell-type specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit + cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2 CreERT2 transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. Results -Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2 CreERT2 transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artefact of greater leukocyte-cardiomyocyte cellular fusion due to defective endothelial cell differentiation in the absence of Gata4 Conclusions -Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit + cells using Kit allele lineage tracing appears to be an artefact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit + endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and capillary network integrity.

Myelin and oligodendrocyte lineage cells in white matter pathology and plasticity after traumatic brain injury.

PubMed

Armstrong, Regina C; Mierzwa, Amanda J; Sullivan, Genevieve M; Sanchez, Maria A

2016-11-01

Impact to the head or rapid head acceleration-deceleration can cause traumatic brain injury (TBI) with a characteristic pathology of traumatic axonal injury (TAI) and secondary damage in white matter tracts. Myelin and oligodendrocyte lineage cells have significant roles in the progression of white matter pathology after TBI and in the potential for plasticity and subsequent recovery. The myelination pattern of specific brain regions, such as frontal cortex, may also increase susceptibility to neurodegeneration and psychiatric symptoms after TBI. White matter pathology after TBI depends on the extent and distribution of axon damage, microhemorrhages and/or neuroinflammation. TAI occurs in a pattern of damaged axons dispersed among intact axons in white matter tracts. TAI accompanied by bleeding and/or inflammation produces focal regions of overt tissue destruction, resulting in loss of both axons and myelin. White matter regions with TAI may also exhibit demyelination of intact axons. Demyelinated axons that remain viable have the potential for remyelination and recovery of function. Indeed, animal models of TBI have demonstrated demyelination that is associated with evidence of remyelination, including oligodendrocyte progenitor cell proliferation, generation of new oligodendrocytes, and formation of thinner myelin. Changes in neuronal activity that accompany TBI may also involve myelin remodeling, which modifies conduction efficiency along intact myelinated fibers. Thus, effective remyelination and myelin remodeling may be neurobiological substrates of plasticity in neuronal circuits that require long-distance communication. This perspective integrates findings from multiple contexts to propose a model of myelin and oligodendrocyte lineage cell relevance in white matter injury after TBI. This article is part of the Special Issue entitled 'Oligodendrocytes in Health and Disease'. Published by Elsevier Ltd.

Cell fate regulation in early mammalian development

NASA Astrophysics Data System (ADS)

Oron, Efrat; Ivanova, Natalia

2012-08-01

Preimplantation development in mammals encompasses a period from fertilization to implantation and results in formation of a blastocyst composed of three distinct cell lineages: epiblast, trophectoderm and primitive endoderm. The epiblast gives rise to the organism, while the trophectoderm and the primitive endoderm contribute to extraembryonic tissues that support embryo development after implantation. In many vertebrates, such as frog or fish, maternally supplied lineage determinants are partitioned within the egg. Cell cleavage that follows fertilization results in polarization of these factors between the individual blastomeres, which become restricted in their developmental fate. In contrast, the mouse oocyte and zygote lack clear polarity and, until the eight-cell stage, individual blastomeres retain the potential to form all lineages. How are cell lineages specified in the absence of a maternally supplied blueprint? This is a fundamental question in the field of developmental biology. The answer to this question lies in understanding the cell-cell interactions and gene networks involved in embryonic development prior to implantation and using this knowledge to create testable models of the developmental processes that govern cell fates. We provide an overview of classic and contemporary models of early lineage development in the mouse and discuss the emerging body of work that highlights similarities and differences between blastocyst development in the mouse and other mammalian species.

YY1 Regulates Melanocyte Development and Function by Cooperating with MITF

PubMed Central

Bell, Robert J. A.; Tran, Thanh-Nga T.; Haq, Rizwan; Liu, Huifei; Love, Kevin T.; Langer, Robert; Anderson, Daniel G.; Larue, Lionel; Fisher, David E.

2012-01-01

Studies of coat color mutants have greatly contributed to the discovery of genes that regulate melanocyte development and function. Here, we generated Yy1 conditional knockout mice in the melanocyte-lineage and observed profound melanocyte deficiency and premature gray hair, similar to the loss of melanocytes in human piebaldism and Waardenburg syndrome. Although YY1 is a ubiquitous transcription factor, YY1 interacts with M-MITF, the Waardenburg Syndrome IIA gene and a master transcriptional regulator of melanocytes. YY1 cooperates with M-MITF in regulating the expression of piebaldism gene KIT and multiple additional pigmentation genes. Moreover, ChIP–seq identified genome-wide YY1 targets in the melanocyte lineage. These studies mechanistically link genes implicated in human conditions of melanocyte deficiency and reveal how a ubiquitous factor (YY1) gains lineage-specific functions by co-regulating gene expression with a lineage-restricted factor (M-MITF)—a general mechanism which may confer tissue-specific gene expression in multiple lineages. PMID:22570637

Modeling and visualizing cell type switching.

PubMed

Ghaffarizadeh, Ahmadreza; Podgorski, Gregory J; Flann, Nicholas S

2014-01-01

Understanding cellular differentiation is critical in explaining development and for taming diseases such as cancer. Differentiation is conventionally represented using bifurcating lineage trees. However, these lineage trees cannot readily capture or quantify all the types of transitions now known to occur between cell types, including transdifferentiation or differentiation off standard paths. This work introduces a new analysis and visualization technique that is capable of representing all possible transitions between cell states compactly, quantitatively, and intuitively. This method considers the regulatory network of transcription factors that control cell type determination and then performs an analysis of network dynamics to identify stable expression profiles and the potential cell types that they represent. A visualization tool called CellDiff3D creates an intuitive three-dimensional graph that shows the overall direction and probability of transitions between all pairs of cell types within a lineage. In this study, the influence of gene expression noise and mutational changes during myeloid cell differentiation are presented as a demonstration of the CellDiff3D technique, a new approach to quantify and envision all possible cell state transitions in any lineage network.

Amniotic-Fluid Stem Cells: Growth Dynamics and Differentiation Potential after a CD-117-Based Selection Procedure

PubMed Central

Arnhold, S.; Glüer, S.; Hartmann, K.; Raabe, O.; Addicks, K.; Wenisch, S.; Hoopmann, M.

2011-01-01

Amniotic fluid (AF) has become an interesting source of fetal stem cells. However, AF contains heterogeneous and multiple, partially differentiated cell types. After isolation from the amniotic fluid, cells were characterized regarding their morphology and growth dynamics. They were sorted by magnetic associated cell sorting using the surface marker CD 117. In order to show stem cell characteristics such as pluripotency and to evaluate a possible therapeutic application of these cells, AF fluid-derived stem cells were differentiated along the adipogenic, osteogenic, and chondrogenic as well as the neuronal lineage under hypoxic conditions. Our findings reveal that magnetic associated cell sorting (MACS) does not markedly influence growth characteristics as demonstrated by the generation doubling time. There was, however, an effect regarding an altered adipogenic, osteogenic, and chondrogenic differentiation capacity in the selected cell fraction. In contrast, in the unselected cell population neuronal differentiation is enhanced. PMID:21437196

Interferons direct Th2 cell reprogramming to generate a stable GATA-3(+)T-bet(+) cell subset with combined Th2 and Th1 cell functions.

PubMed

Hegazy, Ahmed N; Peine, Michael; Helmstetter, Caroline; Panse, Isabel; Fröhlich, Anja; Bergthaler, Andreas; Flatz, Lukas; Pinschewer, Daniel D; Radbruch, Andreas; Löhning, Max

2010-01-29

Current T cell differentiation models invoke separate T helper 2 (Th2) and Th1 cell lineages governed by the lineage-specifying transcription factors GATA-3 and T-bet. However, knowledge on the plasticity of Th2 cell lineage commitment is limited. Here we show that infection with Th1 cell-promoting lymphocytic choriomeningitis virus (LCMV) reprogrammed otherwise stably committed GATA-3(+) Th2 cells to adopt a GATA-3(+)T-bet(+) and interleukin-4(+)interferon-gamma(+) "Th2+1" phenotype that was maintained in vivo for months. Th2 cell reprogramming required T cell receptor stimulation, concerted type I and type II interferon and interleukin-12 signals, and T-bet. LCMV-triggered T-bet induction in adoptively transferred virus-specific Th2 cells was crucial to prevent viral persistence and fatal immunopathology. Thus, functional reprogramming of unfavorably differentiated Th2 cells may facilitate the establishment of protective immune responses. Stable coexpression of GATA-3 and T-bet provides a molecular concept for the long-term coexistence of Th2 and Th1 cell lineage characteristics in single memory T cells. Copyright 2010 Elsevier Inc. All rights reserved.

Effect of Diabetes Mellitus on Adipocyte-Derived Stem Cells in Rat.

PubMed

Jumabay, Medet; Moon, Jeremiah H; Yeerna, Huwate; Boström, Kristina I

2015-11-01

Diabetes mellitus affects the adipose tissue and mesenchymal stem cells derived from the adipose stroma and other tissues. Previous reports suggest that bone morphogenetic protein 4 (BMP4) is involved in diabetic complications, at the same time playing an important role in the maintenance of stem cells. In this study, we used rats transgenic for human islet amyloid polypeptide (HIP rats), a model of type 2 diabetes, to study the effect of diabetes on adipocyte-derived stem cells, referred to as dedifferentiated fat (DFAT) cells. Our results show that BMP4 expression in inguinal adipose tissue is significantly increased in HIP rats compared to controls, whereas matrix Gla protein (MGP), an inhibitor of BMP4 is decreased as determined by quantitative PCR, and immunofluorescence. In addition, adipose vascularity and expression of multiple endothelial cell markers was increased in the diabetic tissue, visualized by immunofluorescence for endothelial markers. The endothelial markers co-localized with the enhanced BMP4 expression, suggesting that vascular cells play a role BMP4 induction. The DFAT cells are multipotent stem cells derived from white mature adipocytes that undergo endothelial and adipogenic differentiation. DFAT cells prepared from the inguinal adipose tissue in HIP rats exhibited enhanced proliferative capacity compared to wild type. In addition, their ability to undergo both endothelial cell and adipogenic lineage differentiation was enhanced, as well as their response to BMP4, as assessed by lineage marker expression. We conclude that the DFAT cells are affected by diabetic changes and may contribute to the adipose dysfunction in diabetes. © 2015 Wiley Periodicals, Inc.

[B lymphocyte clonal evolution of human reactive lymph nodes revealed by lineage tree analysis].

PubMed

Tabibian-Keissar, Hilla; Schiby, Ginette; Azogui-Rosenthal, Noemie; Hazanov, Helena; Rakovsky, Aviya Shapira; Michaeli, Miri; Rosenblatt, Kinneret; Mehr, Ramit; Barshack, Iris

2013-06-01

Hypermutation and selection processes, characterizing T-dependent B cell responses taking place in germinal centers of lymph nodes, lead to B cell receptor affinity maturation. Those immune responses lead to the development of memory B cells and plasma cells that secrete high amounts of antibody molecules. The dynamics of B cell clonal evolution during affinity maturation has significant importance in infectious and autoimmune diseases, malignancies and aging. Immunoglobulin (Ig) gene mutational Lineage tree construction by comparing variable regions of Ig-gene sequences to the Ig germline gene is an interesting approach for studying B cell cLonal evolution. Lineage tree shapes and Ig gene mutations can be evaluated not only qualitatively and intuitively, but also quantitatively, and thus reveal important information related to hypermutation and selection. In this paper we describe the experimental protocols that we used for PCR amplification of Igvariable region genes from human formalin fixed paraffin embedded reactive lymph node tissues and the subsequent bioinformatical analyses of sequencing data using Ig mutational lineage trees. B cell populations of three out of four reactive Lymph node tissues were composed of several clones. Most of the Ig gene mutational lineage trees were small and narrow. Significant differences were not detected by quantification of Lineage trees. B lymphocyte clones that were detected in human reactive lymph node tissues represent major responding clones in normal polyclonal immune response. This result is in line with the polyclonal profile of B Lymphocyte populations that reside in reactive lymph node tissues.

Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella.

PubMed

Engel, Philipp; Salzburger, Walter; Liesch, Marius; Chang, Chao-Chin; Maruyama, Soichi; Lanz, Christa; Calteau, Alexandra; Lajus, Aurélie; Médigue, Claudine; Schuster, Stephan C; Dehio, Christoph

2011-02-10

Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens. Furthermore, our study highlights the remarkable evolvability of T4SSs and their effector proteins, explaining their broad application in bacterial interactions with the environment.

Parallel Evolution of a Type IV Secretion System in Radiating Lineages of the Host-Restricted Bacterial Pathogen Bartonella

PubMed Central

Engel, Philipp; Salzburger, Walter; Liesch, Marius; Chang, Chao-Chin; Maruyama, Soichi; Lanz, Christa; Calteau, Alexandra; Lajus, Aurélie; Médigue, Claudine; Schuster, Stephan C.; Dehio, Christoph

2011-01-01

Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens. Furthermore, our study highlights the remarkable evolvability of T4SSs and their effector proteins, explaining their broad application in bacterial interactions with the environment. PMID:21347280

A systems approach defining constraints of the genome architecture on lineage selection and evolvability during somatic cancer evolution

PubMed Central

Rübben, Albert; Nordhoff, Ole

2013-01-01

Summary Most clinically distinguishable malignant tumors are characterized by specific mutations, specific patterns of chromosomal rearrangements and a predominant mechanism of genetic instability but it remains unsolved whether modifications of cancer genomes can be explained solely by mutations and selection through the cancer microenvironment. It has been suggested that internal dynamics of genomic modifications as opposed to the external evolutionary forces have a significant and complex impact on Darwinian species evolution. A similar situation can be expected for somatic cancer evolution as molecular key mechanisms encountered in species evolution also constitute prevalent mutation mechanisms in human cancers. This assumption is developed into a systems approach of carcinogenesis which focuses on possible inner constraints of the genome architecture on lineage selection during somatic cancer evolution. The proposed systems approach can be considered an analogy to the concept of evolvability in species evolution. The principal hypothesis is that permissive or restrictive effects of the genome architecture on lineage selection during somatic cancer evolution exist and have a measurable impact. The systems approach postulates three classes of lineage selection effects of the genome architecture on somatic cancer evolution: i) effects mediated by changes of fitness of cells of cancer lineage, ii) effects mediated by changes of mutation probabilities and iii) effects mediated by changes of gene designation and physical and functional genome redundancy. Physical genome redundancy is the copy number of identical genetic sequences. Functional genome redundancy of a gene or a regulatory element is defined as the number of different genetic elements, regardless of copy number, coding for the same specific biological function within a cancer cell. Complex interactions of the genome architecture on lineage selection may be expected when modifications of the genome architecture have multiple and possibly opposed effects which manifest themselves at disparate times and progression stages. Dissection of putative mechanisms mediating constraints exerted by the genome architecture on somatic cancer evolution may provide an algorithm for understanding and predicting as well as modifying somatic cancer evolution in individual patients. PMID:23336076

Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters.

PubMed

Beamish, Christine A; Strutt, Brenda J; Arany, Edith J; Hill, David J

2016-04-18

Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture, but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive, glucose-transporter-2-low (Ins(+)Glut2(LO)) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new, functional β-cells, and which may be potentially exploited for regenerative therapies in the future.

Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters

PubMed Central

Beamish, Christine A.; Strutt, Brenda J.; Arany, Edith J.; Hill, David J.

2016-01-01

ABSTRACT Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP+/+ mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture, but remaining HPAP+ cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP+Ck19+ cells had derived from insulin-positive, glucose-transporter-2-low (Ins+Glut2LO) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin+Glut2LO cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin+Glut2+ cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins+Glut2LO cells may represent a resident population of cells capable of forming new, functional β-cells, and which may be potentially exploited for regenerative therapies in the future. PMID:27010375

Role of monocyte-lineage cells in prostate cancer cell invasion and tissue factor expression.

PubMed

Lindholm, Paul F; Lu, Yi; Adley, Brian P; Vladislav, Tudor; Jovanovic, Borko; Sivapurapu, Neela; Yang, Ximing J; Kajdacsy-Balla, André

2010-11-01

Tissue factor (TF) is a cell surface glycoprotein intricately related to blood coagulation and inflammation. This study was performed to investigate the role of monocyte-lineage cells in prostate cancer cell TF expression and cell invasion. Prostate cancer cell invasion was tested with and without added peripheral blood monocytes or human monocyte-lineage cell lines. TF neutralizing antibodies were used to determine the TF requirement for prostate cancer cell invasion activity. Immunohistochemistry was performed to identify prostate tissue CD68 positive monocyte-derived cells and prostate epithelial TF expression. Co-culture of PC-3, DU145, and LNCaP cells with isolated human monocytes significantly stimulated prostate cancer cell invasion activity. TF expression was greater in highly invasive prostate cancer cells and was induced in PC-3, DU145, and LNCaP cells by co-culture with U-937 cells, but not with THP-1 cells. TF neutralizing antibodies inhibited PC-3 cell invasion in co-cultures with monocyte-lineage U-937 or THP-1 cells. Prostate cancer tissues contained more CD68 positive cells in the stroma and epithelium (145 ± 53/mm(2)) than benign prostate (108 ± 31/mm(2)). Samples from advanced stage prostate cancer tended to contain more CD68 positive cells when compared with lower stage lesions. Prostatic adenocarcinoma demonstrated significantly increased TF expression compared with benign prostatic epithelium. This study shows that co-culture with monocyte-lineage cells induced prostate cancer cell invasion activity. PC-3 invasion and TF expression was induced in co-culture with U-937 cells and partially inhibited with TF neutralizing antibodies.

NS1 codon usage adaptation to humans in pandemic Zika virus.

PubMed

Freire, Caio César de Melo; Palmisano, Giuseppe; Braconi, Carla T; Cugola, Fernanda R; Russo, Fabiele B; Beltrão-Braga, Patricia Cb; Iamarino, Atila; Lima Neto, Daniel Ferreira de; Sall, Amadou Alpha; Rosa-Fernandes, Livia; Larsen, Martin R; Zanotto, Paolo Marinho de Andrade

2018-05-10

Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human infections were infrequently reported until 2007, when the first known epidemic occurred in Micronesia. After 2013, the Asian lineage of ZIKV spread along the Pacific Islands and Americas, causing severe outbreaks with millions of human infections. The recent human infections of ZIKV were also associated with severe complications, such as an increase in cases of Guillain-Barre syndrome and the emergence of congenital Zika syndrome. To better understand the recent and rapid expansion of ZIKV, as well as the presentation of novel complications, we compared the genetic differences between the African sylvatic lineage and the Asian epidemic lineage that caused the recent massive outbreaks. The epidemic lineages have significant codon adaptation in NS1 gene to translate these proteins in human and Aedes aegypti mosquito cells compared to the African zoonotic lineage. Accordingly, a Brazilian epidemic isolate (ZBR) produced more NS1 protein than the MR766 African lineage (ZAF) did, as indicated by proteomic data from infections of neuron progenitor cells-derived neurospheres. Although ZBR replicated more efficiently in these cells, the differences observed in the stoichiometry of ZIKV proteins were not exclusively explained by the differences in viral replication between the lineages. Our findings suggest that natural, silent translational selection in the second half of 20th century could have improved the fitness of Asian ZIKV lineage in human and mosquito cells.

Cell tracing reveals a dorsoventral lineage restriction plane in the mouse limb bud mesenchyme.

PubMed

Arques, Carlos G; Doohan, Roisin; Sharpe, James; Torres, Miguel

2007-10-01

Regionalization of embryonic fields into independent units of growth and patterning is a widespread strategy during metazoan development. Compartments represent a particular instance of this regionalization, in which unit coherence is maintained by cell lineage restriction between adjacent regions. Lineage compartments have been described during insect and vertebrate development. Two common characteristics of the compartments described so far are their occurrence in epithelial structures and the presence of signaling regions at compartment borders. Whereas Drosophila compartmental organization represents a background subdivision of embryonic fields that is not necessarily related to anatomical structures, vertebrate compartment borders described thus far coincide with, or anticipate, anatomical or cell-type discontinuities. Here, we describe a general method for clonal analysis in the mouse and use it to determine the topology of clone distribution along the three limb axes. We identify a lineage restriction boundary at the limb mesenchyme dorsoventral border that is unrelated to any anatomical discontinuity, and whose lineage restriction border is not obviously associated with any signaling center. This restriction is the first example in vertebrates of a mechanism of primordium subdivision unrelated to anatomical boundaries. Furthermore, this is the first lineage compartment described within a mesenchymal structure in any organism, suggesting that lineage restrictions are fundamental not only for epithelial structures, but also for mesenchymal field patterning. No lineage compartmentalization was found along the proximodistal or anteroposterior axes, indicating that patterning along these axes does not involve restriction of cell dispersion at specific axial positions.

Global reorganisation of cis-regulatory units upon lineage commitment of human embryonic stem cells

PubMed Central

Freire-Pritchett, Paula; Schoenfelder, Stefan; Várnai, Csilla; Wingett, Steven W; Cairns, Jonathan; Collier, Amanda J; García-Vílchez, Raquel; Furlan-Magaril, Mayra; Osborne, Cameron S; Fraser, Peter; Rugg-Gunn, Peter J; Spivakov, Mikhail

2017-01-01

Long-range cis-regulatory elements such as enhancers coordinate cell-specific transcriptional programmes by engaging in DNA looping interactions with target promoters. Deciphering the interplay between the promoter connectivity and activity of cis-regulatory elements during lineage commitment is crucial for understanding developmental transcriptional control. Here, we use Promoter Capture Hi-C to generate a high-resolution atlas of chromosomal interactions involving ~22,000 gene promoters in human pluripotent and lineage-committed cells, identifying putative target genes for known and predicted enhancer elements. We reveal extensive dynamics of cis-regulatory contacts upon lineage commitment, including the acquisition and loss of promoter interactions. This spatial rewiring occurs preferentially with predicted changes in the activity of cis-regulatory elements and is associated with changes in target gene expression. Our results provide a global and integrated view of promoter interactome dynamics during lineage commitment of human pluripotent cells. DOI: http://dx.doi.org/10.7554/eLife.21926.001 PMID:28332981

Developmental lineage priming in Dictyostelium by heterogeneous Ras activation.

PubMed

Chattwood, Alex; Nagayama, Koki; Bolourani, Parvin; Harkin, Lauren; Kamjoo, Marzieh; Weeks, Gerald; Thompson, Christopher R L

2013-11-26

In cell culture, genetically identical cells often exhibit heterogeneous behavior, with only 'lineage primed' cells responding to differentiation inducing signals. It has recently been proposed that such heterogeneity exists during normal embryonic development to allow position independent patterning based on 'salt and pepper' differentiation and sorting out. However, the molecular basis of lineage priming and how it leads to reproducible cell type proportioning are poorly understood. To address this, we employed a novel forward genetic approach in the model organism Dictyostelium discoideum. These studies reveal that the Ras-GTPase regulator gefE is required for normal lineage priming and salt and pepper differentiation. This is because Ras-GTPase activity sets the intrinsic response threshold to lineage specific differentiation signals. Importantly, we show that although gefE expression is uniform, transcription of its target, rasD, is both heterogeneous and dynamic, thus providing a novel mechanism for heterogeneity generation and position-independent differentiation. DOI: http://dx.doi.org/10.7554/eLife.01067.001.

Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

NASA Technical Reports Server (NTRS)

Chen, Silvia S.; Revoltella, Roberto P.; Papini, Sandra; Michelini, Monica; Fitzgerald, Wendy; Zimmerberg, Joshua; Margolis, Leonid

2003-01-01

In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice.

PubMed

Tarkowski, Andrzej K; Suwińska, Aneta; Czołowska, Renata; Ożdżeński, Wacław

2010-12-15

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile. Copyright © 2010 Elsevier Inc. All rights reserved.

New Chimeric Antigen Receptor Design for Solid Tumors

PubMed Central

Wang, Yuedi; Luo, Feifei; Yang, Jiao; Zhao, Chujun; Chu, Yiwei

2017-01-01

In recent years, chimeric antigen receptor (CAR) T-cell therapy has become popular in immunotherapy, particularly after its tremendous success in the treatment of lineage-restricted hematologic cancers. However, the application of CAR T-cell therapy for solid tumors has not reached its full potential because of the lack of specific tumor antigens and inhibitory factors in suppressive tumor microenvironment (TME) (e.g., programmed death ligand-1, myeloid-derived suppressor cells, and transforming growth factor-β). In this review, we include some limitations in CAR design, such as tumor heterogeneity, indefinite spatial distance between CAR T-cell and its target cell, and suppressive TME. We also summarize some new approaches to overcome these hurdles, including targeting neoantigens and/or multiple antigens at once and depleting some inhibitory factors. PMID:29312360

A hierarchy of self-renewing tumor-initiating cell types in glioblastoma.

PubMed

Chen, Ruihuan; Nishimura, Merry C; Bumbaca, Stephanie M; Kharbanda, Samir; Forrest, William F; Kasman, Ian M; Greve, Joan M; Soriano, Robert H; Gilmour, Laurie L; Rivers, Celina Sanchez; Modrusan, Zora; Nacu, Serban; Guerrero, Steve; Edgar, Kyle A; Wallin, Jeffrey J; Lamszus, Katrin; Westphal, Manfred; Heim, Susanne; James, C David; VandenBerg, Scott R; Costello, Joseph F; Moorefield, Scott; Cowdrey, Cynthia J; Prados, Michael; Phillips, Heidi S

2010-04-13

The neural stem cell marker CD133 is reported to identify cells within glioblastoma (GBM) that can initiate neurosphere growth and tumor formation; however, instances of CD133(-) cells exhibiting similar properties have also been reported. Here, we show that some PTEN-deficient GBM tumors produce a series of CD133(+) and CD133(-) self-renewing tumor-initiating cell types and provide evidence that these cell types constitute a lineage hierarchy. Our results show that the capacities for self-renewal and tumor initiation in GBM need not be restricted to a uniform population of stemlike cells, but can be shared by a lineage of self-renewing cell types expressing a range of markers of forebrain lineage. Copyright 2010 Elsevier Inc. All rights reserved.

Reprogramming of blood cells into induced pluripotent stem cells as a new cell source for cartilage repair.

PubMed

Li, Yueying; Liu, Tie; Van Halm-Lutterodt, Nicholas; Chen, JiaYu; Su, Qingjun; Hai, Yong

2016-02-17

An attempt was made to reprogram peripheral blood cells into human induced pluripotent stem cell (hiPSCs) as a new cell source for cartilage repair. We generated chondrogenic lineage from human peripheral blood via hiPSCs using an integration-free method. Peripheral blood cells were either obtained from a human blood bank or freshly collected from volunteers. After transforming peripheral blood cells into iPSCs, the newly derived iPSCs were further characterized through karyotype analysis, pluripotency gene expression and cell differentiation ability. iPSCs were differentiated through multiple steps, including embryoid body formation, hiPSC-mesenchymal stem cell (MSC)-like cell expansion, and chondrogenic induction for 21 days. Chondrocyte phenotype was then assessed by morphological, histological and biochemical analysis, as well as the chondrogenic expression. hiPSCs derived from peripheral blood cells were successfully generated, and were characterized by fluorescent immunostaining of pluripotent markers and teratoma formation in vivo. Flow cytometric analysis showed that MSC markers CD73 and CD105 were present in monolayer cultured hiPSC-MSC-like cells. Both alcian blue and toluidine blue staining of hiPSC-MSC-chondrogenic pellets showed as positive. Immunohistochemistry of collagen II and X staining of the pellets were also positive. The sulfated glycosaminoglycan content was significantly increased, and the expression levels of the chondrogenic markers COL2, COL10, COL9 and AGGRECAN were significantly higher in chondrogenic pellets than in undifferentiated cells. These results indicated that peripheral blood cells could be a potential source for differentiation into chondrogenic lineage in vitro via generation of mesenchymal progenitor cells. This study supports the potential applications of utilizing peripheral blood cells in generating seed cells for cartilage regenerative medicine in a patient-specific and cost-effective approach.

TFF2 mRNA transcript expression marks a gland progenitor cell of the gastric oxyntic mucosa

PubMed Central

Quante, Michael; Marrache, Frederic; Goldenring, James R.; Wang, Timothy C.

2010-01-01

Background and Aims Gastric stem cells are located in the isthmus of the gastric glands, and give rise to epithelial progenitors that undergo bipolar migration and differentiation into pit and oxyntic lineages. While gastric mucus neck cells, located below the isthmus, express trefoil factor family 2 (TFF2) protein, TFF2 mRNA transcripts are concentrated in cells above the neck region in normal corpus mucosa, suggesting that TFF2 transcription is a marker of gastric progenitor cells. Methods Using a BAC strategy, we generated a transgenic mouse with a tamoxifen-inducible Cre under the control of the TFF2 promoter (TFF2-BAC-CreERT2) and analyzed the lineage derivation from TFF2 mRNA transcript-expressing (TTE) cells. Results TTE cells were localized to the isthmus, above and distinct from TFF2 protein-expressing mucus neck cells. Lineage tracing revealed that these cells migrated towards the bottom of the gland within 20 days, giving rise to parietal, mucous neck and chief cells, but not to ECL cells. Surface mucus cells were not derived from TTE cells, and the progeny of the TTE lineage did not survive beyond 200 days. TTE cells were localized in the isthmus adjacent to Dclk1+ putative progenitor cells. Induction of spasmolytic polypeptide-expressing metaplasia (SPEM) with DMP-777-induced acute parietal cell loss revealed that this metaplastic phenotype might arise in part through transdiferentiation of chief cells as opposed to expansion of mucus neck or progenitor cells. Conclusion TFF2-transcript-expressing cells are progenitors for mucus neck, parietal and zymogenic, but not for pit or ECL cell lineages in the oxyntic gastric mucosa. PMID:20708616

Japanese encephalitis virus invasion of cell: allies and alleys.

PubMed

Nain, Minu; Abdin, Malik Z; Kalia, Manjula; Vrati, Sudhanshu

2016-03-01

The mosquito-borne flavivirus, Japanese encephalitis virus (JEV), is the leading cause of virus-induced encephalitis globally and a major public health concern of several countries in Southeast Asia, with the potential to become a global pathogen. The virus is neurotropic, and the disease ranges from mild fever to severe hemorrhagic and encephalitic manifestations and death. The early steps of the virus life cycle, binding, and entry into the cell are crucial determinants of infection and are potential targets for the development of antiviral therapies. JEV can infect multiple cell types; however, the key receptor molecule(s) still remains elusive. JEV also has the capacity to utilize multiple endocytic pathways for entry into cells of different lineages. This review not only gives a comprehensive update on what is known about the virus attachment and receptor system (allies) and the endocytic pathways (alleys) exploited by the virus to gain entry into the cell and establish infection but also discusses crucial unresolved issues. We also highlight common themes and key differences between JEV and other flaviviruses in these contexts. Copyright © 2015 John Wiley & Sons, Ltd.

Systemic Mastocytosis with Smoldering Multiple Myeloma: Report of a Case

PubMed Central

Garcia, Gwenalyn; Ying, Liu; Hurford, Matthew; Odaimi, Marcel

2016-01-01

Systemic mastocytosis (SM) is a disease characterized by a clonal infiltration of mast cells affecting various tissues of the body. It is grouped into six different subtypes according to the World Health Organization classification. It is called indolent systemic mastocytosis (ISM) when there is no evidence of end organ dysfunction, while the presence of end organ dysfunction defines aggressive systemic mastocytosis (ASM). When SM coexists with a clonal hematological disorder, it is classified as systemic mastocytosis with associated clonal hematological nonmast cell lineage disease (SM-AHNMD). Over 80% of SM-AHNMD cases involve disorders of the myeloid cell lines. To our knowledge, there are only 8 reported cases to date of SM associated with a plasma cell disorder. We report a patient with ISM who was found to have concomitant smoldering multiple myeloma. His disease later progressed to ASM. We discuss this rare association between SM and a plasma cell disorder, and potential common pathophysiologic mechanisms linking the two disorders will be reviewed. We also discuss prognostic factors in SM as well as the management options considered during the evolution of the patient's disease. PMID:27293930

Cells of Origin of Epithelial Ovarian Cancers

DTIC Science & Technology

2015-09-01

cells in oral squamous cell carcinomas by a novel pathway-based lineage tracing approach in a murine model. ! 13! Specific aims: 1. Determine...SUNDARESAN Lineage tracing and clonal analysis of oral cancer initiating cells The goal of this project is to study cancer stem cells /cancer initiating...whether oral cancer cells genetically marked based on their activities for stem cell -related pathways exhibit cancer stem cell properties in vivo by

Accounting for cell lineage and sex effects in the identification of cell-specific DNA methylation using a Bayesian model selection algorithm.

PubMed

White, Nicole; Benton, Miles; Kennedy, Daniel; Fox, Andrew; Griffiths, Lyn; Lea, Rodney; Mengersen, Kerrie

2017-01-01

Cell- and sex-specific differences in DNA methylation are major sources of epigenetic variation in whole blood. Heterogeneity attributable to cell type has motivated the identification of cell-specific methylation at the CpG level, however statistical methods for this purpose have been limited to pairwise comparisons between cell types or between the cell type of interest and whole blood. We developed a Bayesian model selection algorithm for the identification of cell-specific methylation profiles that incorporates knowledge of shared cell lineage and allows for the identification of differential methylation profiles in one or more cell types simultaneously. Under the proposed methodology, sex-specific differences in methylation by cell type are also assessed. Using publicly available, cell-sorted methylation data, we show that 51.3% of female CpG markers and 61.4% of male CpG markers identified were associated with differential methylation in more than one cell type. The impact of cell lineage on differential methylation was also highlighted. An evaluation of sex-specific differences revealed differences in CD56+NK methylation, within both single and multi- cell dependent methylation patterns. Our findings demonstrate the need to account for cell lineage in studies of differential methylation and associated sex effects.

Self-renewal and multilineage differentiation of mouse dental epithelial stem cells.

PubMed

Chang, Julia Yu Fong; Wang, Cong; Jin, Chengliu; Yang, Chaofeng; Huang, Yanqing; Liu, Junchen; McKeehan, Wallace L; D'Souza, Rena N; Wang, Fen

2013-11-01

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49f(Bright) population was enriched in sphere-forming cells. In addition, the CD49f(Bright) population includes both slow-cycling and Lgr5(+) DESCs. The in vitro sphere culture system and identification of CD49f(Bright) as a DESC marker provide a novel platform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies. © 2013.

Self-renewal and Multilineage Differentiation of Mouse Dental Epithelial Stem Cells

PubMed Central

Chang, Julia Yu Fong; Wang, Cong; Jin, Chengliu; Yang, Chaofeng; Huang, Yanqing; Liu, Junchen; McKeehan, Wallace L.; D’Souza, Rena N.; Wang, Fen

2013-01-01

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49fBright population was enriched in sphere-forming cells. In addition, the CD49fBright population includes both slow-cycling and Lgr5+ DESCs. The in vitro sphere culture system and identification of CD49fBright as a DESC marker provide a novel plateform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies. PMID:23906788

Kif3a Controls Murine Nephron Number Via GLI3 Repressor, Cell Survival, and Gene Expression in a Lineage-Specific Manner

PubMed Central

Chi, Lijun; Galtseva, Alevtina; Chen, Lin; Mo, Rong; Hui, Chi-chung; Rosenblum, Norman D.

2013-01-01

The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms. PMID:23762375

Epigenomic Analysis of Multi-lineage Differentiation of Human Embryonic Stem Cells

PubMed Central

Xie, Wei; Schultz, Matthew D.; Lister, Ryan; Hou, Zhonggang; Rajagopal, Nisha; Ray, Pradipta; Whitaker, John W.; Tian, Shulan; Hawkins, R. David; Leung, Danny; Yang, Hongbo; Wang, Tao; Lee, Ah Young; Swanson, Scott A.; Zhang, Jiuchun; Zhu, Yun; Kim, Audrey; Nery, Joseph R.; Urich, Mark A.; Kuan, Samantha; Yen, Chia-an; Klugman, Sarit; Yu, Pengzhi; Suknuntha, Kran; Propson, Nicholas E.; Chen, Huaming; Edsall, Lee E.; Wagner, Ulrich; Li, Yan; Ye, Zhen; Kulkarni, Ashwinikumar; Xuan, Zhenyu; Chung, Wen-Yu; Chi, Neil C.; Antosiewicz-Bourget, Jessica E.; Slukvin, Igor; Stewart, Ron; Zhang, Michael Q.; Wang, Wei; Thomson, James A.; Ecker, Joseph R.; Ren, Bing

2013-01-01

SUMMARY Epigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells, and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in non-expressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation. PMID:23664764

Patterns of cell death in the embryonic antenna of the grasshopper Schistocerca gregaria.

PubMed

Boyan, George; Graf, Philip; Ehrhardt, Erica

2018-03-01

We have investigated the pattern of apoptosis in the antennal epithelium during embryonic development of the grasshopper Schistocerca gregaria. The molecular labels lachesin and annulin reveal that the antennal epithelium becomes subdivided into segment-like meristal annuli within which sensory cell clusters later differentiate. To determine whether apoptosis is involved in the development of such sensory cell clusters, we examined the expression pattern of the cell death labels acridine orange and TUNEL in the epithelium. We found stereotypic, age-dependent, wave-like patterns of cell death in the antenna. Early in embryogenesis, apoptosis is restricted to the most basal meristal annuli but subsequently spreads to encompass almost the entire antenna. Cell death then declines in more basal annuli and is only found in the tip region later in embryogenesis. Apoptosis is restricted throughout to the midregion of a given annulus and away from its border with neighboring annuli, arguing against a causal role in annular formation. Double-labeling for cell death and sensory cell differentiation reveals apoptosis occurring within bands of differentiating sensory cell clusters, matching the meristal organization of the apical antenna. Examination of the individual epithelial lineages which generate sensory cells reveals that apoptosis begins peripherally within a lineage and with age expands to encompass the differentiated sensory cell at the base. We conclude that complete lineages can undergo apoptosis and that the youngest cells in these lineages appear to die first, with the sensory neuron dying last. Lineage-based death in combination with cell death patterns in different regions of the antenna may contribute to odor-mediated behaviors in the grasshopper.

Directed Differentiation of Embryonic Stem Cells Using a Bead-Based Combinatorial Screening Method

PubMed Central

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J.; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported. PMID:25251366

Materials for stem cell factories of the future

NASA Astrophysics Data System (ADS)

Celiz, Adam D.; Smith, James G. W.; Langer, Robert; Anderson, Daniel G.; Winkler, David A.; Barrett, David A.; Davies, Martyn C.; Young, Lorraine E.; Denning, Chris; Alexander, Morgan R.

2014-06-01

Polymeric substrates are being identified that could permit translation of human pluripotent stem cells from laboratory-based research to industrial-scale biomedicine. Well-defined materials are required to allow cell banking and to provide the raw material for reproducible differentiation into lineages for large-scale drug-screening programs and clinical use. Yet more than 1 billion cells for each patient are needed to replace losses during heart attack, multiple sclerosis and diabetes. Producing this number of cells is challenging, and a rethink of the current predominant cell-derived substrates is needed to provide technology that can be scaled to meet the needs of millions of patients a year. In this Review, we consider the role of materials discovery, an emerging area of materials chemistry that is in large part driven by the challenges posed by biologists to materials scientists.

Glimpse into Hox and tale regulation of cell differentiation and reprogramming.

PubMed

Cerdá-Esteban, Nuria; Spagnoli, Francesca M

2014-01-01

During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state. Hox genes play a central role in specifying regional identities, thereby providing the cell with critical information on positional value along its differentiation path. The exquisite DNA-binding specificity of the Hox proteins is frequently dependent upon their interaction with members of the TALE family of homeodomain proteins. In addition to their function as Hox-cofactors, TALE homeoproteins control multiple crucial developmental processes through Hox-independent mechanisms. Here, we will review recent findings on the function of both Hox and TALE proteins in cell differentiation, referring mostly to vertebrate species. In addition, we will discuss the direct implications of this knowledge on cell plasticity and cell reprogramming. Copyright © 2013 Wiley Periodicals, Inc.

PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes.

PubMed

Soto-Feliciano, Yadira M; Bartlebaugh, Jordan M E; Liu, Yunpeng; Sánchez-Rivera, Francisco J; Bhutkar, Arjun; Weintraub, Abraham S; Buenrostro, Jason D; Cheng, Christine S; Regev, Aviv; Jacks, Tyler E; Young, Richard A; Hemann, Michael T

2017-05-15

Developmental and lineage plasticity have been observed in numerous malignancies and have been correlated with tumor progression and drug resistance. However, little is known about the molecular mechanisms that enable such plasticity to occur. Here, we describe the function of the plant homeodomain finger protein 6 (PHF6) in leukemia and define its role in regulating chromatin accessibility to lineage-specific transcription factors. We show that loss of Phf6 in B-cell leukemia results in systematic changes in gene expression via alteration of the chromatin landscape at the transcriptional start sites of B-cell- and T-cell-specific factors. Additionally, Phf6 KO cells show significant down-regulation of genes involved in the development and function of normal B cells, show up-regulation of genes involved in T-cell signaling, and give rise to mixed-lineage lymphoma in vivo. Engagement of divergent transcriptional programs results in phenotypic plasticity that leads to altered disease presentation in vivo, tolerance of aberrant oncogenic signaling, and differential sensitivity to frontline and targeted therapies. These findings suggest that active maintenance of a precise chromatin landscape is essential for sustaining proper leukemia cell identity and that loss of a single factor (PHF6) can cause focal changes in chromatin accessibility and nucleosome positioning that render cells susceptible to lineage transition. © 2017 Soto-Feliciano et al.; Published by Cold Spring Harbor Laboratory Press.

Lamin A/C Haploinsufficiency Modulates the Differentiation Potential of Mouse Embryonic Stem Cells

PubMed Central

Sehgal, Poonam; Chaturvedi, Pankaj; Kumaran, R. Ileng; Kumar, Satish; Parnaik, Veena K.

2013-01-01

Background Lamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways. Methodology and Principal Findings We have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by in vitro culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (Lmna). Undifferentiated wild-type and Lmna+/− embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as β-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of Lmna+/− cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with Lmna+/− embryonic stem cells. Conclusions We have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon depletion of lamin A/C. Hence our results implicate lamin A/C level as an important determinant of lineage-specific differentiation during embryonic development. PMID:23451281

The role of lin-22, a hairy/enhancer of split homolog, in patterning the peripheral nervous system of C. elegans.

PubMed

Wrischnik, L A; Kenyon, C J

1997-08-01

In C. elegans, six lateral epidermal stem cells, the seam cells V1-V6, are located in a row along the anterior-posterior (A/P) body axis. Anterior seam cells (V1-V4) undergo a fairly simple sequence of stem cell divisions and generate only epidermal cells. Posterior seam cells (V5 and V6) undergo a more complicated sequence of cell divisions that include additional rounds of stem cell proliferation and the production of neural as well as epidermal cells. In the wild type, activity of the gene lin-22 allows V1-V4 to generate their normal epidermal lineages rather than V5-like lineages. lin-22 activity is also required to prevent additional neurons from being produced by one branch of the V5 lineage. We find that the lin-22 gene exhibits homology to the Drosophila gene hairy, and that lin-22 activity represses neural development within the V5 lineage by blocking expression of the posterior-specific Hox gene mab-5 in specific cells. In addition, in order to prevent anterior V cells from generating V5-like lineages, wild-type lin-22 gene activity must inhibit (directly or indirectly) at least five downstream regulatory gene activities. In anterior body regions, lin-22(+) inhibits expression of the Hox gene mab-5. It also inhibits the activity of the achaete-scute homolog lin-32 and an unidentified gene that we postulate regulates stem cell division. Each of these three genes is required for the expression of a different piece of the ectopic V5-like lineages generated in lin-22 mutants. In addition, lin-22 activity prevents two other Hox genes, lin-39 and egl-5, from acquiring new activities within their normal domains of function along the A/P body axis. Some, but not all, of the patterning activities of lin-22 in C. elegans resemble those of hairy in Drosophila.

Development of Polypeptide-based Nanoparticles for Non-viral Delivery of CD22 RNA Trans-splicing Molecule as a New Precision Medicine Candidate Against B-lineage ALL

PubMed Central

Uckun, Fatih M.; Mitchell, Lloyd G.; Qazi, Sanjive; Liu, Yang; Zheng, Nan; Myers, Dorothea E.; Song, Ziyuan; Ma, Hong; Cheng, Jianjun

2015-01-01

CD22ΔE12 has emerged as a driver lesion in the pathogenesis of pediatric B-lineage acute lymphoblastic leukemia (ALL) and a new molecular target for RNA therapeutics. Here we report a 43-gene CD22ΔE12 signature transcriptome that shows a striking representation in primary human leukemia cells from patients with relapsed BPL. Our data uniquely indicate that CD22ΔE12 is a candidate driver lesion responsible for the activation of MAPK and PI3-K pathways in aggressive forms of B-lineage ALL. We also show that the forced expression of a CD22 RNA trans-splicing molecule (RTM) markedly reduces the capacity of the leukemic stem cell fraction of CD22ΔE12+ B-lineage ALL cells to engraft and cause overt leukemia in NOD/SCID mice. We have successfully complexed our rationally designed lead CD22-RTM with PVBLG-8 to prepare a non-viral nanoscale formulation of CD22ΔE12-RTM with potent anti-cancer activity against CD22ΔE12+ B-lineage leukemia and lymphoma cells. CD22-RTM nanoparticles effectively delivered the CD22-RTM cargo into B-lineage ALL cells and exhibited significant anti-leukemic activity in vitro. PMID:26288837

Development of Polypeptide-based Nanoparticles for Non-viral Delivery of CD22 RNA Trans-splicing Molecule as a New Precision Medicine Candidate Against B-lineage ALL.

PubMed

Uckun, Fatih M; Mitchell, Lloyd G; Qazi, Sanjive; Liu, Yang; Zheng, Nan; Myers, Dorothea E; Song, Ziyuan; Ma, Hong; Cheng, Jianjun

2015-07-01

CD22ΔE12 has emerged as a driver lesion in the pathogenesis of pediatric B-lineage acute lymphoblastic leukemia (ALL) and a new molecular target for RNA therapeutics. Here we report a 43-gene CD22ΔE12 signature transcriptome that shows a striking representation in primary human leukemia cells from patients with relapsed BPL. Our data uniquely indicate that CD22ΔE12 is a candidate driver lesion responsible for the activation of MAPK and PI3-K pathways in aggressive forms of B-lineage ALL. We also show that the forced expression of a CD22 RNA trans-splicing molecule (RTM) markedly reduces the capacity of the leukemic stem cell fraction of CD22ΔE12(+) B-lineage ALL cells to engraft and cause overt leukemia in NOD/SCID mice. We have successfully complexed our rationally designed lead CD22-RTM with PVBLG-8 to prepare a non-viral nanoscale formulation of CD22ΔE12-RTM with potent anti-cancer activity against CD22ΔE12(+) B-lineage leukemia and lymphoma cells. CD22-RTM nanoparticles effectively delivered the CD22-RTM cargo into B-lineage ALL cells and exhibited significant anti-leukemic activity in vitro.

Mouse androgenetic embryonic stem cells differentiated to multiple cell lineages in three embryonic germ layers in vitro.

PubMed

Teramura, Takeshi; Onodera, Yuta; Murakami, Hideki; Ito, Syunsuke; Mihara, Toshihiro; Takehara, Toshiyuki; Kato, Hiromi; Mitani, Tasuku; Anzai, Masayuki; Matsumoto, Kazuya; Saeki, Kazuhiro; Fukuda, Kanji; Sagawa, Norimasa; Osoi, Yoshihiko

2009-06-01

The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible tissues for transplantation.

Ablation of Coactivator Med1 Switches the Cell Fate of Dental Epithelia to That Generating Hair

PubMed Central

Nguyen, Thai; Sakai, Kiyoshi; He, Bing; Fong, Chak; Oda, Yuko

2014-01-01

Cell fates are determined by specific transcriptional programs. Here we provide evidence that the transcriptional coactivator, Mediator 1 (Med1), is essential for the cell fate determination of ectodermal epithelia. Conditional deletion of Med1 in vivo converted dental epithelia into epidermal epithelia, causing defects in enamel organ development while promoting hair formation in the incisors. We identified multiple processes by which hairs are generated in Med1 deficient incisors: 1) dental epithelial stem cells lacking Med 1 fail to commit to the dental lineage, 2) Sox2-expressing stem cells extend into the differentiation zone and remain multi-potent due to reduced Notch1 signaling, and 3) epidermal fate is induced by calcium as demonstrated in dental epithelial cell cultures. These results demonstrate that Med1 is a master regulator in adult stem cells to govern epithelial cell fate. PMID:24949995

Interleukin-7 Availability Is Maintained by a Hematopoietic Cytokine Sink Comprising Innate Lymphoid Cells and T Cells.

PubMed

Martin, Christopher E; Spasova, Darina S; Frimpong-Boateng, Kwesi; Kim, Hee-Ok; Lee, Minji; Kim, Kwang Soon; Surh, Charles D

2017-07-18

Interleukin-7 (IL-7) availability determines the size and proliferative state of the resting T cell pool. However, the mechanisms that regulate steady-state IL-7 amounts are unclear. Using experimental lymphopenic mouse models and IL-7-induced homeostatic proliferation to measure IL-7 availability in vivo, we found that radioresistant cells were the source of IL-7 for both CD4 + and CD8 + T cells. Hematopoietic lineage cells, although irrelevant as a source of IL-7, were primarily responsible for limiting IL-7 availability via their expression of IL-7R. Unexpectedly, innate lymphoid cells were found to have a potent influence on IL-7 amounts in the primary and secondary lymphoid tissues. These results demonstrate that IL-7 homeostasis is achieved through consumption by multiple subsets of innate and adaptive immune cells. Copyright © 2017 Elsevier Inc. All rights reserved.

A comprehensive model of the spatio-temporal stem cell and tissue organisation in the intestinal crypt.

PubMed

Buske, Peter; Galle, Jörg; Barker, Nick; Aust, Gabriela; Clevers, Hans; Loeffler, Markus

2011-01-06

We introduce a novel dynamic model of stem cell and tissue organisation in murine intestinal crypts. Integrating the molecular, cellular and tissue level of description, this model links a broad spectrum of experimental observations encompassing spatially confined cell proliferation, directed cell migration, multiple cell lineage decisions and clonal competition.Using computational simulations we demonstrate that the model is capable of quantitatively describing and predicting the dynamic behaviour of the intestinal tissue during steady state as well as after cell damage and following selective gain or loss of gene function manipulations affecting Wnt- and Notch-signalling. Our simulation results suggest that reversibility and flexibility of cellular decisions are key elements of robust tissue organisation of the intestine. We predict that the tissue should be able to fully recover after complete elimination of cellular subpopulations including subpopulations deemed to be functional stem cells. This challenges current views of tissue stem cell organisation.

RNAi as a Routine Route Toward Breast Cancer Therapy

DTIC Science & Technology

2014-05-01

hematopoietic stem/ progenitor cells (HSPCs) and mature cells from the myeloid and lymphoid lineages. Hypomethylated regions (HMRs) associated with...Hematopoietic Cells (A and B) Genome browser tracks depict methylation profiles across a lymphoid (A) and myeloid (B) specific locus in blood cells ...multipotent populations, and two derived, mature cell types from the lymphoid and myeloid lineages, respectively. For comparison, we generated methylomes

VpreB gene expression in hematopoietic malignancies: a lineage- and stage-restricted marker for B-cell precursor leukemias.

PubMed

Bauer, S R; Kubagawa, H; Maclennan, I; Melchers, F

1991-09-15

We show here that analysis of VpreB gene transcription can be a specific way to identify acute leukemias of cells at very early stages of B-cell development. Northern blot analysis of RNAs from 63 leukemia samples showed that VpreB RNA was present in malignancies of precursor B cells, the expression being a feature of both common acute lymphoblastic leukemia (ALL) (CD10+) and null ALL (CD10-). It was absent from malignancies of mature B cells (surface Ig positive), from acute leukemias of the T-cell lineage and granulocyte-macrophage lineages, and from normal tonsil B and T lymphocytes. Chronic myeloid leukemia blast crises of the B-precursor-cell type expressed the VpreB gene while myeloid blast crises did not. VpreB RNA was also expressed in the neoplastic cells of one of three patients with acute undifferentiated leukemias. These data show that VpreB RNA expression is a marker of the malignant forms of precursor B cells, and that it appears at least as early as cytoplasmic CD22 and CD19 in tumors of the B-cell lineage.

Redundant miR-3077-5p and miR-705 mediate the shift of mesenchymal stem cell lineage commitment to adipocyte in osteoporosis bone marrow.

PubMed

Liao, L; Yang, X; Su, X; Hu, C; Zhu, X; Yang, N; Chen, X; Shi, S; Shi, S; Jin, Y

2013-04-18

During the process of aging, especially for postmenopausal females, the cell lineage commitment of mesenchymal stem cells (MSCs) shift to adipocyte in bone marrow, resulting in osteoporosis. However, the cell-intrinsic mechanism of this cell lineage commitment switch is poorly understood. As the post-transcription regulation by microRNAs (miRNAs) has a critical role in MSCs differentiation and bone homeostasis, we performed comprehensive miRNAs profiling and found miR-705 and miR-3077-5p were significantly enhanced in MSCs from osteoporosis bone marrow. Both miR-705 and miR-3077-5p acted as inhibitors of MSCs osteoblast differentiation and promoters of adipocyte differentiation, by targeting on the 3'untranslated region (3'UTR) of HOXA10 and RUNX2 mRNA separately. Combined inhibition of miR-705 and miR-3077-5p rescued the cell lineage commitment disorder of MSCs through restoring HOXA10 and RUNX2 protein level. Furthermore, we found excessive TNFα and reactive oxygen species caused by estrogen deficiency led to the upregulation of both miRNAs through NF-κB pathway. In conclusion, our findings showed that redundant miR-705 and miR-3077-5p synergistically mediated the shift of MSCs cell lineage commitment to adipocyte in osteoporosis bone marrow, providing new insight into the etiology of osteoporosis at the post-transcriptional level. Moreover, the rescue of MSCs lineage commitment disorder by regulating miRNAs expression suggested a novel potential therapeutic target for osteoporosis as well as stem cell-mediated regenerative medicine.

Fgf10-positive cells represent a progenitor cell population during lung development and postnatally

PubMed Central

El Agha, Elie; Herold, Susanne; Alam, Denise Al; Quantius, Jennifer; MacKenzie, BreAnne; Carraro, Gianni; Moiseenko, Alena; Chao, Cho-Ming; Minoo, Parviz; Seeger, Werner; Bellusci, Saverio

2014-01-01

The lung mesenchyme consists of a widely heterogeneous population of cells that play crucial roles during development and homeostasis after birth. These cells belong to myogenic, adipogenic, chondrogenic, neuronal and other lineages. Yet, no clear hierarchy for these lineages has been established. We have previously generated a novel Fgf10iCre knock-in mouse line that allows lineage tracing of Fgf10-positive cells during development and postnatally. Using these mice, we hereby demonstrate the presence of two waves of Fgf10 expression during embryonic lung development: the first wave, comprising Fgf10-positive cells residing in the submesothelial mesenchyme at early pseudoglandular stage (as well as their descendants); and the second wave, comprising Fgf10-positive cells from late pseudoglandular stage (as well as their descendants). Our lineage-tracing data reveal that the first wave contributes to the formation of parabronchial and vascular smooth muscle cells as well as lipofibroblasts at later developmental stages, whereas the second wave does not give rise to smooth muscle cells but to lipofibroblasts as well as an Nkx2.1- E-Cad- Epcam+ Pro-Spc+ lineage that requires further in-depth analysis. During alveologenesis, Fgf10-positive cells give rise to lipofibroblasts rather than alveolar myofibroblasts, and during adult life, a subpopulation of Fgf10-expressing cells represents a pool of resident mesenchymal stromal (stem) cells (MSCs) (Cd45- Cd31- Sca-1+). Taken together, we show for the first time that Fgf10-expressing cells represent a pool of mesenchymal progenitors in the embryonic and postnatal lung. Our findings suggest that Fgf10-positive cells could be useful for developing stem cell-based therapies for treating interstitial lung diseases. PMID:24353064

Nuclei of Tsuga canadensis: Role of Flavanols in Chromatin Organization

PubMed Central

Feucht, Walter; Schmid, Markus; Treutter, Dieter

2011-01-01

Needle primordia of Tsuga canadensis (hemlock) arising from flank meristems of a shoot apex, form cell lineages consisting of four or eight cells. Within a recently established lineage there is striking uniformity in the pattern of nuclear flavanols. This fact points to an identical transcriptional expression of these flavanols during cell cycling. However two lineages, even if located close together within the same meristem, can be very different in the expression of both cell shape and nuclear flavanol pattern, indicating that epigenetic positional signals are operating in a collective specification of cell lineage development. There is a wide range of nuclear flavanol patterning from a mosaic-like distribution in an activated cell type to a homogenous appearance in silenced cell types. Single cells deriving from lineages are desynchronized because they underlie a signaling network at a higher tissue level which results in stronger epigenetic modifications of their nuclear flavanols. As an extreme case of epigenetic modulation, transient drought conditions caused a drastic reduction of nuclear flavanols. Upon treatment with sucrose or cytokinin, these nuclear flavanols could be fully restored. Analytical determination of the flavanols revealed 3.4 mg/g DW for newly sprouting needles and 19.6 mg/g DW for anthers during meiosis. The roughly 6-fold difference in flavanols is apparently a reflection of the highly diverging organogenetic processes. Collectively, the studies provide strong evidence for combinatorial interplay between cell fate and nuclear flavanols. PMID:22072922

Wnt and FGF signals interact to coordinate growth with cell fate specification during limb development.

PubMed

ten Berge, Derk; Brugmann, Samantha A; Helms, Jill A; Nusse, Roel

2008-10-01

A fundamental question in developmental biology is how does an undifferentiated field of cells acquire spatial pattern and undergo coordinated differentiation? The development of the vertebrate limb is an important paradigm for understanding these processes. The skeletal and connective tissues of the developing limb all derive from a population of multipotent progenitor cells located in its distal tip. During limb outgrowth, these progenitors segregate into a chondrogenic lineage, located in the center of the limb bud, and soft connective tissue lineages located in its periphery. We report that the interplay of two families of signaling proteins, fibroblast growth factors (FGFs) and Wnts, coordinate the growth of the multipotent progenitor cells with their simultaneous segregation into these lineages. FGF and Wnt signals act together to synergistically promote proliferation while maintaining the cells in an undifferentiated, multipotent state, but act separately to determine cell lineage specification. Withdrawal of both signals results in cell cycle withdrawal and chondrogenic differentiation. Continued exposure to Wnt, however, maintains proliferation and re-specifies the cells towards the soft connective tissue lineages. We have identified target genes that are synergistically regulated by Wnts and FGFs, and show how these factors actively suppress differentiation and promote growth. Finally, we show how the spatial restriction of Wnt and FGF signals to the limb ectoderm, and to a specialized region of it, the apical ectodermal ridge, controls the distribution of cell behaviors within the growing limb, and guides the proper spatial organization of the differentiating tissues.

Lineage-negative Progenitors Mobilize to Regenerate Lung Epithelium after Major Injury

PubMed Central

Vaughan, Andrew E.; Brumwell, Alexis N.; Xi, Ying; Gotts, Jeffrey; Brownfield, Doug G.; Treutlein, Barbara; Tan, Kevin; Tan, Victor; Liu, Fengchun; Looney, Mark R.; Matthay, Michael; Rock, Jason R.; Chapman, Harold A.

2014-01-01

Broadly, tissue regeneration is achieved in two ways: by proliferation of common differentiated cells and/or by deployment of specialized stem/progenitor cells. Which of these pathways applies is both organ and injury-specific1–4. Current paradigms in the lung posit that epithelial repair can be attributed to cells expressing mature lineage markers5–8. In contrast we here define the regenerative role of previously uncharacterized, rare lineage-negative epithelial stem/progenitor (LNEPs) cells present within normal distal lung. Quiescent LNEPs activate a ΔNp63/cytokeratin 5 (Krt5+) remodeling program after influenza or bleomycin injury. Activated cells proliferate and migrate widely to occupy heavily injured areas depleted of mature lineages, whereupon they differentiate toward mature epithelium. Lineage tracing revealed scant contribution of pre-existing mature epithelial cells in such repair, whereas orthotopic transplantation of LNEPs, isolated by a definitive surface profile identified through single cell sequencing, directly demonstrated the proliferative capacity and multipotency of this population. LNEPs require Notch signaling to activate the ΔNp63/Krt5+ program whereas subsequent Notch blockade promotes an alveolar cell fate. Persistent Notch signaling post-injury led to parenchymal micro-honeycombing, indicative of failed regeneration. Lungs from fibrosis patients show analogous honeycomb cysts with evidence of hyperactive Notch signaling. Our findings indicate distinct stem/progenitor cell pools repopulate injured tissue depending on the extent of injury, and the outcomes of regeneration or fibrosis may ride in part on the dynamics of LNEP Notch signaling. PMID:25533958

Overexpression of Trophoblast Stem Cell-Enriched MicroRNAs Promotes Trophoblast Fate in Embryonic Stem Cells.

PubMed

Nosi, Ursula; Lanner, Fredrik; Huang, Tsu; Cox, Brian

2017-05-09

The first cell fate choice of the preimplantation embryo generates the extraembryonic trophoblast and embryonic epiblast lineages. Embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) can be utilized to investigate molecular mechanisms of this first cell fate decision. It has been established that ESCs can be induced to acquire trophoblast lineage characteristics upon manipulation of lineage-determining transcription factors. Here, we have interrogated the potential of microRNAs (miRNAs) to drive trans-differentiation of ESCs into the trophoblast lineage. Analysis of gene expression data identified a network of TSC-enriched miRNAs that were predicted to target mRNAs enriched in ESCs. Ectopic expression of these miRNAs in ESCs resulted in a stable trophoblast phenotype, supported by gene expression changes and in vivo contribution potential. This process is highly miRNA-specific and dependent on Hdac2 inhibition. Our experimental evidence suggests that these miRNAs promote a mural trophectoderm (TE)-like cell fate with physiological properties that differentiate them from the polar TE. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Stepwise renal lineage differentiation of mouse embryonic stem cells tracing in vivo development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishikawa, Masaki, E-mail: masakiwestriver@gmail.com; University of California at Los Angeles, David Geffen School of Medicine, Los Angeles, CA 91343; Yanagawa, Naomi

2012-01-13

Highlights: Black-Right-Pointing-Pointer We induced renal lineages from mESCs by following the in vivo developmental cues. Black-Right-Pointing-Pointer We induced nephrogenic intermediate mesoderm by stepwise addition of factors. Black-Right-Pointing-Pointer We induced two types of renal progenitor cells by reciprocal conditioned media. Black-Right-Pointing-Pointer We propose the potential role of CD24 for the enrichment of renal lineage cells. -- Abstract: The in vitro derivation of renal lineage progenitor cells is essential for renal cell therapy and regeneration. Despite extensive studies in the past, a protocol for renal lineage induction from embryonic stem cells remains unestablished. In this study, we aimed to induce renal lineagesmore » from mouse embryonic stem cells (mESC) by following in vivo developmental stages, i.e., the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For stage I induction, in accordance with known signaling pathways involved in mesoderm development in vivo, i.e., Nodal, bone morphogenic proteins (BMPs) and Wnt, we found that the sequential addition of three factors, i.e., Activin-A (A), a surrogate for Nodal signaling, during days 0-2, A plus BMP-4 (4) during days 2-4, and A4 plus lithium (L), a surrogate for Wnt signaling, during days 4-6, was most effective to induce the mesodermal marker, Brachyury. For stage II induction, the addition of retinoic acid (R) in the continuous presence of A4L during days 6-8 was most effective to induce nephrogenic intermediate mesodermal markers, such as Pax2 and Lim1. Under this condition, more than 30% of cells were stained positive for Pax2, and there was a concomitant decrease in the expression of non-mesodermal markers. For stage III induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, we found that the exposure to conditioned media derived from UB and MM cells was effective in inducing MM and UB markers, respectively. We also observed the emergence and gradual increase of cell populations expressing progenitor cell marker CD24 from Stage I to Stage III. These CD24{sup +} cells correlated with higher levels of expression of Brachyury at stage I, Pax2 and Lim1 at stage II and MM markers, such as WT1 and Cadherin 11, after exposure to UB-conditioned media at stage III. In conclusion, our results show that stepwise induction by tracing in vivo developmental stages was effective to generate renal lineage progenitor cells from mESC, and CD24 may serve as a useful surface marker for renal lineage cells at stage II and MM cells at stage III.« less

Persistence of Multiple Genetic Lineages within Intrahost Populations of Ross River Virus▿

PubMed Central

Liu, Wen J.; Rourke, Michelle F.; Holmes, Edward C.; Aaskov, John G.

2011-01-01

We examined the structure and extent of genetic diversity in intrahost populations of Ross River virus (RRV) in samples from six human patients, focusing on the nonstructural (nsP3) and structural (E2) protein genes. Strikingly, although the samples were collected from contrasting ecological settings 3,000 kilometers apart in Australia, we observed multiple viral lineages in four of the six individuals, which is indicative of widespread mixed infections. In addition, a comparison with previously published RRV sequences revealed that these distinct lineages have been in circulation for at least 5 years, and we were able to document their long-term persistence over extensive geographical distances. PMID:21430052

Hacking cell differentiation: transcriptional rerouting in reprogramming, lineage infidelity and metaplasia

PubMed Central

Regalo, Gonçalo; Leutz, Achim

2013-01-01

Initiating neoplastic cell transformation events are of paramount importance for the comprehension of regeneration and vanguard oncogenic processes but are difficult to characterize and frequently clinically overlooked. In epithelia, pre-neoplastic transformation stages are often distinguished by the appearance of phenotypic features of another differentiated tissue, termed metaplasia. In haemato/lymphopoietic malignancies, cell lineage ambiguity is increasingly recorded. Both, metaplasia and biphenotypic leukaemia/lymphoma represent examples of dysregulated cell differentiation that reflect a history of trans-differentiation and/or epigenetic reprogramming. Here we compare the similarity between molecular events of experimental cell trans-differentiation as an emerging therapeutic concept, with lineage confusion, as in metaplasia and dysplasia forecasting tumour development. PMID:23828660

Fitness cost of reassortment in human influenza.

PubMed

Villa, Mara; Lässig, Michael

2017-11-01

Reassortment, which is the exchange of genome sequence between viruses co-infecting a host cell, plays an important role in the evolution of segmented viruses. In the human influenza virus, reassortment happens most frequently between co-existing variants within the same lineage. This process breaks genetic linkage and fitness correlations between viral genome segments, but the resulting net effect on viral fitness has remained unclear. In this paper, we determine rate and average selective effect of reassortment processes in the human influenza lineage A/H3N2. For the surface proteins hemagglutinin and neuraminidase, reassortant variants with a mean distance of at least 3 nucleotides to their parent strains get established at a rate of about 10-2 in units of the neutral point mutation rate. Our inference is based on a new method to map reassortment events from joint genealogies of multiple genome segments, which is tested by extensive simulations. We show that intra-lineage reassortment processes are, on average, under substantial negative selection that increases in strength with increasing sequence distance between the parent strains. The deleterious effects of reassortment manifest themselves in two ways: there are fewer reassortment events than expected from a null model of neutral reassortment, and reassortant strains have fewer descendants than their non-reassortant counterparts. Our results suggest that influenza evolves under ubiquitous epistasis across proteins, which produces fitness barriers against reassortment even between co-circulating strains within one lineage.

Lineage analysis of quiescent regenerative stem cells in the adult brain by genetic labelling reveals spatially restricted neurogenic niches in the olfactory bulb.

PubMed

Giachino, Claudio; Taylor, Verdon

2009-07-01

The subventricular zone (SVZ) of the lateral ventricles is the major neurogenic region in the adult mammalian brain, harbouring neural stem cells within defined niches. The identity of these stem cells and the factors regulating their fate are poorly understood. We have genetically mapped a population of Nestin-expressing cells during postnatal development to study their potential and fate in vivo. Taking advantage of the recombination characteristics of a nestin::CreER(T2) allele, we followed a subpopulation of neural stem cells and traced their fate in a largely unrecombined neurogenic niche. Perinatal nestin::CreER(T2)-expressing cells give rise to multiple glial cell types and neurons, as well as to stem cells of the adult SVZ. In the adult SVZ nestin::CreER(T2)-expressing neural stem cells give rise to several neuronal subtypes in the olfactory bulb (OB). We addressed whether the same population of neural stem cells play a role in SVZ regeneration. Following anti-mitotic treatment to eliminate rapidly dividing progenitors, relatively quiescent nestin::CreER(T2)-targeted cells are spared and contribute to SVZ regeneration, generating new proliferating precursors and neuroblasts. Finally, we have identified neurogenic progenitors clustered in ependymal-like niches within the rostral migratory stream (RMS) of the OB. These OB-RMS progenitors generate neuroblasts that, upon transplantation, graft, migrate and differentiate into granule and glomerular neurons. In summary, using conditional lineage tracing we have identified neonatal cells that are the source of neurogenic and regenerative neural stem cells in the adult SVZ and occupy a novel neurogenic niche in the OB.

Mixed Lineage Kinases as Novel Targets for the Treatment of Endocrine-Resistant, ER-Positive Breast Cancer

DTIC Science & Technology

2016-06-01

a small molecule inhibitor of Mixed Lineage Kinases (MLKs) induces a cell cycle arrest and apoptosis in estrogen receptor (ER) - positive breast...breast cancer, mixed lineage kinases , patient derived xenografts, kinase inhibitor , pre-clinical models 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF...very successful, resistance to these treatments is a serious clinical problem. We previously demonstrated in cell culture that a small molecule

Quantitative Raman spectral changes of the differentiation of mesenchymal stem cells into islet-like cells by biochemical component analysis and multiple peak fitting

NASA Astrophysics Data System (ADS)

Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; He, Yingtian; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

2015-12-01

Mesenchymal stem cells (MSCs) differentiate into islet-like cells, providing a possible solution for type I diabetes treatment. To search for the precise molecular mechanism of the directional differentiation of MSC-derived islet-like cells, biomolecular composition, and structural conformation information during MSC differentiation, is required. Because islet-like cells lack specific surface markers, the commonly employed immunostaining technique is not suitable for their identification, physical separation, and enrichment. Combining Raman spectroscopic data, a fitting accuracy-improved biochemical component analysis, and multiple peaks fitting approach, we identified the quantitative biochemical and intensity change of Raman peaks that show the differentiation of MSCs into islet-like cells. Along with increases in protein and glycogen content, and decreases in deoxyribonucleic acid and ribonucleic acid content, in islet-like cells relative to MSCs, it was found that a characteristic peak of insulin (665 cm-1) has twice the intensity in islet-like cells relative to MSCs, indicating differentiation of MSCs into islet-like cells was successful. Importantly, these Raman signatures provide useful information on the structural and pathological states during MSC differentiation and help to develop noninvasive and label-free Raman sorting methods for stem cells and their lineages.

A post-transcriptional mechanism pacing expression of neural genes with precursor cell differentiation status.

PubMed

Dai, Weijun; Li, Wencheng; Hoque, Mainul; Li, Zhuyun; Tian, Bin; Makeyev, Eugene V

2015-07-06

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3'-untranslated regions (3' UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36), an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly, TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand, inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons.

Requirement for sustained MAPK signaling in both CD4 and CD8 lineage commitment: a threshold model.

PubMed

Wilkinson, B; Kaye, J

2001-08-01

Although there is general agreement that the RAS/MAPK signaling pathway is required for positive selection of CD4 T cells in the thymus, the role of this pathway in CD8 lineage commitment remains controversial. We show here that the differentiation of isolated cultured thymocytes to the CD8 as well as CD4 T cell lineage is sensitive to MEK inhibition and that both CD4 and CD8 thymocyte differentiation requires sustained MEK signaling. However, CD4 lineage commitment is promoted by a stronger stimulus for longer duration than required for CD8 lineage commitment. Interestingly, CD4 lineage commitment is not irreversibly set even after 10 h of signaling, well past early changes in gene expression. These findings are presented in the context of a model of lineage commitment in which a default pathway of CD8 lineage commitment is altered to CD4 commitment if the thymocyte achieves a threshold level of active MAPK within a certain time frame. Copyright 2001 Academic Press.

Viral immunogenicity determines epidemiological fitness in a cohort of DENV-1 infection in Brazil.

PubMed

Pinheiro, Tauyne Menegaldo; Mota, Mânlio Tasso de Oliveira; Watanabe, Aripuanã Sakurada Aranha; Biselli-Périco, Joice Matos; Drumond, Betânia Paiva; Ribeiro, Milene Rocha; Vedovello, Danila; Araújo, João Pessoa; Pimenta, Paulo Filemon Paolucci; Chaves, Bárbara Aparecida; Silva, Mayara Marques Carneiro da; Batista, Izabella Cristina Andrade; Papa, Michelle Premazzi; Meuren, Lana Monteiro; Lucas, Carolina Gonçalves de Oliveira; Matassoli, Flavio Lemos; Gil, Laura Helena Vega Gonzales; Bozzi, Adriana; Calzavara-Silva, Carlos Eduardo; Arruda, Luciana Barros de; Souza, Danielle da Glória de; Teixeira, Mauro Martins; Vasilakis, Nikos; Nogueira, Maurício Lacerda

2018-05-01

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto.

Viral immunogenicity determines epidemiological fitness in a cohort of DENV-1 infection in Brazil

PubMed Central

Pinheiro, Tauyne Menegaldo; Mota, Mânlio Tasso de Oliveira; Biselli-Périco, Joice Matos; Ribeiro, Milene Rocha; Araújo, João Pessoa; Pimenta, Paulo Filemon Paolucci; da Silva, Mayara Marques Carneiro; Batista, Izabella Cristina Andrade; Meuren, Lana Monteiro; Lucas, Carolina Gonçalves de Oliveira; Matassoli, Flavio Lemos; Gil, Laura Helena Vega Gonzales; Bozzi, Adriana; Calzavara-Silva, Carlos Eduardo; de Souza, Danielle da Glória; Teixeira, Mauro Martins; Vasilakis, Nikos

2018-01-01

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto. PMID:29813061

BONE MARROW MESENCHYMAL STEM CELLS ARE PROGENITORS IN VITRO FOR INNER EAR HAIR CELLS

PubMed Central

Jeon, Sang-Jun; Oshima, Kazuo; Heller, Stefan; Edge, Albert S.B.

2011-01-01

Stem cells have been demonstrated in the inner ear but they do not spontaneously divide to replace damaged sensory cells. Mesenchymal stem cells (MSC) from bone marrow have been reported to differentiate into multiple lineages including neurons, and we therefore asked whether MSCs could generate sensory cells. Overexpression of the prosensory transcription factor, Math1, in sensory epithelial precursor cells induced expression of myosin VIIa, espin, Brn3c, p27Kip, and jagged2, indicating differentiation to inner ear sensory cells. Some of the cells displayed F-actin positive protrusions in the morphology characteristic of hair cell stereociliary bundles. Hair cell markers were also induced by culture of mouse MSC-derived cells in contact with embryonic chick inner ear cells, and this induction was not due to a cell fusion event, because the chick hair cells could be identified with a chick-specific antibody and chick and mouse antigens were never found in the same cell. PMID:17113786

Mixed - Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states.

PubMed

Craige, Siobhan M; Reif, Michaella M; Kant, Shashi

2016-09-01

Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival.

PubMed

Tinkum, Kelsey L; Stemler, Kristina M; White, Lynn S; Loza, Andrew J; Jeter-Jones, Sabrina; Michalski, Basia M; Kuzmicki, Catherine; Pless, Robert; Stappenbeck, Thaddeus S; Piwnica-Worms, David; Piwnica-Worms, Helen

2015-12-22

Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.

Reconstructing Cell Lineages from Single-Cell Gene Expression Data: A Pilot Study

DTIC Science & Technology

2016-08-30

Reconstructing cell lineages from single- cell gene expression data: a pilot study The goal of this pilot study is to develop novel mathematical...methods, by leveraging tools developed in the bifurcation theory, to infer the underlying cell -state dynamics from single- cell gene expression data. Our...proposed method contains two steps. The first step is to reconstruct the temporal order of the cells from gene expression data, whereas the second

Development, differentiation and diversity of innate lymphoid cells

PubMed Central

Diefenbach, Andreas; Colonna, Marco; Koyasu, Shigeo

2014-01-01

Recent years have witnessed the discovery of an unprecedented complexity in innate lymphocyte lineages, now collectively referred to as innate lymphoid cells (ILC). ILC are preferentially located at barrier surfaces and are important for protection against pathogens and for the maintenance of organ homeostasis. Inappropriate activation of ILC has been linked to the pathogenesis of inflammatory and autoimmune disorders. Recent evidence suggests that ILC can be grouped into two separate lineages, cytotoxic ILC represented by conventional natural killer (cNK) cells and cytokine-producing helper-like ILC (i.e., ILC1, ILC2, ILC3). We will focus here on current work in humans and mice that has identified core transcriptional circuitry required for the commitment of lymphoid progenitors to the ILC lineage. The striking similarities in transcriptional control of ILC and T cell lineages reveal important insights into the evolution of transcriptional programs required to protect multicellular organisms against infections and to fortify barrier surfaces. PMID:25238093

Multigenerational memory and adaptive adhesion in early bacterial biofilm communities.

PubMed

Lee, Calvin K; de Anda, Jaime; Baker, Amy E; Bennett, Rachel R; Luo, Yun; Lee, Ernest Y; Keefe, Joshua A; Helali, Joshua S; Ma, Jie; Zhao, Kun; Golestanian, Ramin; O'Toole, George A; Wong, Gerard C L

2018-04-24

Using multigenerational, single-cell tracking we explore the earliest events of biofilm formation by Pseudomonas aeruginosa During initial stages of surface engagement (≤20 h), the surface cell population of this microbe comprises overwhelmingly cells that attach poorly (∼95% stay <30 s, well below the ∼1-h division time) with little increase in surface population. If we harvest cells previously exposed to a surface and direct them to a virgin surface, we find that these surface-exposed cells and their descendants attach strongly and then rapidly increase the surface cell population. This "adaptive," time-delayed adhesion requires determinants we showed previously are critical for surface sensing: type IV pili (TFP) and cAMP signaling via the Pil-Chp-TFP system. We show that these surface-adapted cells exhibit damped, coupled out-of-phase oscillations of intracellular cAMP levels and associated TFP activity that persist for multiple generations, whereas surface-naïve cells show uncorrelated cAMP and TFP activity. These correlated cAMP-TFP oscillations, which effectively impart intergenerational memory to cells in a lineage, can be understood in terms of a Turing stochastic model based on the Pil-Chp-TFP framework. Importantly, these cAMP-TFP oscillations create a state characterized by a suppression of TFP motility coordinated across entire lineages and lead to a drastic increase in the number of surface-associated cells with near-zero translational motion. The appearance of this surface-adapted state, which can serve to define the historical classification of "irreversibly attached" cells, correlates with family tree architectures that facilitate exponential increases in surface cell populations necessary for biofilm formation.

Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chow, Paik Wah; Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur; Abdul Hamid, Zariyantey, E-mail: zyantey@ukm.edu.my

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24 h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-formingmore » unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p < 0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e{sup +} cells but reduced the total counts of Sca-1{sup +}, CD11b{sup +}, Gr-1{sup +}, and CD45{sup +} cells at 7 and 12 μM (p < 0.05). Furthermore, the CFU assay showed reduced (p < 0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5 μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12 μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage. - Highlights: • We examine 1,4-BQ toxicity targeting mouse hematopoietic cell lineages. • 1,4-BQ induces concentration-dependent cytotoxicity in bone marrow (BM) cells. • 1,4-BQ shows lineage-related toxicity on hematopoietic stem and progenitors. • 1,4-BQ toxicity is greater in single- than multilineage committed progenitors.« less

Epigenetic Marks Define the Lineage and Differentiation Potential of Two Distinct Neural Crest-Derived Intermediate Odontogenic Progenitor Populations

PubMed Central

Gopinathan, Gokul; Kolokythas, Antonia

2013-01-01

Epigenetic mechanisms, such as histone modifications, play an active role in the differentiation and lineage commitment of mesenchymal stem cells. In the present study, epigenetic states and differentiation profiles of two odontogenic neural crest-derived intermediate progenitor populations were compared: dental pulp (DP) and dental follicle (DF). ChIP on chip assays revealed substantial H3K27me3-mediated repression of odontoblast lineage genes DSPP and dentin matrix protein 1 (DMP1) in DF cells, but not in DP cells. Mineralization inductive conditions caused steep increases of mineralization and patterning gene expression levels in DP cells when compared to DF cells. In contrast, mineralization induction resulted in a highly dynamic histone modification response in DF cells, while there was only a subdued effect in DP cells. Both DF and DP progenitors featured H3K4me3-active marks on the promoters of early mineralization genes RUNX2, MSX2, and DLX5, while OSX, IBSP, and BGLAP promoters were enriched for H3K9me3 or H3K27me3. Compared to DF cells, DP cells expressed higher levels of three pluripotency-associated genes, OCT4, NANOG, and SOX2. Finally, gene ontology comparison of bivalent marks unique for DP and DF cells highlighted cell–cell attachment genes in DP cells and neurogenesis genes in DF cells. In conclusion, the present study indicates that the DF intermediate odontogenic neural crest lineage is distinguished from its DP counterpart by epigenetic repression of DSPP and DMP1 genes and through dynamic histone enrichment responses to mineralization induction. Findings presented here highlight the crucial role of epigenetic regulatory mechanisms in the terminal differentiation of odontogenic neural crest lineages. PMID:23379639

The Modern Primitives: Applying New Technological Approaches to Explore the Biology of the Earliest Red Blood Cells

PubMed Central

Fraser, Stuart T.

2013-01-01

One of the most critical stages in mammalian embryogenesis is the independent production of the embryo's own circulating, functional red blood cells. Correspondingly, erythrocytes are the first cell type to become functionally mature during embryogenesis. Failure to achieve this invariably leads to in utero lethality. The recent application of technologies such as transcriptome analysis, flow cytometry, mutant embryo analysis, and transgenic fluorescent gene expression reporter systems has shed new light on the distinct erythroid lineages that arise early in development. Here, I will describe the similarities and differences between the distinct erythroid populations that must form for the embryo to survive. While much of the focus of this review will be the poorly understood primitive erythroid lineage, a discussion of other erythroid and hematopoietic lineages, as well as the cell types making up the different niches that give rise to these lineages, is essential for presenting an appropriate developmental context of these cells. PMID:24222861

Notch-dependent T-lineage commitment occurs at extrathymic sites following bone marrow transplantation

PubMed Central

Maillard, Ivan; Schwarz, Benjamin A.; Sambandam, Arivazhagan; Fang, Terry; Shestova, Olga; Xu, Lanwei; Bhandoola, Avinash; Pear, Warren S.

2006-01-01

Early T-lineage progenitors (ETPs) arise after colonization of the thymus by multipotent bone marrow progenitors. ETPs likely serve as physiologic progenitors of T-cell development in adult mice, although alternative T-cell differentiation pathways may exist. While we were investigating mechanisms of T-cell reconstitution after bone marrow transplantation (BMT), we found that efficient donor-derived thymopoiesis occurred before the pool of ETPs had been replenished. Simultaneously, T lineage–restricted progenitors were generated at extrathymic sites, both in the spleen and in peripheral lymph nodes, but not in the bone marrow or liver. The generation of these T lineage–committed cells occurred through a Notch-dependent differentiation process. Multipotent bone marrow progenitors efficiently gave rise to extrathymic T lineage–committed cells, whereas common lymphoid progenitors did not. Our data show plasticity of T-lineage commitment sites in the post-BMT environment and indicate that Notch-driven extrathymic Tlineage commitment from multipotent progenitors may contribute to early T-lineage reconstitution after BMT. PMID:16397133

Molecular changes associated with heat-shock treatment in avian mononuclear and lymphoid lineage cells.

PubMed

Miller, L; Qureshi, M A

1992-03-01

The induction of heat-shock protein (HSP) synthesis in avian cells of the mononuclear phagocytic system (MPS) and lymphoid system (LS) lineage was investigated by exposure to in vitro heat-shock conditions. In addition, the kinetics of HSP90 mRNA expression was examined in chicken peritoneal macrophages (PM) as well as heat-shock-induced HSP synthesis in PM from chickens, turkeys, quail, and ducks. Each MPS and LS cell type expressed three major (23, 70, and 90 kDa) HSP following a 1-h heat shock at 45 C. However, a unique heat-induced 32-kDa protein (P32) was expressed only by cells of MPS lineage. The expression of HSP90 mRNA in chicken PM was temperature- and time-dependent. These findings imply that avian PM undergo molecular changes in response to elevated environmental temperatures and that the pattern of HSP expression appears to be distinct for cells of the MPS and LS lineages in chickens.

Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics.

PubMed

Martinovich, Kelly M; Iosifidis, Thomas; Buckley, Alysia G; Looi, Kevin; Ling, Kak-Ming; Sutanto, Erika N; Kicic-Starcevich, Elizabeth; Garratt, Luke W; Shaw, Nicole C; Montgomery, Samuel; Lannigan, Francis J; Knight, Darryl A; Kicic, Anthony; Stick, Stephen M

2017-12-21

Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range.

Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology.

PubMed

Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

2016-03-17

Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated.

Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujimura, Juri; Department of Pediatrics, Nippon Medical School, Tokyo; E-mail: juri-f@nms.ac.jp

2005-07-22

Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have recently reported that adipose-derived stromal cells (ASCs) could differentiate into mesenchymal lineages in vitro. In this study, we performed neural induction using ASCs from GFP transgenic mice and were able to induce these ASCs into neuronal and glial cell lineages. Most of the neurally induced cells showed bipolar or multipolar appearance morphologically and expressed neuronal markers. Electron microscopy revealed their neuronal morphology. Some cells also showed glial phenotypes, as shown immunocytochemically. The present study clearly shows that ASCs derived from GFP transgenic mice differentiate intomore » neural lineages in vitro, suggesting that these cells might provide an ideal source for further neural stem cell research with possible therapeutic application for neurological disorders.« less

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Patient-specific cardiovascular progenitor cells derived from integration-free induced pluripotent stem cells for vascular tissue regeneration.

PubMed

Hu, Jiang; Wang, Yongyu; Jiao, Jiao; Liu, Zhongning; Zhao, Chao; Zhou, Zhou; Zhang, Zhanpeng; Forde, Kaitlynn; Wang, Lunchang; Wang, Jiangang; Baylink, David J; Zhang, Xiao-Bing; Gao, Shaorong; Yang, Bo; Chen, Y Eugene; Ma, Peter X

2015-12-01

Tissue-engineered blood vessels (TEBVs) are promising in regenerating a live vascular replacement. However, the vascular cell source is limited, and it is crucial to develop a scaffold that accommodates new type of vascular progenitor cells and facilitates in vivo lineage specification of the cells into functional vascular smooth muscle cells (VSMCs) to regenerate vascular tissue. In the present study, integration-free human induced pluripotent stem cells (hiPSCs) were established from patient peripheral blood mononuclear cells through episomal vector nucleofection of reprogramming factors. The established hiPSCs were then induced into mesoderm-originated cardiovascular progenitor cells (CVPCs) with a highly efficient directed lineage specification method. The derived CVPCs were demonstrated to be able to differentiate into functional VSMCs. Subcutaneous implantation of CVPCs seeded on macroporous nanofibrous poly(l-lactide) scaffolds led to in vivo VSMC lineage specification and matrix deposition inside the scaffolds. In summary, we established integration-free patient-specific hiPSCs from peripheral blood mononuclear cells, derived CVPCs through directed lineage specification, and developed an advanced scaffold for these progenitor cells to further differentiate in vivo into VSMCs and regenerate vascular tissue in a subcutaneous implantation model. This study has established an efficient patient-specific approach towards in vivo regeneration of vascular tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular evolution of lineage 2 West Nile virus.

PubMed

McMullen, Allison R; Albayrak, Harun; May, Fiona J; Davis, C Todd; Beasley, David W C; Barrett, Alan D T

2013-02-01

Since the 1990s West Nile virus (WNV) has become an increasingly important public health problem and the cause of outbreaks of neurological disease. Genetic analyses have identified multiple lineages with many studies focusing on lineage 1 due to its emergence in New York in 1999 and its neuroinvasive phenotype. Until recently, viruses in lineage 2 were not thought to be of public health importance due to few outbreaks of disease being associated with viruses in this lineage. However, recent epidemics of lineage 2 in Europe (Greece and Italy) and Russia have shown the increasing importance of this lineage. There are very few genetic studies examining isolates belonging to lineage 2. We have sequenced the full-length genomes of four older lineage 2 WNV isolates, compared them to 12 previously published genomic sequences and examined the evolution of this lineage. Our studies show that this lineage has evolved over the past 300-400 years and appears to correlate with a change from mouse attenuated to virulent phenotype based on previous studies by our group. This evolution mirrors that which is seen in lineage 1 isolates, which have also evolved to a virulent phenotype over the same period of time.

Biomaterial delivery of morphogens to mimic the natural healing cascade in bone

PubMed Central

Mehta, Manav; Schmidt-Bleek, Katharina; Duda, Georg N; Mooney, David J

2012-01-01

Complications in treatment of large bone defects using bone grafting still remain. Our understanding of the endogenous bone regeneration cascade has inspired the exploration of a wide variety of growth factors (GFs) in an effort to mimic the natural signaling that controls bone healing. Biomaterial-based delivery of single exogenous GFs has shown therapeutic efficacy, and this likely relates to its ability to recruit and promote replication of cells involved in tissue development and the healing process. However, as the natural bone healing cascade involves the action of multiple factors, each acting in a specific spatiotemporal pattern, strategies aiming to mimic the critical aspects of this process will likely benefit from the usage of multiple therapeutic agents. This article reviews the current status of approaches to deliver single GFs, as well as ongoing efforts to develop sophisticated delivery platforms to deliver multiple lineage-directing morphogens (multiple GFs) during bone healing. PMID:22626978

GM-CSF: An Immune Modulatory Cytokine that can Suppress Autoimmunity

PubMed Central

Bhattacharya, Palash; Thiruppathi, Muthusamy; Elshabrawy, Hatem A.; Alharshawi, Khaled; Kumar, Prabhakaran; Prabhakar, Bellur S.

2015-01-01

GM-CSF was originally identified as a colony stimulating factor (CSF) because of its ability to induce granulocyte and macrophage populations from precursor cells. Multiple studies have demonstrated that GM-CSF is also an immune-modulatory cytokine, capable of affecting not only the phenotype of myeloid lineage cells, but also T-cell activation through various myeloid intermediaries. This property has been implicated in the sustenance of several autoimmune diseases like arthritis and multiple sclerosis. In contrast, several studies using animal models have shown that GM-CSF is also capable of suppressing many autoimmune diseases like Crohn's disease, Type-1 diabetes, Myasthenia gravis and experimental autoimmune thyroiditis. Knockout mouse studies have suggested that the role of GM-CSF in maintaining granulocyte and macrophage populations in the physiological steady state is largely redundant. Instead, its immune-modulatory role plays a significant role in the development or resolution of autoimmune diseases. This is mediated either through the differentiation of precursor cells into specialized non-steady state granulocytes, macrophages and dendritic cells, or through the modulation of the phenotype of mature myeloid cells. Thus, outside of myelopoiesis, GM-CSF has a profound role in regulating the immune response and maintaining immunological tolerance. PMID:26113402

miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction

PubMed Central

Guda, Swaroopa; Brendel, Christian; Renella, Raffaele; Du, Peng; Bauer, Daniel E; Canver, Matthew C; Grenier, Jennifer K; Grimson, Andrew W; Kamran, Sophia C; Thornton, James; de Boer, Helen; Root, David E; Milsom, Michael D; Orkin, Stuart H; Gregory, Richard I; Williams, David A

2015-01-01

RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNAmiR) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNAmiRs resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNAmiRs for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences. PMID:26080908

Kupffer cells induce Notch-mediated hepatocyte conversion in a common mouse model of intrahepatic cholangiocarcinoma

PubMed Central

Terada, Maiko; Horisawa, Kenichi; Miura, Shizuka; Takashima, Yasuo; Ohkawa, Yasuyuki; Sekiya, Sayaka; Matsuda-Ito, Kanae; Suzuki, Atsushi

2016-01-01

Intrahepatic cholangiocarcinoma (ICC) is a malignant epithelial neoplasm composed of cells resembling cholangiocytes that line the intrahepatic bile ducts in portal areas of the hepatic lobule. Although ICC has been defined as a tumor arising from cholangiocyte transformation, recent evidence from genetic lineage-tracing experiments has indicated that hepatocytes can be a cellular origin of ICC by directly changing their fate to that of biliary lineage cells. Notch signaling has been identified as an essential factor for hepatocyte conversion into biliary lineage cells at the onset of ICC. However, the mechanisms underlying Notch signal activation in hepatocytes remain unclear. Here, using a mouse model of ICC, we found that hepatic macrophages called Kupffer cells transiently congregate around the central veins in the liver and express the Notch ligand Jagged-1 coincident with Notch activation in pericentral hepatocytes. Depletion of Kupffer cells prevents the Notch-mediated cell-fate conversion of hepatocytes to biliary lineage cells, inducing hepatocyte apoptosis and increasing mortality in mice. These findings will be useful for uncovering the pathogenic mechanism of ICC and developing prevenient and therapeutic strategies for this refractory disease. PMID:27698452

Protein kinase inhibitors against malignant lymphoma

PubMed Central

D’Cruz, Osmond J; Uckun, Fatih M

2013-01-01

Introduction Tyrosine kinases (TKs) are intimately involved in multiple signal transduction pathways regulating survival, activation, proliferation and differentiation of lymphoid cells. Deregulation or overexpression of specific oncogenic TKs is implicated in maintaining the malignant phenotype in B-lineage lymphoid malignancies. Several novel targeted TK inhibitors (TKIs) have recently emerged as active in the treatment of relapsed or refractory B-cell lymphomas that inhibit critical signaling pathways, promote apoptotic mechanisms or modulate the tumor microenvironment. Areas covered In this review, the authors summarize the clinical outcomes of newer TKIs in various B-cell lymphomas from published and ongoing clinical studies and abstracts from major cancer and hematology conferences. Expert opinion Multiple clinical trials have demonstrated that robust antitumor activity can be obtained with TKIs directed toward specific oncogenic TKs that are genetically deregulated in various subtypes of B-cell lymphomas. Clinical success of targeting TKIs is dependent upon on identifying reliable molecular and clinical markers associated with select cohorts of patients. Further understanding of the signaling pathways should stimulate the identification of novel molecular targets and expand the development of new therapeutic options and individualized therapies. PMID:23496343

Evolution of cytokinesis-related protein localization during the emergence of multicellularity in volvocine green algae.

PubMed

Arakaki, Yoko; Fujiwara, Takayuki; Kawai-Toyooka, Hiroko; Kawafune, Kaoru; Featherston, Jonathan; Durand, Pierre M; Miyagishima, Shin-Ya; Nozaki, Hisayoshi

2017-12-06

The volvocine lineage, containing unicellular Chlamydomonas reinhardtii and differentiated multicellular Volvox carteri, is a powerful model for comparative studies aiming at understanding emergence of multicellularity. Tetrabaena socialis is the simplest multicellular volvocine alga and belongs to the family Tetrabaenaceae that is sister to more complex multicellular volvocine families, Goniaceae and Volvocaceae. Thus, T. socialis is a key species to elucidate the initial steps in the evolution of multicellularity. In the asexual life cycle of C. reinhardtii and multicellular volvocine species, reproductive cells form daughter cells/colonies by multiple fission. In embryogenesis of the multicellular species, daughter protoplasts are connected to one another by cytoplasmic bridges formed by incomplete cytokinesis during multiple fission. These bridges are important for arranging the daughter protoplasts in appropriate positions such that species-specific integrated multicellular individuals are shaped. Detailed comparative studies of cytokinesis between unicellular and simple multicellular volvocine species will help to elucidate the emergence of multicellularity from the unicellular ancestor. However, the cytokinesis-related genes between closely related unicellular and multicellular species have not been subjected to a comparative analysis. Here we focused on dynamin-related protein 1 (DRP1), which is known for its role in cytokinesis in land plants. Immunofluorescence microscopy using an antibody against T. socialis DRP1 revealed that volvocine DRP1 was localized to division planes during cytokinesis in unicellular C. reinhardtii and two simple multicellular volvocine species T. socialis and Gonium pectorale. DRP1 signals were mainly observed in the newly formed division planes of unicellular C. reinhardtii during multiple fission, whereas in multicellular T. socialis and G. pectorale, DRP1 signals were observed in all division planes during embryogenesis. These results indicate that the molecular mechanisms of cytokinesis may be different in unicellular and multicellular volvocine algae. The localization of DRP1 during multiple fission might have been modified in the common ancestor of multicellular volvocine algae. This modification may have been essential for the re-orientation of cells and shaping colonies during the emergence of multicellularity in this lineage.

c-Myc-Induced Survivin Is Essential for Promoting the Notch-Dependent T Cell Differentiation from Hematopoietic Stem Cells

PubMed Central

Haque, Rizwanul; Song, Jianyong; Haque, Mohammad; Lei, Fengyang; Sandhu, Praneet; Ni, Bing; Zheng, Songguo; Fang, Deyu; Yang, Jin-Ming; Song, Jianxun

2017-01-01

Notch is indispensable for T cell lineage commitment, and is needed for thymocyte differentiation at early phases. During early stages of T cell development, active Notch prevents other lineage potentials including B cell lineage and myeloid cell (e.g., dendritic cell) lineage. Nevertheless, the precise intracellular signaling pathways by which Notch promotes T cell differentiation remain unclear. Here we report that the transcription factor c-Myc is a key mediator of the Notch signaling–regulated T cell differentiation. In a well-established in vitro differentiation model of T lymphocytes from hematopoietic stem cells, we showed that Notch1 and 4 directly promoted c-Myc expression; dominant-negative (DN) c-Myc inhibited early T cell differentiation. Moreover, the c-Myc expression activated by Notch signaling increased the expression of survivin, an inhibitor of apoptosis (IAP) protein. We further demonstrated that over-expression of c-Myc increased the abundance of survivin and the T cell differentiation thereof, whereas dn c-Myc reduced survivin levels and concomitantly retarded the differentiation. The c-Myc–dependent survivin induction is functionally germane, because Notch-dependent T cell differentiation was canceled by the depletion of survivin. These results identify both c-Myc and survivin as important mediators of the Notch signaling–regulated differentiation of T lymphocytes from hematopoietic stem cells. PMID:28272325

Positive and negative peptide signals control stomatal density.

PubMed

Shimada, Tomoo; Sugano, Shigeo S; Hara-Nishimura, Ikuko

2011-06-01

The stoma is a micro valve found on aerial plant organs that promotes gas exchange between the atmosphere and the plant body. Each stoma is formed by a strict cell lineage during the early stages of leaf development. Molecular genetics research using the model plant Arabidopsis has revealed the genes involved in stomatal differentiation. Cysteine-rich secretory peptides of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family play crucial roles as extracellular signaling factors. Stomatal development is orchestrated by the positive factor STOMAGEN/EPFL9 and the negative factors EPF1, EPF2, and CHALLAH/EPFL6 in combination with multiple receptors. EPF1 and EPF2 are produced in the stomatal lineage cells of the epidermis, whereas STOMAGEN and CHALLAH are derived from the inner tissues. These findings highlight the complex cell-to-cell and intertissue communications that regulate stomatal development. To optimize gas exchange, particularly the balance between the uptake of carbon dioxide (CO(2)) and loss of water, plants control stomatal activity in response to environmental conditions. The CO(2) level and light intensity influence stomatal density. Plants sense environmental cues in mature leaves and adjust the stomatal density of newly forming leaves, indicating the involvement of long-distance systemic signaling. This review summarizes recent research progress in the peptide signaling of stomatal development and discusses the evolutionary model of the signaling machinery.

Generation of Xeroderma Pigmentosum-A Patient-Derived Induced Pluripotent Stem Cell Line for Use As Future Disease Model.

PubMed

Ohnishi, Hiroe; Kawasaki, Takashi; Deguchi, Tomonori; Yuba, Shunsuke

2015-08-01

Xeroderma pigmentosum group A (XP-A) is a genetic disorder in which there is an abnormality in nucleotide excision repair that causes hypersensitivity to sunlight and multiple skin cancers. The development of central and peripheral neurological disorders not correlated to ultraviolet light exposure is associated with XP-A. The genes responsible for XP-A have been identified and a XPA knockout mouse has been generated. These knockout mice exhibit cutaneous symptoms, but they do not show neurological disorders. The mechanism of pathogenesis of neurological disorders is still unclear and therapeutic methods have not been established. Therefore, we generated XP-A patient-derived human induced pluripotent stem cells (XPA-iPSCs) to produce in vitro models of neurological disorders. We obtained iPSC lines from fibroblasts of two patients carrying different mutations. Drugs screened using XPA-iPSC lines can be helpful for treating XP-A patients in Japan. Additionally, we revealed that these iPSCs have the potential to differentiate into neural lineage cells, including dopaminergic neurons, which decrease in XP-A patients. Our results indicate that expression of the normal XPA gene without mutations is not required for generation of iPSCs and differentiation of iPSCs into neural lineage cells. XPA-iPSCs may become useful models that clarify our understanding of neurological pathogenesis and help to establish therapeutic methods.

Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting.

PubMed

Suzuki, Atsushi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2004-08-01

During pancreatic development, neogenesis, and regeneration, stem cells might act as a central player to generate endocrine, acinar, and duct cells. Although these cells are well known as pancreatic stem cells (PSCs), indisputable proof of their existence has not been reported. Identification of phenotypic markers for PSCs leads to their prospective isolation and precise characterization to clear whether stem cells exist in the pancreas. By combining flow cytometry and clonal analysis, we show here that a possible pancreatic stem or progenitor cell candidate that resides in the developing and adult mouse pancreas expresses the receptor for the hepatocyte growth factor (HGF) c-Met, but does not express hematopoietic and vascular endothelial antigens such as CD45, TER119, c-Kit, and Flk-1. These cells formed clonal colonies in vitro and differentiated into multiple pancreatic lineage cells from single cells. Some of them could largely expand with self-renewing cell divisions in culture, and, following cell transplantation, they differentiated into pancreatic endocrine and acinar cells in vivo. Furthermore, they produced cells expressing multiple markers of nonpancreatic organs including liver, stomach, and intestine in vitro. Our data strongly suggest that c-Met/HGF signaling plays an important role in stem/progenitor cell function in both developing and adult pancreas. By using this antigen, PSCs could be isolated prospectively, enabling a detailed investigation of stem cell markers and application toward regenerative therapies for diabetes.

Small Molecules Affect Human Dental Pulp Stem Cell Properties Via Multiple Signaling Pathways

PubMed Central

Al-Habib, Mey; Yu, Zongdong

2013-01-01

One fundamental issue regarding stem cells for regenerative medicine is the maintenance of stem cell stemness. The purpose of the study was to test whether small molecules can enhance stem cell properties of mesenchymal stem cells (MSCs) derived from human dental pulp (hDPSCs), which have potential for multiple clinical applications. We identified the effects of small molecules (Pluripotin (SC1), 6-bromoindirubin-3-oxime and rapamycin) on the maintenance of hDPSC properties in vitro and the mechanisms involved in exerting the effects. Primary cultures of hDPSCs were exposed to optimal concentrations of these small molecules. Treated hDPSCs were analyzed for their proliferation, the expression levels of pluripotent and MSC markers, differentiation capacities, and intracellular signaling activations. We found that small molecule treatments decreased cell proliferation and increased the expression of STRO-1, NANOG, OCT4, and SOX2, while diminishing cell differentiation into odonto/osteogenic, adipogenic, and neurogenic lineages in vitro. These effects involved Ras-GAP-, ERK1/2-, and mTOR-signaling pathways, which may preserve the cell self-renewal capacity, while suppressing differentiation. We conclude that small molecules appear to enhance the immature state of hDPSCs in culture, which may be used as a strategy for adult stem cell maintenance and extend their capacity for regenerative applications. PMID:23573877

Size distribution of retrovirally marked lineages matches prediction from population measurements of cell cycle behavior

NASA Technical Reports Server (NTRS)

Cai, Li; Hayes, Nancy L.; Takahashi, Takao; Caviness, Verne S Jr; Nowakowski, Richard S.

2002-01-01

Mechanisms that regulate neuron production in the developing mouse neocortex were examined by using a retroviral lineage marking method to determine the sizes of the lineages remaining in the proliferating population of the ventricular zone during the period of neuron production. The distribution of clade sizes obtained experimentally in four different injection-survival paradigms (E11-E13, E11-E14, E11-E15, and E12-E15) from a total of over 500 labeled lineages was compared with that obtained from three models in which the average behavior of the proliferating population [i.e., the proportion of cells remaining in the proliferative population (P) vs. that exiting the proliferative population (Q)] was quantitatively related to lineage size distribution. In model 1, different proportions of asymmetric, symmetric terminal, and symmetric nonterminal cell divisions coexisted during the entire developmental period. In model 2, the developmental period was divided into two epochs: During the first, asymmetric and symmetric nonterminal cell divisions occurred, but, during the second, asymmetric and symmetric terminal cell divisions occurred. In model 3, the shifts in P and Q are accounted for by changes in the proportions of the two types of symmetric cell divisions without the inclusion of any asymmetric cell divisions. The results obtained from the retroviral experiments were well accounted for by model 1 but not by model 2 or 3. These findings demonstrate that: 1) asymmetric and both types of symmetric cell divisions coexist during the entire period of neurogenesis in the mouse, 2) neuron production is regulated in the proliferative population by the independent decisions of the two daughter cells to reenter S phase, and 3) neurons are produced by both asymmetric and symmetric terminal cell divisions. In addition, the findings mean that cell death and/or tangential movements of cells in the proliferative population occur at only a low rate and that there are no proliferating lineages "reserved" to make particular laminae or cell types. Copyright 2002 Wiley-Liss, Inc.

Aging, mortality, and the fast growth trade-off of Schizosaccharomyces pombe

PubMed Central

Nakaoka, Hidenori; Wakamoto, Yuichi

2017-01-01

Replicative aging has been demonstrated in asymmetrically dividing unicellular organisms, seemingly caused by unequal damage partitioning. Although asymmetric segregation and inheritance of potential aging factors also occur in symmetrically dividing species, it nevertheless remains controversial whether this results in aging. Based on large-scale single-cell lineage data obtained by time-lapse microscopy with a microfluidic device, in this report, we demonstrate the absence of replicative aging in old-pole cell lineages of Schizosaccharomyces pombe cultured under constant favorable conditions. By monitoring more than 1,500 cell lineages in 7 different culture conditions, we showed that both cell division and death rates are remarkably constant for at least 50–80 generations. Our measurements revealed that the death rate per cellular generation increases with the division rate, pointing to a physiological trade-off with fast growth under balanced growth conditions. We also observed the formation and inheritance of Hsp104-associated protein aggregates, which are a potential aging factor in old-pole cell lineages, and found that these aggregates exhibited a tendency to preferentially remain at the old poles for several generations. However, the aggregates were eventually segregated from old-pole cells upon cell division and probabilistically allocated to new-pole cells. We found that cell deaths were typically preceded by sudden acceleration of protein aggregation; thus, a relatively large amount of protein aggregates existed at the very ends of the dead cell lineages. Our lineage tracking analyses, however, revealed that the quantity and inheritance of protein aggregates increased neither cellular generation time nor cell death initiation rates. Furthermore, our results demonstrated that unusually large amounts of protein aggregates induced by oxidative stress exposure did not result in aging; old-pole cells resumed normal growth upon stress removal, despite the fact that most of them inherited significant quantities of aggregates. These results collectively indicate that protein aggregates are not a major determinant of triggering cell death in S. pombe and thus cannot be an appropriate molecular marker or index for replicative aging under both favorable and stressful environmental conditions. PMID:28632741

The Vascular Wall: a Plastic Hub of Activity in Cardiovascular Homeostasis and Disease.

PubMed

Awgulewitsch, Cassandra P; Trinh, Linh T; Hatzopoulos, Antonis K

2017-06-01

This review aims to summarize recent findings regarding the plasticity and fate switching among somatic and progenitor cells residing in the vascular wall of blood vessels in health and disease. Cell lineage tracing methods have identified multiple origins of stem cells, macrophages, and matrix-producing cells that become mobilized after acute or chronic injury of cardiovascular tissues. These studies also revealed that in the disease environment, resident somatic cells become plastic, thereby changing their stereotypical identities to adopt proinflammatory and profibrotic phenotypes. Currently, the functional significance of this heterogeneity among reparative cells is unknown. Furthermore, mechanisms that control cellular plasticity and fate decisions in the disease environment are poorly understood. Cardiovascular diseases are responsible for the majority of deaths worldwide. From a therapeutic perspective, these novel discoveries may identify new targets to improve the repair and regeneration of the cardiovascular system.

Cnidarian Cell Type Diversity and Regulation Revealed by Whole-Organism Single-Cell RNA-Seq.

PubMed

Sebé-Pedrós, Arnau; Saudemont, Baptiste; Chomsky, Elad; Plessier, Flora; Mailhé, Marie-Pierre; Renno, Justine; Loe-Mie, Yann; Lifshitz, Aviezer; Mukamel, Zohar; Schmutz, Sandrine; Novault, Sophie; Steinmetz, Patrick R H; Spitz, François; Tanay, Amos; Marlow, Heather

2018-05-31

The emergence and diversification of cell types is a leading factor in animal evolution. So far, systematic characterization of the gene regulatory programs associated with cell type specificity was limited to few cell types and few species. Here, we perform whole-organism single-cell transcriptomics to map adult and larval cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal with complex tissue-level body-plan organization. We uncover eight broad cell classes in Nematostella, including neurons, cnidocytes, and digestive cells. Each class comprises different subtypes defined by the expression of multiple specific markers. In particular, we characterize a surprisingly diverse repertoire of neurons, which comparative analysis suggests are the result of lineage-specific diversification. By integrating transcription factor expression, chromatin profiling, and sequence motif analysis, we identify the regulatory codes that underlie Nematostella cell-specific expression. Our study reveals cnidarian cell type complexity and provides insights into the evolution of animal cell-specific genomic regulation. Copyright © 2018 Elsevier Inc. All rights reserved.

Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak

PubMed Central

Saelens, Joseph W.; Lau-Bonilla, Dalia; Moller, Anneliese; Medina, Narda; Guzmán, Brenda; Calderón, Maylena; Herrera, Raúl; Sisk, Dana M.; Xet-Mull, Ana M.; Stout, Jason E.; Arathoon, Eduardo; Samayoa, Blanca; Tobin, David M.

2015-01-01

Summary Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City. PMID:26542222

Clinically viable magnetic poly(lactide-co-glycolide) (PLGA) particles for MRI-based cell tracking

PubMed Central

Granot, Dorit; Nkansah, Michael K.; Bennewitz, Margaret F.; Tang, Kevin S.; Markakis, Eleni A.; Shapiro, Erik M.

2013-01-01

Purpose To design, fabricate, characterize and in vivo assay clinically viable magnetic particles for MRI-based cell tracking. Methods PLGA encapsulated magnetic nano- and microparticles were fabricated. Multiple biologically relevant experiments were performed to assess cell viability, cellular performance and stem cell differentiation. In vivo MRI experiments were performed to separately test cell transplantation and cell migration paradigms, as well as in vivo biodegradation. Results Highly magnetic nano- (~100 nm) and microparticles (~1–2 μm) were fabricated. Magnetic cell labeling in culture occurred rapidly achieving 3–50 pg Fe/cell at 3 hrs for different particles types, and >100 pg Fe/cell after 10 hours, without the requirement of a transfection agent, and with no effect on cell viability. The capability of magnetically labeled mesenchymal or neural stem cells to differentiate down multiple lineages, or for magnetically labeled immune cells to release cytokines following stimulation, was uncompromised. An in vivo biodegradation study revealed that NPs degraded ~80% over the course of 12 weeks. MRI detected as few as 10 magnetically labeled cells, transplanted into the brains of rats. Also, these particles enabled the in vivo monitoring of endogenous neural progenitor cell migration in rat brains over 2 weeks. Conclusion The robust MRI properties and benign safety profile of these particles make them promising candidates for clinical translation for MRI-based cell tracking. PMID:23568825

A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes

PubMed Central

Zhao, Xiangshan; Malhotra, Gautam K.; Band, Hamid; Band, Vimla

2011-01-01

Introduction: Emerging evidence suggests a direct role of cancer stem cells (CSCs) in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs) immortalized with human telomerase reverse transcriptase (hTERT) possess properties of mammary stem / progenitor cells. Materials and Methods: In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with γ-radiation), share with hTERT, the ability to maintain mammary stem / progenitor cells. Results: The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Conclusions: Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers. PMID:22279424

A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes.

PubMed

Zhao, Xiangshan; Malhotra, Gautam K; Band, Hamid; Band, Vimla

2011-01-01

Emerging evidence suggests a direct role of cancer stem cells (CSCs) in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs) immortalized with human telomerase reverse transcriptase (hTERT) possess properties of mammary stem / progenitor cells. In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with γ-radiation), share with hTERT, the ability to maintain mammary stem / progenitor cells. The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers.

PU.1 regulates TCR expression by modulating GATA-3 activity

PubMed Central

Chang, Hua-Chen; Han, Ling; Jabeen, Rukhsana; Carotta, Sebastian; Nutt, Stephen L.; Kaplan, Mark H.

2009-01-01

The Ets transcription factor PU.1 is a master regulator for the development of multiple lineages during hematopoiesis. The expression pattern of PU.1 is dynamically regulated during early T lineage development in the thymus. We previously revealed that PU.1 delineates heterogeneity of effector Th2 populations. In this study, we further define the function of PU.1 on the Th2 phenotype using mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1lck-/-). While deletion of PU.1 by the lck-Cre transgene does not affect T cell development, Sfpi1lck-/- T cells have a lower activation threshold than wild type T cells. When TCR engagement is limiting, Sfpi1lck-/- T cells cultured in Th2 polarizing conditions secrete higher levels of Th2 cytokines and have greater cytokine homogeneity than wild type cells. We show that PU.1 modulates the levels of TCR expression in CD4+ T cells by regulating the DNA-binding activity of GATA-3 and limiting GATA-3 regulation of TCR gene expression. GATA-3 dependent regulation of TCR expression is also observed in Th1 and Th2 cells. In CD4+ T cells, PU.1 expression segregates into subpopulations of cells that have lower levels of surface TCR, suggesting that PU.1 contributes to the heterogeneity of TCR expression. Thus, we have identified a mechanism whereby increased GATA-3 function in the absence of the antagonizing activity of PU.1 leads to increased TCR expression, a reduced activation threshold and increased homogeneity in Th2 populations. PMID:19801513

Evidence of two distinct functionally specialized fibroblast lineages in breast stroma.

PubMed

Morsing, Mikkel; Klitgaard, Marie Christine; Jafari, Abbas; Villadsen, René; Kassem, Moustapha; Petersen, Ole William; Rønnov-Jessen, Lone

2016-11-03

The terminal duct lobular unit (TDLU) is the most dynamic structure in the human breast and the putative site of origin of human breast cancer. Although stromal cells contribute to a specialized microenvironment in many organs, this component remains largely understudied in the human breast. We here demonstrate the impact on epithelium of two lineages of breast stromal fibroblasts, one of which accumulates in the TDLU while the other resides outside the TDLU in the interlobular stroma. The two lineages are prospectively isolated by fluorescence activated cell sorting (FACS) based on different expression levels of CD105 and CD26. The characteristics of the two fibroblast lineages are assessed by immunocytochemical staining and gene expression analysis. The differentiation capacity of the two fibroblast populations is determined by exposure to specific differentiating conditions followed by analysis of adipogenic and osteogenic differentiation. To test whether the two fibroblast lineages are functionally imprinted by their site of origin, single cell sorted CD271 low /MUC1 high normal breast luminal epithelial cells are plated on fibroblast feeders for the observation of morphological development. Epithelial structure formation and polarization is shown by immunofluorescence and digitalized quantification of immunoperoxidase-stained cultures. Lobular fibroblasts are CD105 high /CD26 low while interlobular fibroblasts are CD105 low /CD26 high . Once isolated the two lineages remain phenotypically stable and functionally distinct in culture. Lobular fibroblasts have properties in common with bone marrow derived mesenchymal stem cells and they specifically convey growth and branching morphogenesis of epithelial progenitors. Two distinct functionally specialized fibroblast lineages exist in the normal human breast, of which the lobular fibroblasts have properties in common with mesenchymal stem cells and support epithelial growth and morphogenesis. We propose that lobular fibroblasts constitute a specialized microenvironment for human breast luminal epithelial progenitors, i.e. the putative precursors of breast cancer.

Cell death and morphogenesis during early mouse development: Are they interconnected?

PubMed Central

Bedzhov, Ivan; Zernicka-Goetz, Magdalena

2015-01-01

Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage. PMID:25640415

Molecular diversity and distribution of marine fungi across 130 European environmental samples.

PubMed

Richards, Thomas A; Leonard, Guy; Mahé, Frédéric; Del Campo, Javier; Romac, Sarah; Jones, Meredith D M; Maguire, Finlay; Dunthorn, Micah; De Vargas, Colomban; Massana, Ramon; Chambouvet, Aurélie

2015-11-22

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal 'OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments. © 2015 The Authors.

Molecular diversity and distribution of marine fungi across 130 European environmental samples

PubMed Central

Richards, Thomas A.; Leonard, Guy; Mahé, Frédéric; del Campo, Javier; Romac, Sarah; Jones, Meredith D. M.; Maguire, Finlay; Dunthorn, Micah; De Vargas, Colomban; Massana, Ramon; Chambouvet, Aurélie

2015-01-01

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal ‘OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments. PMID:26582030

Cytomegalovirus immune evasion of myeloid lineage cells.

PubMed

Brinkmann, Melanie M; Dağ, Franziska; Hengel, Hartmut; Messerle, Martin; Kalinke, Ulrich; Čičin-Šain, Luka

2015-06-01

Cytomegalovirus (CMV) evades the immune system in many different ways, allowing the virus to grow and its progeny to spread in the face of an adverse environment. Mounting evidence about the antiviral role of myeloid immune cells has prompted the research of CMV immune evasion mechanisms targeting these cells. Several cells of the myeloid lineage, such as monocytes, dendritic cells and macrophages, play a role in viral control, but are also permissive for CMV and are naturally infected by it. Therefore, CMV evasion of myeloid cells involves mechanisms that qualitatively differ from the evasion of non-CMV-permissive immune cells of the lymphoid lineage. The evasion of myeloid cells includes effects in cis, where the virus modulates the immune signaling pathways within the infected myeloid cell, and those in trans, where the virus affects somatic cells targeted by cytokines released from myeloid cells. This review presents an overview of CMV strategies to modulate and evade the antiviral activity of myeloid cells in cis and in trans.

Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage.

PubMed

Balic, Adam; Garcia-Morales, Carla; Vervelde, Lonneke; Gilhooley, Hazel; Sherman, Adrian; Garceau, Valerie; Gutowska, Maria W; Burt, David W; Kaiser, Pete; Hume, David A; Sang, Helen M

2014-08-01

We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens. © 2014. Published by The Company of Biologists Ltd.

Multipotent Caudal Neural Progenitors Derived from Human Pluripotent Stem Cells That Give Rise to Lineages of the Central and Peripheral Nervous System

PubMed Central

Hasegawa, Kouichi; Menheniott, Trevelyan; Rollo, Ben; Zhang, Dongcheng; Hough, Shelley; Alshawaf, Abdullah; Febbraro, Fabia; Ighaniyan, Samiramis; Leung, Jessie; Elliott, David A.; Newgreen, Donald F.; Pera, Martin F.

2015-01-01

Abstract The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named “caudal neural progenitors” (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube. Stem Cells 2015;33:1759–1770 PMID:25753817

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed Central

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-01-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes. PMID:9121442

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-04-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

[Monoclonal antibodies in diagnosis of acute leukemias].

PubMed

Krawczyńska, A; Robak, T

1996-01-01

Immunophenotyping has become an essential component for the study of acute myeloblastic (AML) and lymphoblastic (ALL) leukaemias. The recent development of highly specific monoclonal antibodies (Mc Ab) to differentiation antigens (CD) of haematopoetic cells have made it readily available to clinical laboratories in most major hospitals. Immunophenotyping complements standard morphology by providing information on lineage, stage of differentiation and clonality. In addition some of the flow cytometry findings have independent prognostic significance. Monoclonal antibodies useful in defining lineage (B-cell versus T-cell) and stages of differentiation of ALL. It can be also used in identifying characteristic feature of AML and aiding in lineage determination in acute leukaemias that are morphologically undifferentiated. Surface immunophenotyping is especially helpful for recognizing mixed lineage acute leukaemia and diagnosing certain rare entities such as erythroleukaemia (M6), acute megakaryocytic leukaemia (M7) and minimally differentiation acute myeloid leukaemia.

Bhlhb5 is Required for the Subtype Development of Retinal Amacrine and Bipolar Cells in Mice

PubMed Central

Huang, Liang; Hu, Fang; Feng, Liang; Luo, Xiong-Jian; Liang, Guoqing; Zeng, Xiang-Yun; Yi, Jing-Lin; Gan, Lin

2014-01-01

Background BHLHB5, an OLIG-related basic helix-loop-helix transcription factor, is required for the development of a subset of gamma-amino butyric acid–releasing (GABAergic) amacrine cells and OFF-cone bipolar (CB) cells in mouse retinas. In order to determine BHLHB5’s functional mechanism in retinogenesis, we used the Cre-loxP recombination system to genetically trace the lineage of BHLHB5+ cells in normal and Bhlhb5-null retinas. The Bhlhb5-Cre knock-in allele was used to activate the constitutive expression of a GFP reporter in the Bhlhb5-expressing cells, and the cell fates of Bhlhb5-lineage cells were identified by using specific cell markers and were compared between normal and Bhlhb5-null retinas. Results In addition to GABAergic amacrine and OFF-CB cells, Bhlhb5 lineage cells give rise to ganglion, glycinergic amacrine, rod bipolar, ON-bipolar, and rod photoreceptor cells during normal retinal development. Targeted deletion of Bhlhb5 resulted in the loss of GABAergic amacrine, glycinergic amacrine, dopaminergic amacrine, and Type 2 OFF-CB cells. Furthermore, in the absence of BHLHB5, a portion of Bhlhb5 lineage cells switch their fate and differentiate into cholinergic amacrine cells. Conclusions Our data reveal a broad expression pattern of Bhlhb5 throughout retinogenesis and demonstrate the cell-autonomous as well as non-cell-autonomous role of Bhlhb5 in the specification of amacrine and bipolar subtypes. PMID:24123365

Bhlhb5 is required for the subtype development of retinal amacrine and bipolar cells in mice.

PubMed

Huang, Liang; Hu, Fang; Feng, Liang; Luo, Xiong-Jian; Liang, Guoqing; Zeng, Xiang-Yun; Yi, Jing-Lin; Gan, Lin

2014-02-01

BHLHB5, an OLIG-related basic helix-loop-helix transcription factor, is required for the development of a subset of gamma-amino butyric acid-releasing (GABAergic) amacrine cells and OFF-cone bipolar (CB) cells in mouse retinas. In order to determine BHLHB5's functional mechanism in retinogenesis, we used the Cre-loxP recombination system to genetically trace the lineage of BHLHB5+ cells in normal and Bhlhb5-null retinas. The Bhlhb5-Cre knock-in allele was used to activate the constitutive expression of a GFP reporter in the Bhlhb5-expressing cells, and the cell fates of Bhlhb5-lineage cells were identified by using specific cell markers and were compared between normal and Bhlhb5-null retinas. In addition to GABAergic amacrine and OFF-CB cells, Bhlhb5 lineage cells give rise to ganglion, glycinergic amacrine, rod bipolar, ON-bipolar, and rod photoreceptor cells during normal retinal development. Targeted deletion of Bhlhb5 resulted in the loss of GABAergic amacrine, glycinergic amacrine, dopaminergic amacrine, and Type 2 OFF-CB cells. Furthermore, in the absence of BHLHB5, a portion of Bhlhb5 lineage cells switch their fate and differentiate into cholinergic amacrine cells. Our data reveal a broad expression pattern of Bhlhb5 throughout retinogenesis and demonstrate the cell-autonomous as well as non-cell-autonomous role of Bhlhb5 in the specification of amacrine and bipolar subtypes. Copyright © 2013 Wiley Periodicals, Inc.

Genome-Wide Analysis Reveals the Unique Stem Cell Identity of Human Amniocytes

PubMed Central

Maguire, Colin T.; Demarest, Bradley L.; Hill, Jonathon T.; Palmer, James D.; Brothman, Arthur R.; Yost, H. Joseph; Condic, Maureen L.

2013-01-01

Human amniotic fluid contains cells that potentially have important stem cell characteristics, yet the programs controlling their developmental potency are unclear. Here, we provide evidence that amniocytes derived from multiple patients are marked by heterogeneity and variability in expression levels of pluripotency markers. Clonal analysis from multiple patients indicates that amniocytes have large pools of self-renewing cells that have an inherent property to give rise to a distinct amniocyte phenotype with a heterogeneity of pluripotent markers. Significant to their therapeutic potential, genome-wide profiles are distinct at different gestational ages and times in culture, but do not differ between genders. Based on hierarchical clustering and differential expression analyses of the entire transcriptome, amniocytes express canonical regulators associated with pluripotency and stem cell repression. Their profiles are distinct from human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and newborn foreskin fibroblasts. Amniocytes have a complex molecular signature, coexpressing trophoblastic, ectodermal, mesodermal, and endodermal cell-type-specific regulators. In contrast to the current view of the ground state of stem cells, ESCs and iPSCs also express high levels of a wide range of cell-type-specific regulators. The coexpression of multilineage differentiation markers combined with the strong expression of a subset of ES cell repressors in amniocytes suggests that these cells have a distinct phenotype that is unlike any other known cell-type or lineage. PMID:23326421

Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

PubMed Central

Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

2016-01-01

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793

In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Armstrong, R.; Friedrich, V.L. Jr.; Holmes, K.V.

1990-09-01

A demyelinating disease induced in C57B1/6N mice by intracranial injection of a coronavirus (murine hepatitis virus strain A59) is followed by functional recovery and efficient CNS myelin repair. To study the biological properties of the cells involved in this repair process, glial cells were isolated and cultured from spinal cords of these young adult mice during demyelination and remyelination. Using three-color immunofluorescence combined with (3H)thymidine autoradiography, we have analyzed the antigenic phenotype and mitotic potential of individual glial cells. We identified oligodendrocytes with an antibody to galactocerebroside, astrocytes with an antibody to glial fibrillary acidic protein, and oligodendrocyte-type 2 astrocytemore » (O-2A) progenitor cells with the O4 antibody. Cultures from demyelinated tissue differed in several ways from those of age-matched controls: first, the total number of O-2A lineage cells was strikingly increased; second, the O-2A population consisted of a higher proportion of O4-positive astrocytes and cells of mixed oligodendrocyte-astrocyte phenotype; and third, all the cell types within the O-2A lineage showed enhanced proliferation. This proliferation was not further enhanced by adding PDGF, basic fibroblast growth factor (bFGF), or insulin-like growth factor I (IGF-I) to the defined medium. However, bFGF and IGF-I seemed to influence the fate of O-2A lineage cells in cultures of demyelinated tissue. Basic FGF decreased the percentage of cells expressing galactocerebroside. In contrast, IGF-I increased the relative proportion of oligodendrocytes. Thus, O-2A lineage cells from adult mice display greater phenotypic plasticity and enhanced mitotic potential in response to an episode of demyelination. These properties may be linked to the efficient remyelination achieved in this demyelinating disease.« less

Opposing activities of Notch and Wnt signaling regulate intestinal stem cells and gut homeostasis

PubMed Central

Tian, Hua; Biehs, Brian; Chiu, Cecilia; Siebel, Chris; Wu, Yan; Costa, Mike; de Sauvage, Frederic J.; Klein, Ophir D.

2015-01-01

Summary Proper organ homeostasis requires tight control of adult stem cells and differentiation through integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a de-repression of the Wnt signaling pathway, leading to mis-expression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology. PMID:25818302

Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events.

PubMed

Wong, Vanessa K; Baker, Stephen; Pickard, Derek J; Parkhill, Julian; Page, Andrew J; Feasey, Nicholas A; Kingsley, Robert A; Thomson, Nicholas R; Keane, Jacqueline A; Weill, François-Xavier; Edwards, David J; Hawkey, Jane; Harris, Simon R; Mather, Alison E; Cain, Amy K; Hadfield, James; Hart, Peter J; Thieu, Nga Tran Vu; Klemm, Elizabeth J; Glinos, Dafni A; Breiman, Robert F; Watson, Conall H; Kariuki, Samuel; Gordon, Melita A; Heyderman, Robert S; Okoro, Chinyere; Jacobs, Jan; Lunguya, Octavie; Edmunds, W John; Msefula, Chisomo; Chabalgoity, Jose A; Kama, Mike; Jenkins, Kylie; Dutta, Shanta; Marks, Florian; Campos, Josefina; Thompson, Corinne; Obaro, Stephen; MacLennan, Calman A; Dolecek, Christiane; Keddy, Karen H; Smith, Anthony M; Parry, Christopher M; Karkey, Abhilasha; Mulholland, E Kim; Campbell, James I; Dongol, Sabina; Basnyat, Buddha; Dufour, Muriel; Bandaranayake, Don; Naseri, Take Toleafoa; Singh, Shalini Pravin; Hatta, Mochammad; Newton, Paul; Onsare, Robert S; Isaia, Lupeoletalalei; Dance, David; Davong, Viengmon; Thwaites, Guy; Wijedoru, Lalith; Crump, John A; De Pinna, Elizabeth; Nair, Satheesh; Nilles, Eric J; Thanh, Duy Pham; Turner, Paul; Soeng, Sona; Valcanis, Mary; Powling, Joan; Dimovski, Karolina; Hogg, Geoff; Farrar, Jeremy; Holt, Kathryn E; Dougan, Gordon

2015-06-01

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species.

Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events

PubMed Central

Wong, Vanessa K; Baker, Stephen; Pickard, Derek J; Parkhill, Julian; Page, Andrew J; Feasey, Nicholas A; Kingsley, Robert A; Thomson, Nicholas R; Keane, Jacqueline A; Weill, François-Xavier; Edwards, David J; Hawkey, Jane; Harris, Simon R; Mather, Alison E; Cain, Amy K; Hadfield, James; Hart, Peter J; Thieu, Nga Tran Vu; Klemm, Elizabeth J; Glinos, Dafni A; Breiman, Robert F; Watson, Conall H; Kariuki, Samuel; Gordon, Melita A; Heyderman, Robert S; Okoro, Chinyere; Jacobs, Jan; Lunguya, Octavie; Edmunds, W John; Msefula, Chisomo; Chabalgoity, Jose A; Kama, Mike; Jenkins, Kylie; Dutta, Shanta; Marks, Florian; Campos, Josefina; Thompson, Corinne; Obaro, Stephen; MacLennan, Calman A; Dolecek, Christiane; Keddy, Karen H; Smith, Anthony M; Parry, Christopher M; Karkey, Abhilasha; Mulholland, E Kim; Campbell, James I; Dongol, Sabina; Basnyat, Buddha; Dufour, Muriel; Bandaranayake, Don; Naseri, Take Toleafoa; Singh, Shalini Pravin; Hatta, Mochammad; Newton, Paul; Onsare, Robert S; Isaia, Lupeoletalalei; Dance, David; Davong, Viengmon; Thwaites, Guy; Wijedoru, Lalith; Crump, John A; De Pinna, Elizabeth; Nair, Satheesh; Nilles, Eric J; Thanh, Duy Pham; Turner, Paul; Soeng, Sona; Valcanis, Mary; Powling, Joan; Dimovski, Karolina; Hogg, Geoff; Farrar, Jeremy; Holt, Kathryn E; Dougan, Gordon

2016-01-01

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species. PMID:25961941

Pancreas lineage allocation and specification are regulated by sphingosine-1-phosphate signalling.

PubMed

Serafimidis, Ioannis; Rodriguez-Aznar, Eva; Lesche, Mathias; Yoshioka, Kazuaki; Takuwa, Yoh; Dahl, Andreas; Pan, Duojia; Gavalas, Anthony

2017-03-01

During development, progenitor expansion, lineage allocation, and implementation of differentiation programs need to be tightly coordinated so that different cell types are generated in the correct numbers for appropriate tissue size and function. Pancreatic dysfunction results in some of the most debilitating and fatal diseases, including pancreatic cancer and diabetes. Several transcription factors regulating pancreas lineage specification have been identified, and Notch signalling has been implicated in lineage allocation, but it remains unclear how these processes are coordinated. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata, and genomics, we found that sphingosine-1-phosphate (S1p), signalling through the G protein coupled receptor (GPCR) S1pr2, plays a key role in pancreas development linking lineage allocation and specification. S1pr2 signalling promotes progenitor survival as well as acinar and endocrine specification. S1pr2-mediated stabilisation of the yes-associated protein (YAP) is essential for endocrine specification, thus linking a regulator of progenitor growth with specification. YAP stabilisation and endocrine cell specification rely on Gαi subunits, revealing an unexpected specificity of selected GPCR intracellular signalling components. Finally, we found that S1pr2 signalling posttranscriptionally attenuates Notch signalling levels, thus regulating lineage allocation. Both S1pr2-mediated YAP stabilisation and Notch attenuation are necessary for the specification of the endocrine lineage. These findings identify S1p signalling as a novel key pathway coordinating cell survival, lineage allocation, and specification and linking these processes by regulating YAP levels and Notch signalling. Understanding lineage allocation and specification in the pancreas will shed light in the origins of pancreatic diseases and may suggest novel therapeutic approaches.

Pancreas lineage allocation and specification are regulated by sphingosine-1-phosphate signalling

PubMed Central

Serafimidis, Ioannis; Rodriguez-Aznar, Eva; Lesche, Mathias; Yoshioka, Kazuaki; Takuwa, Yoh; Dahl, Andreas; Pan, Duojia; Gavalas, Anthony

2017-01-01

During development, progenitor expansion, lineage allocation, and implementation of differentiation programs need to be tightly coordinated so that different cell types are generated in the correct numbers for appropriate tissue size and function. Pancreatic dysfunction results in some of the most debilitating and fatal diseases, including pancreatic cancer and diabetes. Several transcription factors regulating pancreas lineage specification have been identified, and Notch signalling has been implicated in lineage allocation, but it remains unclear how these processes are coordinated. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata, and genomics, we found that sphingosine-1-phosphate (S1p), signalling through the G protein coupled receptor (GPCR) S1pr2, plays a key role in pancreas development linking lineage allocation and specification. S1pr2 signalling promotes progenitor survival as well as acinar and endocrine specification. S1pr2-mediated stabilisation of the yes-associated protein (YAP) is essential for endocrine specification, thus linking a regulator of progenitor growth with specification. YAP stabilisation and endocrine cell specification rely on Gαi subunits, revealing an unexpected specificity of selected GPCR intracellular signalling components. Finally, we found that S1pr2 signalling posttranscriptionally attenuates Notch signalling levels, thus regulating lineage allocation. Both S1pr2-mediated YAP stabilisation and Notch attenuation are necessary for the specification of the endocrine lineage. These findings identify S1p signalling as a novel key pathway coordinating cell survival, lineage allocation, and specification and linking these processes by regulating YAP levels and Notch signalling. Understanding lineage allocation and specification in the pancreas will shed light in the origins of pancreatic diseases and may suggest novel therapeutic approaches. PMID:28248965

Raman spectroscopy for discrimination of neural progenitor cells and their lineages (Conference Presentation)

NASA Astrophysics Data System (ADS)

Chen, Keren; Ong, William; Chew, Sing Yian; Liu, Quan

2017-02-01

Neurological diseases are one of the leading causes of adult disability and they are estimated to cause more deaths than cancer in the elderly population by 2040. Stem cell therapy has shown great potential in treating neurological diseases. However, before cell therapy can be widely adopted in the long term, a number of challenges need to be addressed, including the fundamental research about cellular development of neural progenitor cells. To facilitate the fundamental research of neural progenitor cells, many methods have been developed to identify neural progenitor cells. Although great progress has been made, there is still lack of an effective method to achieve fast, label-free and noninvasive differentiation of neural progenitor cells and their lineages. As a fast, label-free and noninvasive technique, spontaneous Raman spectroscopy has been conducted to characterize many types of stem cells including neural stem cells. However, to our best knowledge, it has not been studied for the discrimination of neural progenitor cells from specific lineages. Here we report the differentiation of neural progenitor cell from their lineages including astrocytes, oligodendrocytes and neurons using spontaneous Raman spectroscopy. Moreover, we also evaluate the influence of system parameters during spectral acquisition on the quality of measured Raman spectra and the accuracy of classification using the spectra, which yield a set of optimal system parameters facilitating future studies.

Multiple introductions of divergent genetic lineages in an invasive fungal pathogen, Cryphonectria parasitica, in France.

PubMed

Dutech, C; Fabreguettes, O; Capdevielle, X; Robin, C

2010-08-01

The occurrence of multiple introductions may be a crucial factor in the successful establishment of invasive species, but few studies focus on the introduction of fungal pathogens, despite their significant effect on invaded habitats. Although Cryphonectria parasitica, the chestnut blight fungus introduced in North America and Europe from Asia during the 20th century, caused dramatic changes in its new range, the history of its introduction is not well retraced in Europe. Using 10 microsatellite loci, we investigated the genetic diversity of 583 isolates in France, where several introductions have been hypothesized. Our analyses showed that the seven most frequent multilocus genotypes belonged to three genetic lineages, which had a different and geographically limited distribution. These results suggest that different introduction events occurred in France. Genetic recombination was low among these lineages, despite the presence of the two mating types in each chestnut stand analysed. The spatial distribution of lineages suggests that the history of introductions in France associated with the slow expansion of the disease has contributed to the low observed rate of recombination among the divergent lineages. However, we discuss the possibility that environmental conditions or viral interactions could locally reduce recombination among genotypes.

Silent subtype 3 pituitary adenomas are not always silent and represent poorly differentiated monomorphous plurihormonal Pit-1 lineage adenomas.

PubMed

Mete, Ozgur; Gomez-Hernandez, Karen; Kucharczyk, Walter; Ridout, Rowena; Zadeh, Gelareh; Gentili, Fred; Ezzat, Shereen; Asa, Sylvia L

2016-02-01

Originally classified as a variant of silent corticotroph adenoma, silent subtype 3 adenomas are a distinct histologic variant of pituitary adenoma of unknown cytogenesis. We reviewed the clinical, biochemical, radiological, immunohistochemical and ultrastructural features of 31 silent subtype 3 adenomas to clarify their cellular origin. Among 25 with clinical and/or radiological data, all were macroadenomas; there was cavernous sinus invasion in 30% of cases and involvement of the clivus in 17% of cases. Almost 90% of patients were symptomatic; 67% had mass effect symptoms, 37% were hypogonadal and 8% had secondary adrenal insufficiency. Significant hormonal excess in 29% of cases included hyperthyroidism in 17%, acromegaly in 8% and hyperprolactinemia above 150 μg/l in 4%. Two individuals with hyperprolactinemia who were younger than 30 years had multiple endocrine neoplasia type 1. Immunohistochemically, all 31 tumors were diffusely positive for the pituitary lineage-specific transcription factor Pit-1. Although three only expressed Pit-1, others revealed variable positivity for one or more hormones of Pit-1 cell lineage (growth hormone, prolactin, thyroid-stimulating hormone), as well as alpha-subunit and estrogen receptor. Most tumors exhibited perinuclear reactivity for keratins with the CAM5.2 antibody; scattered fibrous bodies were noted in five (16%) tumors. The mean MIB-1 labeling index was 4% (range, 1-9%). Fourteen cases examined by electron microscopy were composed of a monomorphous population of large polygonal or elongated cells with nuclear spheridia. Sixty-five percent of patients had residual disease after surgery; after a mean follow-up of 48.4 months (median 41.5; range=2-171) disease progression was documented in 53% of those cases. These data identify silent subtype 3 adenomas as aggressive monomorphous plurihormonal adenomas of Pit-1 lineage that may be associated with hyperthyroidism, acromegaly or galactorrhea and amenorrhea. Our findings argue against the use of the nomenclature 'silent' for these tumors. To better reflect the characteristics of these tumors, we propose that they be classified as 'poorly differentiated Pit-1 lineage adenomas'.

Hacking cell differentiation: transcriptional rerouting in reprogramming, lineage infidelity and metaplasia.

PubMed

Regalo, Gonçalo; Leutz, Achim

2013-08-01

Initiating neoplastic cell transformation events are of paramount importance for the comprehension of regeneration and vanguard oncogenic processes but are difficult to characterize and frequently clinically overlooked. In epithelia, pre-neoplastic transformation stages are often distinguished by the appearance of phenotypic features of another differentiated tissue, termed metaplasia. In haemato/lymphopoietic malignancies, cell lineage ambiguity is increasingly recorded. Both, metaplasia and biphenotypic leukaemia/lymphoma represent examples of dysregulated cell differentiation that reflect a history of trans-differentiation and/or epigenetic reprogramming. Here we compare the similarity between molecular events of experimental cell trans-differentiation as an emerging therapeutic concept, with lineage confusion, as in metaplasia and dysplasia forecasting tumour development. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury.

PubMed

Llorens-Bobadilla, Enric; Zhao, Sheng; Baser, Avni; Saiz-Castro, Gonzalo; Zwadlo, Klara; Martin-Villalba, Ana

2015-09-03

Heterogeneous pools of adult neural stem cells (NSCs) contribute to brain maintenance and regeneration after injury. The balance of NSC activation and quiescence, as well as the induction of lineage-specific transcription factors, may contribute to diversity of neuronal and glial fates. To identify molecular hallmarks governing these characteristics, we performed single-cell sequencing of an unbiased pool of adult subventricular zone NSCs. This analysis identified a discrete, dormant NSC subpopulation that already expresses distinct combinations of lineage-specific transcription factors during homeostasis. Dormant NSCs enter a primed-quiescent state before activation, which is accompanied by downregulation of glycolytic metabolism, Notch, and BMP signaling and a concomitant upregulation of lineage-specific transcription factors and protein synthesis. In response to brain ischemia, interferon gamma signaling induces dormant NSC subpopulations to enter the primed-quiescent state. This study unveils general principles underlying NSC activation and lineage priming and opens potential avenues for regenerative medicine in the brain. Copyright © 2015 Elsevier Inc. All rights reserved.

Regenerative medicine using dental pulp stem cells for liver diseases.

PubMed

Ohkoshi, Shogo; Hara, Hajime; Hirono, Haruka; Watanabe, Kazuhiko; Hasegawa, Katsuhiko

2017-02-06

Acute liver failure is a refractory disease and its prognosis, if not treated using liver transplantation, is extremely poor. It is a good candidate for regenerative medicine, where stem cell-based therapies play a central role. Mesenchymal stem cells (MSCs) are known to differentiate into multiple cell lineages including hepatocytes. Autologous cell transplant without any foreign gene induction is feasible using MSCs, thereby avoiding possible risks of tumorigenesis and immune rejection. Dental pulp also contains an MSC population that differentiates into hepatocytes. A point worthy of special mention is that dental pulp can be obtained from deciduous teeth during childhood and can be subsequently harvested when necessary after deposition in a tooth bank. MSCs have not only a regenerative capacity but also act in an anti-inflammatory manner via paracrine mechanisms. Promising efficacies and difficulties with the use of MSC derived from teeth are summarized in this review.

Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland.

PubMed

Lilja, Anna M; Rodilla, Veronica; Huyghe, Mathilde; Hannezo, Edouard; Landragin, Camille; Renaud, Olivier; Leroy, Olivier; Rulands, Steffen; Simons, Benjamin D; Fre, Silvia

2018-06-01

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.

Rearrangements of genes for the antigen receptor on T cells as markers of lineage and clonality in human lymphoid neoplasms.

PubMed

Waldmann, T A; Davis, M M; Bongiovanni, K F; Korsmeyer, S J

1985-09-26

The T alpha and T beta chains of the heterodimeric T-lymphocyte antigen receptor are encoded by separated DNA segments that recombine during T-cell development. We have used rearrangements of the T beta gene as a widely applicable marker of clonality in the T-cell lineage. We show that the T beta genes are used in both the T8 and T4 subpopulations of normal T cells and that Sézary leukemia, adult T-cell leukemia, and the non-B-lineage acute lymphoblastic leukemias are clonal expansions of T cells. Furthermore, circulating T cells from a patient with the T8-cell-predominantly lymphocytosis associated with granulocytopenia are shown to be monoclonal. Finally, the sensitivity and specificity of this tumor-associated marker have been exploited to monitor the therapy of a patient with adult T-cell leukemia. These unique DNA rearrangements provide insights into the cellular origin, clonality, and natural history of T-cell neoplasia.

Developmental Origin Governs CD8+ T Cell Fate Decisions during Infection.

PubMed

Smith, Norah L; Patel, Ravi K; Reynaldi, Arnold; Grenier, Jennifer K; Wang, Jocelyn; Watson, Neva B; Nzingha, Kito; Yee Mon, Kristel J; Peng, Seth A; Grimson, Andrew; Davenport, Miles P; Rudd, Brian D

2018-06-06

Heterogeneity is a hallmark feature of the adaptive immune system in vertebrates. Following infection, naive T cells differentiate into various subsets of effector and memory T cells, which help to eliminate pathogens and maintain long-term immunity. The current model suggests there is a single lineage of naive T cells that give rise to different populations of effector and memory T cells depending on the type and amounts of stimulation they encounter during infection. Here, we have discovered that multiple sub-populations of cells exist in the naive CD8 + T cell pool that are distinguished by their developmental origin, unique transcriptional profiles, distinct chromatin landscapes, and different kinetics and phenotypes after microbial challenge. These data demonstrate that the naive CD8 + T cell pool is not as homogeneous as previously thought and offers a new framework for explaining the remarkable heterogeneity in the effector and memory T cell subsets that arise after infection. Copyright © 2018 Elsevier Inc. All rights reserved.

Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.

PubMed

Hoban, Megan D; Cost, Gregory J; Mendel, Matthew C; Romero, Zulema; Kaufman, Michael L; Joglekar, Alok V; Ho, Michelle; Lumaquin, Dianne; Gray, David; Lill, Georgia R; Cooper, Aaron R; Urbinati, Fabrizia; Senadheera, Shantha; Zhu, Allen; Liu, Pei-Qi; Paschon, David E; Zhang, Lei; Rebar, Edward J; Wilber, Andrew; Wang, Xiaoyan; Gregory, Philip D; Holmes, Michael C; Reik, Andreas; Hollis, Roger P; Kohn, Donald B

2015-04-23

Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers. © 2015 by The American Society of Hematology.

CDC42 is required for epicardial and pro-epicardial development by mediating FGF receptor trafficking to the plasma membrane

PubMed Central

Li, Jingjing; Miao, Lianjie; Zhao, Chen; Shaikh Qureshi, Wasay Mohiuddin; Shieh, David; Guo, Hua; Lu, Yangyang; Hu, Saiyang; Huang, Alice; Zhang, Lu; Cai, Chen-leng; Wan, Leo Q.; Xin, Hongbo; Vincent, Peter; Singer, Harold A.; Zheng, Yi; Cleaver, Ondine; Fan, Zhen-Chuan

2017-01-01

The epicardium contributes to multiple cardiac lineages and is essential for cardiac development and regeneration. However, the mechanism of epicardium formation is unclear. This study aimed to establish the cellular and molecular mechanisms underlying the dissociation of pro-epicardial cells (PECs) from the pro-epicardium (PE) and their subsequent translocation to the heart to form the epicardium. We used lineage tracing, conditional deletion, mosaic analysis and ligand stimulation in mice to determine that both villous protrusions and floating cysts contribute to PEC translocation to myocardium in a CDC42-dependent manner. We resolved a controversy by demonstrating that physical contact of the PE with the myocardium constitutes a third mechanism for PEC translocation to myocardium, and observed a fourth mechanism in which PECs migrate along the surface of the inflow tract to reach the ventricles. Epicardial-specific Cdc42 deletion disrupted epicardium formation, and Cdc42 null PECs proliferated less, lost polarity and failed to form villous protrusions and floating cysts. FGF signaling promotes epicardium formation in vivo, and biochemical studies demonstrated that CDC42 is involved in the trafficking of FGF receptors to the cell membrane to regulate epicardium formation. PMID:28465335

The Antiviral Drug Arbidol Inhibits Zika Virus.

PubMed

Fink, Susan L; Vojtech, Lucia; Wagoner, Jessica; Slivinski, Natalie S J; Jackson, Konner J; Wang, Ruofan; Khadka, Sudip; Luthra, Priya; Basler, Christopher F; Polyak, Stephen J

2018-06-12

There are many emerging and re-emerging globally prevalent viruses for which there are no licensed vaccines or antiviral medicines. Arbidol (ARB, umifenovir), used clinically for decades in several countries as an anti-influenza virus drug, inhibits many other viruses. In the current study, we show that ARB inhibits six different isolates of Zika virus (ZIKV), including African and Asian lineage viruses in multiple cell lines and primary human vaginal and cervical epithelial cells. ARB protects against ZIKV-induced cytopathic effects. Time of addition studies indicate that ARB is most effective at suppressing ZIKV when added to cells prior to infection. Moreover, ARB inhibits pseudoviruses expressing the ZIKV Envelope glycoprotein. Thus, ARB, a broadly acting anti-viral agent with a well-established safety profile, inhibits ZIKV, likely by blocking viral entry.

Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak.

PubMed

Saelens, Joseph W; Lau-Bonilla, Dalia; Moller, Anneliese; Medina, Narda; Guzmán, Brenda; Calderón, Maylena; Herrera, Raúl; Sisk, Dana M; Xet-Mull, Ana M; Stout, Jason E; Arathoon, Eduardo; Samayoa, Blanca; Tobin, David M

2015-12-01

Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Assessment of Erythroid and Granulocytic Hematopoietic Lineages in Patients with Non-Small-Cell Lung Carcinoma.

PubMed

Goldberg, V E; Polyakova, T Yu; Popova, N O; Vysotskaya, V V; Simolina, E I; Belevich, Yu V; Tuzikova, T P; Goldberg, A V; Zhdanov, V V; Miroshnichenko, L A; Udut, E V; Simanina, E V; Dygai, A M; Zyuz'kov, G N

2017-08-01

The toxic effects of combined cisplatin/docetaxel therapy cycles on erythroid and granulocytic hematopoietic lineages as well as their intercycle recovery were examined in patients with stage III-IV non-small-cell lung carcinoma. Responsiveness of the blood system to this therapy remained at a high level. Combined therapy pronouncedly activated the key elements of the erythroid and granulocytic hematopoietic lineages leading to accumulation of immature and mature myelokaryocytes in the bone marrow, enlargement of the medullary pool of mature neutrophils, and increase in the count of medullary erythroid and granulocytic precursor cells under conditions of their accelerated maturation.

Loss of CXCR6 coreceptor usage characterizes pathogenic lentiviruses

PubMed Central

Wetzel, Katherine S.; Yi, Yanjie; Bauer, Anya M.; Bibollet-Ruche, Frederic; Hahn, Beatrice H.; Paiardini, Mirko; Silvestri, Guido; Peeters, Martine; Collman, Ronald G.

2018-01-01

Pandemic HIV-1 originated from the cross-species transmission of SIVcpz, which infects chimpanzees, while SIVcpz itself emerged following the cross-species transmission and recombination of monkey SIVs, with env contributed by the SIVgsn/mus/mon lineage that infects greater spot-nosed, mustached and mona monkeys. SIVcpz and HIV-1 are pathogenic in their respective hosts, while the phenotype of their SIVgsn/mus/mon ancestors is unknown. However, two well-studied SIV infected natural hosts, sooty mangabeys (SMs) and African green monkeys (AGMs), typically remain healthy despite high viral loads; these species express low levels of the canonical coreceptor CCR5, and recent work shows that CXCR6 is a major coreceptor for SIV in these hosts. It is not known what coreceptors were used by the precursors of SIVcpz, whether coreceptor use changed during emergence of the SIVcpz/HIV-1 lineage, and what T cell subsets express CXCR6 in natural hosts. Using species-matched coreceptors and CD4, we show here that SIVcpz uses only CCR5 for entry and, like HIV-1, cannot use CXCR6. In contrast, SIVmus efficiently uses both CXCR6 and CCR5. Coreceptor selectivity was determined by Env, with CXCR6 use abrogated by Pro326 in the V3 crown, which is absent in monkey SIVs but highly conserved in SIVcpz/HIV-1. To characterize which cells express CXCR6, we generated a novel antibody that recognizes CXCR6 of multiple primate species. Testing lymphocytes from SM, the best-studied natural host, we found that CXCR6 is restricted to CD4+ effector memory cells, and is expressed by a sub-population distinct from those expressing CCR5. Thus, efficient CXCR6 use, previously identified in SM and AGM infection, also characterizes a member of the SIV lineage that gave rise to SIVcpz/HIV-1. Loss of CXCR6 usage by SIVcpz may have altered its cell tropism, shifting virus from CXCR6-expressing cells that may support replication without disrupting immune function or homeostasis, towards CCR5-expressing cells with pathogenic consequences. PMID:29659623

Loss of CXCR6 coreceptor usage characterizes pathogenic lentiviruses.

PubMed

Wetzel, Katherine S; Yi, Yanjie; Yadav, Anjana; Bauer, Anya M; Bello, Ezekiel A; Romero, Dino C; Bibollet-Ruche, Frederic; Hahn, Beatrice H; Paiardini, Mirko; Silvestri, Guido; Peeters, Martine; Collman, Ronald G

2018-04-01

Pandemic HIV-1 originated from the cross-species transmission of SIVcpz, which infects chimpanzees, while SIVcpz itself emerged following the cross-species transmission and recombination of monkey SIVs, with env contributed by the SIVgsn/mus/mon lineage that infects greater spot-nosed, mustached and mona monkeys. SIVcpz and HIV-1 are pathogenic in their respective hosts, while the phenotype of their SIVgsn/mus/mon ancestors is unknown. However, two well-studied SIV infected natural hosts, sooty mangabeys (SMs) and African green monkeys (AGMs), typically remain healthy despite high viral loads; these species express low levels of the canonical coreceptor CCR5, and recent work shows that CXCR6 is a major coreceptor for SIV in these hosts. It is not known what coreceptors were used by the precursors of SIVcpz, whether coreceptor use changed during emergence of the SIVcpz/HIV-1 lineage, and what T cell subsets express CXCR6 in natural hosts. Using species-matched coreceptors and CD4, we show here that SIVcpz uses only CCR5 for entry and, like HIV-1, cannot use CXCR6. In contrast, SIVmus efficiently uses both CXCR6 and CCR5. Coreceptor selectivity was determined by Env, with CXCR6 use abrogated by Pro326 in the V3 crown, which is absent in monkey SIVs but highly conserved in SIVcpz/HIV-1. To characterize which cells express CXCR6, we generated a novel antibody that recognizes CXCR6 of multiple primate species. Testing lymphocytes from SM, the best-studied natural host, we found that CXCR6 is restricted to CD4+ effector memory cells, and is expressed by a sub-population distinct from those expressing CCR5. Thus, efficient CXCR6 use, previously identified in SM and AGM infection, also characterizes a member of the SIV lineage that gave rise to SIVcpz/HIV-1. Loss of CXCR6 usage by SIVcpz may have altered its cell tropism, shifting virus from CXCR6-expressing cells that may support replication without disrupting immune function or homeostasis, towards CCR5-expressing cells with pathogenic consequences.

A reporter mouse model for in vivo tracing and in vitro molecular studies of melanocytic lineage cells and their diseases.

PubMed

Crawford, Melissa; Leclerc, Valerie; Dagnino, Lina

2017-08-15

Alterations in melanocytic lineage cells give rise to a plethora of distinct human diseases, including neurocristopathies, cutaneous pigmentation disorders, loss of vision and hearing, and melanoma. Understanding the ontogeny and biology of melanocytic cells, as well as how they interact with their surrounding environment, are key steps in the development of therapies for diseases that involve this cell lineage. Efforts to culture and characterize primary melanocytes from normal or genetically engineered mouse models have at times yielded contrasting observations. This is due, in part, to differences in the conditions used to isolate, purify and culture these cells in individual studies. By breeding ROSA mT/mG and Tyr::CreER T2 mice, we generated animals in which melanocytic lineage cells are identified through expression of green fluorescent protein. We also used defined conditions to systematically investigate the proliferation and migration responses of primary melanocytes on various extracellular matrix (ECM) substrates. Under our culture conditions, mouse melanocytes exhibit doubling times in the range of 10 days, and retain exponential proliferative capacity for 50-60 days. In culture, these melanocytes showed distinct responses to different ECM substrates. Specifically, laminin-332 promoted cell spreading, formation of dendrites, random motility and directional migration. In contrast, low or intermediate concentrations of collagen I promoted adhesion and acquisition of a bipolar morphology, and interfered with melanocyte forward movements. Our systematic evaluation of primary melanocyte responses emphasizes the importance of clearly defining culture conditions for these cells. This, in turn, is essential for the interpretation of melanocyte responses to extracellular cues and to understand the molecular basis of disorders involving the melanocytic cell lineage. © 2017. Published by The Company of Biologists Ltd.

A reporter mouse model for in vivo tracing and in vitro molecular studies of melanocytic lineage cells and their diseases

PubMed Central

Crawford, Melissa; Leclerc, Valerie

2017-01-01

ABSTRACT Alterations in melanocytic lineage cells give rise to a plethora of distinct human diseases, including neurocristopathies, cutaneous pigmentation disorders, loss of vision and hearing, and melanoma. Understanding the ontogeny and biology of melanocytic cells, as well as how they interact with their surrounding environment, are key steps in the development of therapies for diseases that involve this cell lineage. Efforts to culture and characterize primary melanocytes from normal or genetically engineered mouse models have at times yielded contrasting observations. This is due, in part, to differences in the conditions used to isolate, purify and culture these cells in individual studies. By breeding ROSAmT/mG and Tyr::CreERT2 mice, we generated animals in which melanocytic lineage cells are identified through expression of green fluorescent protein. We also used defined conditions to systematically investigate the proliferation and migration responses of primary melanocytes on various extracellular matrix (ECM) substrates. Under our culture conditions, mouse melanocytes exhibit doubling times in the range of 10 days, and retain exponential proliferative capacity for 50-60 days. In culture, these melanocytes showed distinct responses to different ECM substrates. Specifically, laminin-332 promoted cell spreading, formation of dendrites, random motility and directional migration. In contrast, low or intermediate concentrations of collagen I promoted adhesion and acquisition of a bipolar morphology, and interfered with melanocyte forward movements. Our systematic evaluation of primary melanocyte responses emphasizes the importance of clearly defining culture conditions for these cells. This, in turn, is essential for the interpretation of melanocyte responses to extracellular cues and to understand the molecular basis of disorders involving the melanocytic cell lineage. PMID:28642245

Hybridization and restricted gene flow between native and introduced stocks of Alpine whitefish (Coregonus sp.) across multiple environments

PubMed Central

Winkler, Kathrin A; Pamminger-Lahnsteiner, Barbara; Wanzenböck, Josef; Weiss, Steven

2011-01-01

Translocations of Baltic whitefish (Coregonus sp.) into Austrian Alpine lakes have created ‘artificial hybrid zones’, threatening the genetic integrity of native lineages. We evaluate the genetic structure of Coregonus in Austrian lakes and characterize hybridization and introgression between native and introduced lineages. Fifteen populations (N= 747) were assessed for allelic variation at eight microsatellite loci and a reduced set (N= 253) for variation across two mtDNA genes (cyt b and NADH-3). Bayesian approaches were used to estimate individual admixture proportions (q-values) and classify genotypes as native, introduced or hybrids. q-value distributions varied among populations highlighting differential hybridization and introgression histories. Many lakes revealed a clear distinction between native and introduced genotypes despite hybridization, whereas some locations revealed hybrid swarms. Genetic structure among lakes was congruent with morphological divergence and novelty raising speculation of multiple taxa, including a population south of the Alps, outside the putative native range of Coregonus. Although statistically congruent with inferences based on nuclear markers, mitochondrial haplotype data was not diagnostic with respect to native and non-native lineages, supporting that the Alpine region was colonized post-glacially by an admixture of mtDNA lineages, which coalesce >1 Ma. Mechanisms promoting or eroding lineage isolation are discussed, as well as a high potential to conserve native Alpine lineages despite the extensive historical use of introduced Baltic stocks. PMID:21199024

First report of multiple lineages of dengue viruses type 1 in Rio de Janeiro, Brazil.

PubMed

dos Santos, Flavia B; Nogueira, Fernanda B; Castro, Márcia G; Nunes, Priscila Cg; de Filippis, Ana Maria B; Faria, Nieli Rc; Simões, Jaqueline Bs; Sampaio, Simone A; Santos, Clarice R; Nogueira, Rita Maria R

2011-08-03

In Brazil dengue has been a major public health problem since DENV-1 introduction and spread in 1986. After a low or silent co-circulation, DENV-1 re-emerged in 2009 causing a major epidemic in the country in 2010 and 2011. In this study, the phylogeny of DENV-1 strains isolated in RJ after its first introduction in 1986 and after its emergence in 2009 and 2010 was performed in order to document possible evolutionary patterns or introductions in a re-emergent virus. The analysis of the E gene sequences demonstrated that DENV-1 isolated during 2009/2010 still belong to genotype V (Americas/Africa) but grouping in a distinct clade (lineage II) of that represented by earlier DENV-1 (lineage I). However, strains isolated in 2011 grouped together forming another distinct clade (lineage III). The monitoring of DENV is important to observe the spread of potentially virulent strains as well to evaluate its impact over the population during an outbreak. Whether explosive epidemics reported in Brazil caused mainly by DENV-1 was due to lineage replacement, or due the population susceptibility to this serotype which has not circulated for almost a decade or even due to the occurrence of secondary infections in a hyperendemic country, is not clear. This is the first report of multiple lineages of DENV-1 detected in Brazil.

Fitness cost of reassortment in human influenza

PubMed Central

Lässig, Michael

2017-01-01

Reassortment, which is the exchange of genome sequence between viruses co-infecting a host cell, plays an important role in the evolution of segmented viruses. In the human influenza virus, reassortment happens most frequently between co-existing variants within the same lineage. This process breaks genetic linkage and fitness correlations between viral genome segments, but the resulting net effect on viral fitness has remained unclear. In this paper, we determine rate and average selective effect of reassortment processes in the human influenza lineage A/H3N2. For the surface proteins hemagglutinin and neuraminidase, reassortant variants with a mean distance of at least 3 nucleotides to their parent strains get established at a rate of about 10−2 in units of the neutral point mutation rate. Our inference is based on a new method to map reassortment events from joint genealogies of multiple genome segments, which is tested by extensive simulations. We show that intra-lineage reassortment processes are, on average, under substantial negative selection that increases in strength with increasing sequence distance between the parent strains. The deleterious effects of reassortment manifest themselves in two ways: there are fewer reassortment events than expected from a null model of neutral reassortment, and reassortant strains have fewer descendants than their non-reassortant counterparts. Our results suggest that influenza evolves under ubiquitous epistasis across proteins, which produces fitness barriers against reassortment even between co-circulating strains within one lineage. PMID:29112968

Bone marrow mesenchymal stem cells are abnormal in multiple myeloma

PubMed Central

Corre, Jill; Mahtouk, Karène; Attal, Michel; Gadelorge, Mélanie; Huynh, Anne; Fleury-Cappellesso, Sandrine; Danho, Clotaire; Laharrague, Patrick; Klein, Bernard; Rème, Thierry; Bourin, Philippe

2007-01-01

Recent literature suggested that cell of the microenvironment of solid tumors could be abnormal as well. To address this hypothesis in multiple myeloma (MM), we studied bone marrow mesenchymal stem cells (BMMSCs), the only long-lived cells of the bone marrow microenvironment, by gene expression with Affymetrix arrays and phenotypic and functional study in 3 groups of individuals: patients with MM and those with monoclonal gamopathy of undefined significance (MGUS), and healthy aged-matched subjects. Gene expression profile independently classified the BMMSCs of these individuals in a normal and in a MM group. MGUS BMMSCs were interspersed between those 2 groups. Among the 145 distinct genes differentially expressed in MM and normal BMMSCs 46% were involved in tumor-microenvironment cross-talk. Known soluble factors involved in MM pathophysiologic features, (interleukin (IL)-6, IL-1β, DKK1 and amphiregulin, were revealed and new ones found. In particular, GDF-15 was found to induce dose-dependant growth of MOLP-6, a stromal cell-dependent myeloma cell line. Functionally, MM BMMSCs induced an over-growth of MOLP-6, and their capacity to differentiate into an osteoblastic lineage was impaired. Thus, BMMSCs from MM patients could create a very efficient niche to support the survival and proliferation of the myeloma stem cells. PMID:17344918

Defined three-dimensional culture conditions mediate efficient induction of definitive endoderm lineage from human umbilical cord Wharton's jelly mesenchymal stem cells.

PubMed

Al Madhoun, Ashraf; Ali, Hamad; AlKandari, Sarah; Atizado, Valerie Lopez; Akhter, Nadeem; Al-Mulla, Fahd; Atari, Maher

2016-11-16

Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are gaining increasing interest as an alternative source of stem cells for regenerative medicine applications. Definitive endoderm (DE) specification is a prerequisite for the development of vital organs such as liver and pancreas. Hence, efficient induction of the DE lineage from stem cells is crucial for subsequent generation of clinically relevant cell types. Here we present a defined 3D differentiation protocol of WJ-MSCs into DE cells. WJ-MSCs were cultured in suspension to generate spheroids, about 1500 cells each, for 7 days. The serum-free differentiation media contained specific growth factors, cytokines, and small molecules that specifically regulate signaling pathways including sonic hedgehog, bone morphogenetic protein, Activin/Wnt, and Notch. We obtained more than 85 % DE cells as shown with FACS analysis using antibodies directed against the DE marker CXCR4. In addition, biochemical and molecular analysis of bona-fide DE markers revealed a time-course induction of Sox17, CXCR4, and FoxA2. Focused PCR-based array also indicated a specific induction into the DE lineage. In this study, we report an efficient serum-free protocol to differentiate WJ-MSCs into DE cells utilizing 3D spheroid formation. Our approach might aid in the development of new protocols to obtain DE-derivative lineages including liver-like and pancreatic insulin-producing cells.

Transcriptional Regulation of Th17 Cell Differentiation

PubMed Central

Ivanov, Ivaylo I.; Zhou, Liang; Littman, Dan R.

2009-01-01

The paradigm of effector T helper cell differentiation into either Th1 or Th2 lineages has been profoundly shaken by the discovery of T cells that secrete IL-17 and other inflammatory cytokines. This subset, referred to as Th17, is centrally involved in autoimmune disease and is important in host defense at mucosal surfaces. In mouse, a series of cytokines, including IL-6, IL-21, IL-23, and TGF-β, function sequentially or synergistically to induce the Th17 lineage. Other cytokines, including IL-2, IL-4, IFNγ, and IL-27, inhibit differentiation of this lineage. Here we review how the nuclear orphan receptor RORγt functions to coordinate the diverse cytokine-induced signals and thus control Th17 cell differentiation. PMID:18053739

Cell death and morphogenesis during early mouse development: are they interconnected?

PubMed

Bedzhov, Ivan; Zernicka-Goetz, Magdalena

2015-04-01

Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage. © 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.

Oligodendrocyte progenitor programming and reprogramming: Toward myelin regeneration.

PubMed

Lopez Juarez, Alejandro; He, Danyang; Richard Lu, Q

2016-05-01

Demyelinating diseases such as multiple sclerosis (MS) are among the most disabling and cost-intensive neurological disorders. The loss of myelin in the central nervous system, produced by oligodendrocytes (OLs), impairs saltatory nerve conduction, leading to motor and cognitive deficits. Immunosuppression therapy has a limited efficacy in MS patients, arguing for a paradigm shift to strategies that target OL lineage cells to achieve myelin repair. The inhibitory microenvironment in MS lesions abrogates the expansion and differentiation of resident OL precursor cells (OPCs) into mature myelin-forming OLs. Recent studies indicate that OPCs display a highly plastic ability to differentiate into alternative cell lineages under certain circumstances. Thus, understanding the mechanisms that maintain and control OPC fate and differentiation into mature OLs in a hostile, non-permissive lesion environment may open new opportunities for regenerative therapies. In this review, we will focus on 1) the plasticity of OPCs in terms of their developmental origins, distribution, and differentiation potentials in the normal and injured brain; 2) recent discoveries of extrinsic and intrinsic factors and small molecule compounds that control OPC specification and differentiation; and 3) therapeutic potential for motivation of neural progenitor cells and reprogramming of differentiated cells into OPCs and their likely impacts on remyelination. OL-based therapies through activating regenerative potentials of OPCs or cell replacement offer exciting opportunities for innovative strategies to promote remyelination and neuroprotection in devastating demyelinating diseases like MS. This article is part of a Special Issue entitled SI:NG2-glia(Invited only). Copyright © 2015 Elsevier B.V. All rights reserved.

Biphasic Role of Chondroitin Sulfate in Cardiac Differentiation of Embryonic Stem Cells through Inhibition of Wnt/β-Catenin Signaling

PubMed Central

Prinz, Robert D.; Willis, Catherine M.; van Kuppevelt, Toin H.; Klüppel, Michael

2014-01-01

The glycosaminoglycan chondroitin sulfate is a critical component of proteoglycans on the cell surface and in the extracellular matrix. As such, chondroitin sulfate side chains and the sulfation balance of chondroitin play important roles in the control of signaling pathways, and have a functional importance in human disease. In contrast, very little is known about the roles of chondroitin sulfate molecules and sulfation patterns during mammalian development and cell lineage specification. Here, we report a novel biphasic role of chondroitin sulfate in the specification of the cardiac cell lineage during embryonic stem cell differentiation through modulation of Wnt/beta-catenin signaling. Lineage marker analysis demonstrates that enzymatic elimination of endogenous chondroitin sulfates leads to defects specifically in cardiac differentiation. This is accompanied by a reduction in the number of beating cardiac foci. Mechanistically, we show that endogenous chondroitin sulfate controls cardiac differentiation in a temporal biphasic manner through inhibition of the Wnt/beta-catenin pathway, a known regulatory pathway for the cardiac lineage. Treatment with a specific exogenous chondroitin sulfate, CS-E, could mimic these biphasic effects on cardiac differentiation and Wnt/beta-catenin signaling. These results establish chondroitin sulfate and its sulfation balance as important regulators of cardiac cell lineage decisions through control of the Wnt/beta-catenin pathway. Our work suggests that targeting the chondroitin biosynthesis and sulfation machinery is a novel promising avenue in regenerative strategies after heart injury. PMID:24667694

Biphasic role of chondroitin sulfate in cardiac differentiation of embryonic stem cells through inhibition of Wnt/β-catenin signaling.

PubMed

Prinz, Robert D; Willis, Catherine M; van Kuppevelt, Toin H; Klüppel, Michael

2014-01-01

The glycosaminoglycan chondroitin sulfate is a critical component of proteoglycans on the cell surface and in the extracellular matrix. As such, chondroitin sulfate side chains and the sulfation balance of chondroitin play important roles in the control of signaling pathways, and have a functional importance in human disease. In contrast, very little is known about the roles of chondroitin sulfate molecules and sulfation patterns during mammalian development and cell lineage specification. Here, we report a novel biphasic role of chondroitin sulfate in the specification of the cardiac cell lineage during embryonic stem cell differentiation through modulation of Wnt/beta-catenin signaling. Lineage marker analysis demonstrates that enzymatic elimination of endogenous chondroitin sulfates leads to defects specifically in cardiac differentiation. This is accompanied by a reduction in the number of beating cardiac foci. Mechanistically, we show that endogenous chondroitin sulfate controls cardiac differentiation in a temporal biphasic manner through inhibition of the Wnt/beta-catenin pathway, a known regulatory pathway for the cardiac lineage. Treatment with a specific exogenous chondroitin sulfate, CS-E, could mimic these biphasic effects on cardiac differentiation and Wnt/beta-catenin signaling. These results establish chondroitin sulfate and its sulfation balance as important regulators of cardiac cell lineage decisions through control of the Wnt/beta-catenin pathway. Our work suggests that targeting the chondroitin biosynthesis and sulfation machinery is a novel promising avenue in regenerative strategies after heart injury.

Genetic Ablation of Parietal Cells in Transgenic Mice: A New Model for Analyzing Cell Lineage Relationships in the Gastric Mucosa

NASA Astrophysics Data System (ADS)

Canfield, Victor; West, A. Brian; Goldenring, James R.; Levenson, Robert

1996-03-01

The gastric mucosa of mammalian stomach contains several differentiated cell types specialized for the secretion of acid, digestive enzymes, mucus, and hormones. Understanding whether each of these cell lineages is derived from a common stem cell has been a challenging problem. We have used a genetic approach to analyze the ontogeny of progenitor cells within mouse stomach. Herpes simplex virus 1 thymidine kinase was targeted to parietal cells within the gastric mucosa of transgenic mice, and parietal cells were ablated by treatment of animals with the antiherpetic drug ganciclovir. Ganciclovir treatment produced complete ablation of parietal cells, dissolution of gastric glands, and loss of chief and mucus-producing cells. Termination of drug treatment led to the reemergence of all major gastric epithelial cell types and restoration of glandular architecture. Our results imply the existence of a pluripotent stem cell for the gastric mucosa. Parietal cell ablation should provide a model for analyzing cell lineage relationships within the stomach as well as mechanisms underlying gastric injury and repair.

Next-generation sequencing reveals cryptic mtDNA diversity of Plasmodium relictum in the Hawaiian Islands

USGS Publications Warehouse

Jarvi, S.I.; Farias, M.E.; Lapointe, D.A.; Belcaid, M.; Atkinson, C.T.

2013-01-01

Next-generation 454 sequencing techniques were used to re-examine diversity of mitochondrial cytochrome b lineages of avian malaria (Plasmodium relictum) in Hawaii. We document a minimum of 23 variant lineages of the parasite based on single nucleotide transitional changes, in addition to the previously reported single lineage (GRW4). A new, publicly available portal (Integroomer) was developed for initial parsing of 454 datasets. Mean variant prevalence and frequency was higher in low elevation Hawaii Amakihi (Hemignathus virens) with Avipoxvirus-like lesions (P = 0·001), suggesting that the variants may be biologically distinct. By contrast, variant prevalence and frequency did not differ significantly among mid-elevation Apapane (Himatione sanguinea) with or without lesions (P = 0·691). The low frequency and the lack of detection of variants independent of GRW4 suggest that multiple independent introductions of P. relictum to Hawaii are unlikely. Multiple variants may have been introduced in heteroplasmy with GRW4 or exist within the tandem repeat structure of the mitochondrial genome. The discovery of multiple mitochondrial lineages of P. relictum in Hawaii provides a measure of genetic diversity within a geographically isolated population of this parasite and suggests the origins and evolution of parasite diversity may be more complicated than previously recognized.

Next-generation sequencing reveals cryptic mtDNA diversity of Plasmodium relictum in the Hawaiian Islands.

PubMed

Jarvi, S I; Farias, M E; Lapointe, D A; Belcaid, M; Atkinson, C T

2013-12-01

Next-generation 454 sequencing techniques were used to re-examine diversity of mitochondrial cytochrome b lineages of avian malaria (Plasmodium relictum) in Hawaii. We document a minimum of 23 variant lineages of the parasite based on single nucleotide transitional changes, in addition to the previously reported single lineage (GRW4). A new, publicly available portal (Integroomer) was developed for initial parsing of 454 datasets. Mean variant prevalence and frequency was higher in low elevation Hawaii Amakihi (Hemignathus virens) with Avipoxvirus-like lesions (P = 0·001), suggesting that the variants may be biologically distinct. By contrast, variant prevalence and frequency did not differ significantly among mid-elevation Apapane (Himatione sanguinea) with or without lesions (P = 0·691). The low frequency and the lack of detection of variants independent of GRW4 suggest that multiple independent introductions of P. relictum to Hawaii are unlikely. Multiple variants may have been introduced in heteroplasmy with GRW4 or exist within the tandem repeat structure of the mitochondrial genome. The discovery of multiple mitochondrial lineages of P. relictum in Hawaii provides a measure of genetic diversity within a geographically isolated population of this parasite and suggests the origins and evolution of parasite diversity may be more complicated than previously recognized.

Notch ligands Delta1 and Jagged1 transmit distinct signals to T-cell precursors

PubMed Central

Lehar, Sophie M.; Dooley, James; Farr, Andrew G.; Bevan, Michael J.

2009-01-01

Signaling through the Notch pathway plays an essential role in inducing T-lineage commitment and promoting the maturation of immature thymocytes. Using an in vitro culture system, we show that 2 different classes of Notch ligands, Jagged1 or Delta1, transmit distinct signals to T-cell progenitors. OP9 stromal cells expressing either Jagged1 or Delta1 inhibit the differentiation of DN1 thymocytes into the B-cell lineage, but only the Delta1-expressing stromal cells promote the proliferation and maturation of T-cell progenitors through the early double-negative (DN) stages of thymocyte development. Whereas the majority of bone marrow-derived stem cells do not respond to Jagged1 signals, T-cell progenitors respond to Jagged1 signals during a brief window of their development between the DN1 and DN3 stages of thymic development. During these stages, Jagged1 signals can influence the differentiation of immature thymocytes along the natural killer (NK) and γδ T-cell lineages. PMID:15486060

Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency

PubMed Central

Xie, Yufen; Zhou, Sichang; Jiang, Zhongliang; Dai, Jing; Puscheck, Elizabeth E; Lee, Icksoo; Parker, Graham; Hüttemann, Maik; Rappolee, Daniel A

2014-01-01

Dysfunctional stem cell differentiation into placental lineages is associated with gestational diseases. Of the differentiated lineages available to trophoblast stem cells (TSC), elevated O2 and mitochondrial function are necessary to placental lineages at the maternal-placental surface and important in the etiology of preeclampsia. TSC lineage imbalance leads to embryonic failure during uterine implantation. Stress at implantation exacerbates stem cell depletion by decreasing proliferation and increasing differentiation. Implantation site O2 is normally ~2%. In culture, exposure to 2% O2 and fibroblast growth factor (FGF)4 enabled highest mouse TSC multipotency and proliferation. In contrast, hypoxic stress (0.5% O2) initiated the most TSC differentiation after 24 hr despite FGF4. However, hypoxic stress supported differentiation poorly after 4–7 days, despite FGF4 removal. At all tested O2 levels, FGF4 maintained Warburg metabolism; mitochondrial inactivity and aerobic glycolysis. However, hypoxic stress suppressed mitochondrial membrane potential, maintained low mitochondrial cytochrome c oxidase (oxidative phosphorylation/OxPhos), and high pyruvate kinase M2 (glycolysis) despite FGF4 removal. Inhibiting OxPhos inhibited differentiation at the differentiation optimum at 20% O2. Moreover, adding differentiation-inducing hyperosmolar stress failed to induce differentiation during hypoxia. Thus, differentiation depended on OxPhos at 20% O2; hypoxic and hyperosmolar stresses did not induce differentiation at 0.5% O2. Hypoxia-limited differentiation and mitochondrial inhibition and activation suggest that differentiation into two lineages of the labyrinthine placenta requires O2>0.5–2% and mitochondrial function. Stress-activated protein kinase increases an early lineage and suppresses later lineages in proportion to the deviation from optimal O2 for multipotency, thus it is the first enzyme reported to prioritize differentiation. PMID:25239494

Integrin αv in the mechanical response of osteoblast lineage cells.

PubMed

Kaneko, Keiko; Ito, Masako; Naoe, Yoshinori; Lacy-Hulbert, Adam; Ikeda, Kyoji

2014-05-02

Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src-JNK-YAP/TAZ pathway in response to mechanical stimulation. Copyright © 2014 Elsevier Inc. All rights reserved.

Reconstructing human pancreatic differentiation by mapping specific cell populations during development.

PubMed

Ramond, Cyrille; Glaser, Nicolas; Berthault, Claire; Ameri, Jacqueline; Kirkegaard, Jeannette Schlichting; Hansson, Mattias; Honoré, Christian; Semb, Henrik; Scharfmann, Raphaël

2017-07-21

Information remains scarce on human development compared to animal models. Here, we reconstructed human fetal pancreatic differentiation using cell surface markers. We demonstrate that at 7weeks of development, the glycoprotein 2 (GP2) marks a multipotent cell population that will differentiate into the acinar, ductal or endocrine lineages. Development towards the acinar lineage is paralleled by an increase in GP2 expression. Conversely, a subset of the GP2 + population undergoes endocrine differentiation by down-regulating GP2 and CD142 and turning on NEUROG3 , a marker of endocrine differentiation. Endocrine maturation progresses by up-regulating SUSD2 and lowering ECAD levels. Finally, in vitro differentiation of pancreatic endocrine cells derived from human pluripotent stem cells mimics key in vivo events. Our work paves the way to extend our understanding of the origin of mature human pancreatic cell types and how such lineage decisions are regulated.

mtDNA lineage analysis of mouse L-cell lines reveals the accumulation of multiple mtDNA mutants and intermolecular recombination

PubMed Central

Fan, Weiwei; Lin, Chun Shi; Potluri, Prasanth; Procaccio, Vincent; Wallace, Douglas C.

2012-01-01

The role of mitochondrial DNA (mtDNA) mutations and mtDNA recombination in cancer cell proliferation and developmental biology remains controversial. While analyzing the mtDNAs of several mouse L cell lines, we discovered that every cell line harbored multiple mtDNA mutants. These included four missense mutations, two frameshift mutations, and one tRNA homopolymer expansion. The LA9 cell lines lacked wild-type mtDNAs but harbored a heteroplasmic mixture of mtDNAs, each with a different combination of these variants. We isolated each of the mtDNAs in a separate cybrid cell line. This permitted determination of the linkage phase of each mtDNA and its physiological characteristics. All of the polypeptide mutations inhibited their oxidative phosphorylation (OXPHOS) complexes. However, they also increased mitochondrial reactive oxygen species (ROS) production, and the level of ROS production was proportional to the cellular proliferation rate. By comparing the mtDNA haplotypes of the different cell lines, we were able to reconstruct the mtDNA mutational history of the L–L929 cell line. This revealed that every heteroplasmic L-cell line harbored a mtDNA that had been generated by intracellular mtDNA homologous recombination. Therefore, deleterious mtDNA mutations that increase ROS production can provide a proliferative advantage to cancer or stem cells, and optimal combinations of mutant loci can be generated through recombination. PMID:22345519

Identification of transcript regulatory patterns in cell differentiation.

PubMed

Gusnanto, Arief; Gosling, John Paul; Pope, Christopher

2017-10-15

Studying transcript regulatory patterns in cell differentiation is critical in understanding its complex nature of the formation and function of different cell types. This is done usually by measuring gene expression at different stages of the cell differentiation. However, if the gene expression data available are only from the mature cells, we have some challenges in identifying transcript regulatory patterns that govern the cell differentiation. We propose to exploit the information of the lineage of cell differentiation in terms of correlation structure between cell types. We assume that two different cell types that are close in the lineage will exhibit many common genes that are co-expressed relative to those that are far in the lineage. Current analysis methods tend to ignore this correlation by testing for differential expression assuming some sort of independence between cell types. We employ a Bayesian approach to estimate the posterior distribution of the mean of expression in each cell type, by taking into account the cell formation path in the lineage. This enables us to infer genes that are specific in each cell type, indicating the genes are involved in directing the cell differentiation to that particular cell type. We illustrate the method using gene expression data from a study of haematopoiesis. R codes to perform the analysis are available in http://www1.maths.leeds.ac.uk/∼arief/R/CellDiff/. a.gusnanto@leeds.ac.uk. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Systems Genetic Analysis of Osteoblast-Lineage Cells

PubMed Central

Calabrese, Gina; Bennett, Brian J.; Orozco, Luz; Kang, Hyun M.; Eskin, Eleazar; Dombret, Carlos; De Backer, Olivier; Lusis, Aldons J.; Farber, Charles R.

2012-01-01

The osteoblast-lineage consists of cells at various stages of maturation that are essential for skeletal development, growth, and maintenance. Over the past decade, many of the signaling cascades that regulate this lineage have been elucidated; however, little is known of the networks that coordinate, modulate, and transmit these signals. Here, we identify a gene network specific to the osteoblast-lineage through the reconstruction of a bone co-expression network using microarray profiles collected on 96 Hybrid Mouse Diversity Panel (HMDP) inbred strains. Of the 21 modules that comprised the bone network, module 9 (M9) contained genes that were highly correlated with prototypical osteoblast maker genes and were more highly expressed in osteoblasts relative to other bone cells. In addition, the M9 contained many of the key genes that define the osteoblast-lineage, which together suggested that it was specific to this lineage. To use the M9 to identify novel osteoblast genes and highlight its biological relevance, we knocked-down the expression of its two most connected “hub” genes, Maged1 and Pard6g. Their perturbation altered both osteoblast proliferation and differentiation. Furthermore, we demonstrated the mice deficient in Maged1 had decreased bone mineral density (BMD). It was also discovered that a local expression quantitative trait locus (eQTL) regulating the Wnt signaling antagonist Sfrp1 was a key driver of the M9. We also show that the M9 is associated with BMD in the HMDP and is enriched for genes implicated in the regulation of human BMD through genome-wide association studies. In conclusion, we have identified a physiologically relevant gene network and used it to discover novel genes and regulatory mechanisms involved in the function of osteoblast-lineage cells. Our results highlight the power of harnessing natural genetic variation to generate co-expression networks that can be used to gain insight into the function of specific cell-types. PMID:23300464

Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches

PubMed Central

Wu, Jincheng; Tzanakakis, Emmanuel S.

2014-01-01

Isogenic stem cell populations display cell-to-cell variations in a multitude of attributes including gene or protein expression, epigenetic state, morphology, proliferation and proclivity for differentiation. The origins of the observed heterogeneity and its roles in the maintenance of pluripotency and the lineage specification of stem cells remain unclear. Addressing pertinent questions will require the employment of single-cell analysis methods as traditional cell biochemical and biomolecular assays yield mostly population-average data. In addition to time-lapse microscopy and flow cytometry, recent advances in single-cell genomic, transcriptomic and proteomic profiling are reviewed. The application of multiple displacement amplification, next generation sequencing, mass cytometry and spectrometry to stem cell systems is expected to provide a wealth of information affording unprecedented levels of multiparametric characterization of cell ensembles under defined conditions promoting pluripotency or commitment. Establishing connections between single-cell analysis information and the observed phenotypes will also require suitable mathematical models. Stem cell self-renewal and differentiation are orchestrated by the coordinated regulation of subcellular, intercellular and niche-wide processes spanning multiple time scales. Here, we discuss different modeling approaches and challenges arising from their application to stem cell populations. Integrating single-cell analysis with computational methods will fill gaps in our knowledge about the functions of heterogeneity in stem cell physiology. This combination will also aid the rational design of efficient differentiation and reprogramming strategies as well as bioprocesses for the production of clinically valuable stem cell derivatives. PMID:24035899

Zika Virus Exhibits Lineage-Specific Phenotypes in Cell Culture, in Aedes aegypti Mosquitoes, and in an Embryo Model

PubMed Central

Demakovsky, Leah; Tesla, Blanka; Goodfellow, Forrest T.; Stice, Steven L.; Murdock, Courtney C.

2017-01-01

Zika virus (ZIKV) has quietly circulated in Africa and Southeast Asia for the past 65 years. However, the recent ZIKV epidemic in the Americas propelled this mosquito-borne virus to the forefront of flavivirus research. Based on historical evidence, ZIKV infections in Africa were sporadic and caused mild symptoms such as fever, skin rash, and general malaise. In contrast, recent Asian-lineage ZIKV infections in the Pacific Islands and the Americas are linked to birth defects and neurological disorders. The aim of this study is to compare replication, pathogenicity, and transmission efficiency of two historic and two contemporary ZIKV isolates in cell culture, the mosquito host, and an embryo model to determine if genetic variation between the African and Asian lineages results in phenotypic differences. While all tested isolates replicated at similar rates in Vero cells, the African isolates displayed more rapid viral replication in the mosquito C6/36 cell line, yet they exhibited poor infection rates in Aedes aegypti mosquitoes compared to the contemporary Asian-lineage isolates. All isolates could infect chicken embryos; however, infection with African isolates resulted in higher embryo mortality than infection with Asian-lineage isolates. These results suggest that genetic variation between ZIKV isolates can significantly alter experimental outcomes. PMID:29258204

Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Hedgehog-induced medulloblastoma

PubMed Central

Schüller, Ulrich; Heine, Vivi M.; Mao, Junhao; Kho, Alvin T.; Dillon, Allison K.; Han, Young-Goo; Huillard, Emmanuelle; Sun, Tao; Ligon, Azra H.; Qian, Ying; Ma, Qiufu; Alvarez-Buylla, Arturo; McMahon, Andrew P.; Rowitch, David H.; Ligon, Keith L.

2008-01-01

Origins of the brain tumor, medulloblastoma, from stem cells or restricted progenitor cells are unclear. To investigate this, we activated oncogenic Hedgehog (Hh) signaling in multipotent and lineage-restricted CNS progenitors. We observed that normal unipotent cerebellar granule neuron precursors (CGNP) derive from hGFAP+ and Olig2+ RL progenitors. Hh activation in a spectrum of early and late stage CNS progenitors generated similar medulloblastomas, but not other brain cancers, indicating that acquisition of CGNP identity is essential for tumorigenesis. We show in human and mouse medulloblastoma that cells expressing the glia-associated markers Gfap and Olig2 are neoplastic and that they retain features of embryonic-type granule lineage progenitors. Thus, oncogenic Hh signaling promotes medulloblastoma from lineage-restricted granule cell progenitors. PMID:18691547

Current progress in use of adipose derived stem cells in peripheral nerve regeneration

PubMed Central

Zack-Williams, Shomari DL; Butler, Peter E; Kalaskar, Deepak M

2015-01-01

Unlike central nervous system neurons; those in the peripheral nervous system have the potential for full regeneration after injury. Following injury, recovery is controlled by schwann cells which replicate and modulate the subsequent immune response. The level of nerve recovery is strongly linked to the severity of the initial injury despite the significant advancements in imaging and surgical techniques. Multiple experimental models have been used with varying successes to augment the natural regenerative processes which occur following nerve injury. Stem cell therapy in peripheral nerve injury may be an important future intervention to improve the best attainable clinical results. In particular adipose derived stem cells (ADSCs) are multipotent mesenchymal stem cells similar to bone marrow derived stem cells, which are thought to have neurotrophic properties and the ability to differentiate into multiple lineages. They are ubiquitous within adipose tissue; they can form many structures resembling the mature adult peripheral nervous system. Following early in vitro work; multiple small and large animal in vivo models have been used in conjunction with conduits, autografts and allografts to successfully bridge the peripheral nerve gap. Some of the ADSC related neuroprotective and regenerative properties have been elucidated however much work remains before a model can be used successfully in human peripheral nerve injury (PNI). This review aims to provide a detailed overview of progress made in the use of ADSC in PNI, with discussion on the role of a tissue engineered approach for PNI repair. PMID:25621105

Micropost microenvironments for studying luminal-basal lineage commitment of breast cancer cells

NASA Astrophysics Data System (ADS)

Kesavaraju, Anand; Qing, Bo; Jabart, Eric; Labarge, Mark; Sohn, Lydia

2013-03-01

MCF-7 breast cancer cells were plated onto polydimethylsiloxane (PDMS) microposts in order to examine the effects of the microenvironment on cell lineage. Different stiffnesses and sizes of the microposts are postulated to impact cell surface marker expression levels. We will provide preliminary results analyzing CD271 and focal adhesion markers such as vinculin. 3D shear flow will also be applied to the microposts to study how external mechanical stimuli affect cancer cells within their microenvironment.

Temporal and non-permanent division of labor during sorocarp formation in the social amoeba Acytostelium subglobosum.

PubMed

Mohri, Kurato; Kiyota, Yu; Kuwayama, Hidekazu; Urushihara, Hideko

2013-03-15

Somatic cell differentiation is crucial for the development of multicellular organisms. While the development of a fruiting body in Dictyostelium discoideum represents a simple model of this process with separation of stalk cells from the spore lineage, that of Acytostelium subglobosum is not accompanied by cell type separation. This species produces acellular stalks and, seemingly, all aggregated amoebae become spores; however, it possesses homologs for the stalk-cell marker genes of D. discoideum. In this study, we analyzed the spatio-temporal expression of A. subglobosum orthologs for D. discoideum stalk- or spore-lineage markers to clarify the developmental process of A. subglobosum. We first found that the prespore vesicles, which contained spore coat proteins, started to accumulate in the tip region and were observed in the entire sorogen throughout later development, confirming that all A. subglobosum cells became spores. The expression of a stalk-lineage gene ortholog, As-ecmA, started at the mound stage and was prominent in the protruding sorogen. Although two spore-lineage gene orthologs, As-cotD1 and -cotD2, were likewise detected shortly after cell aggregation and increased in intensity until tip formation, their expression diminished in the protruding sorogen. Double-fluorescence staining of these prestalk and prespore marker genes revealed that the expression of these marker genes was mutually exclusive and that expression switching occurred in the early tip. Our results indicate that A. subglobosum cells become committed to the spore lineage first, and then, while keeping this commitment intact, participate in stalk formation. Instead of the permanent division of labor observed in D. discoideum, A. subglobosum produces fruiting bodies by all cells contributing to the formation of the stalk as well as forming spores. Copyright © 2013 Elsevier Inc. All rights reserved.

Dynamic balance between master transcription factors determines the fates and functions of CD4 T cell and innate lymphoid cell subsets

PubMed Central

2017-01-01

CD4 T cells, including T regulatory cells (Treg cells) and effector T helper cells (Th cells), and recently identified innate lymphoid cells (ILCs) play important roles in host defense and inflammation. Both CD4 T cells and ILCs can be classified into distinct lineages based on their functions and the expression of lineage-specific genes, including those encoding effector cytokines, cell surface markers, and key transcription factors. It was first recognized that each lineage expresses a specific master transcription factor and the expression of these factors is mutually exclusive because of cross-regulation among these factors. However, recent studies indicate that the master regulators are often coexpressed. Furthermore, the expression of master regulators can be dynamic and quantitative. In this review, we will first discuss similarities and differences between the development and functions of CD4 T cell and ILC subsets and then summarize recent literature on quantitative, dynamic, and cell type–specific balance between the master transcription factors in determining heterogeneity and plasticity of these subsets. PMID:28630089

Whole genome sequencing analyses of Listeria monocytogenes that persisted in a milkshake machine for a year and caused illnesses in Washington State.

PubMed

Li, Zhen; Pérez-Osorio, Ailyn; Wang, Yu; Eckmann, Kaye; Glover, William A; Allard, Marc W; Brown, Eric W; Chen, Yi

2017-06-15

In 2015, in addition to a United States multistate outbreak linked to contaminated ice cream, another outbreak linked to ice cream was reported in the Pacific Northwest of the United States. It was a hospital-acquired outbreak linked to milkshakes, made from contaminated ice cream mixes and milkshake maker, served to patients. Here we performed multiple analyses on isolates associated with this outbreak: pulsed-field gel electrophoresis (PFGE), whole genome single nucleotide polymorphism (SNP) analysis, species-specific core genome multilocus sequence typing (cgMLST), lineage-specific cgMLST and whole genome-specific MLST (wgsMLST)/outbreak-specific cgMLST. We also analyzed the prophages and virulence genes. The outbreak isolates belonged to sequence type 1038, clonal complex 101, genetic lineage II. There were no pre-mature stop codons in inlA. Isolates contained Listeria Pathogenicity Island 1 and multiple internalins. PFGE and multiple whole genome sequencing (WGS) analyses all clustered together food, environmental and clinical isolates when compared to outgroup from the same clonal complex, which supported the finding that L. monocytogenes likely persisted in the soft serve ice cream/milkshake maker from November 2014 to November 2015 and caused 3 illnesses, and that the outbreak strain was transmitted between two ice cream production facilities. The whole genome SNP analysis, one of the two species-specific cgMLST, the lineage II-specific cgMLST and the wgsMLST/outbreak-specific cgMLST showed that L. monocytogenes cells persistent in the milkshake maker for a year formed a unique clade inside the outbreak cluster. This clustering was consistent with the cleaning practice after the outbreak was initially recognized in late 2014 and early 2015. Putative prophages were conserved among prophage-containing isolates. The loss of a putative prophage in two isolates resulted in the loss of the AscI restriction site in the prophage, which contributed to their AscI-PFGE banding pattern differences from other isolates. The high resolution of WGS analyses allowed the differentiation of epidemiologically unrelated isolates, as well as the elucidation of the microevolution and persistence of isolates within the scope of one outbreak. We applied a wgsMLST scheme which is essentially the outbreak-specific cgMLST. This scheme can be combined with lineage-specific cgMLST and species-specific cgMLST to maximize the resolution of WGS.

Evidence for the evolution of tenascin and fibronectin early in the chordate lineage.

PubMed

Tucker, Richard P; Chiquet-Ehrismann, Ruth

2009-02-01

Fibronectin and tenascin are extracellular matrix glycoproteins that play important roles in cell adhesion and motility. In a previous study we provided evidence that tenascin first appeared early in the chordate lineage. As tenascin has been proposed to act, in part, through modulation of cell-fibronectin interactions, we sought here to identify fibronectin genes in non-vertebrate chordates and other invertebrates to determine if tenascin and fibronectin evolved separately or together, and to identify phylogenetically conserved features of both proteins. We found that the genome of the urochordate Ciona savignyi contains both a tenascin gene and a gene encoding a fibronectin-like protein with fibronectin type 1, 2 and 3 repeats. The genome of the cephalochordate Branchiostoma floridae (amphioxus) also has a tenascin gene. However, we could not identify a fibronectin-like gene in B. floridae, nor could we identify fibronectin or tenascin genes in echinoderms, protostomes or cnidarians. If urochordates are more closely related to vertebrates, tenascin may have evolved before fibronectin in an ancestor common to tunicates and amphioxus. Alternatively, tenascin and fibronectin may have evolved in an ancestor common to B. floridae and C. savignyi and the fibronectin gene was subsequently lost in the cephalochordate lineage. The fibronectin-like gene from C. savignyi does not encode the RGD motif for integrin binding found in all vertebrate fibronectins, and it lacks most of the fibronectin type 1 domains believed to be critical for fibrillogenesis. In contrast, the tenascin gene in B. floridae encodes multiple RGD motifs, suggesting that integrin binding is fundamental to tenascin function.

Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages.

PubMed

Arsic, Nikola; Mamaeva, Daria; Lamb, Ned J; Fernandez, Anne

2008-04-01

Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal beta III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.

Invariant NKT cells inhibit development of the Th17 lineage

PubMed Central

Mars, Lennart T.; Araujo, Luiza; Kerschen, Philippe; Diem, Séverine; Bourgeois, Elvire; Van, Linh Pham; Carrié, Nadège; Dy, Michel; Liblau, Roland S.; Herbelin, André

2009-01-01

T cells differentiate into functionally distinct effector subsets in response to pathogen encounter. Cells of the innate immune system direct this process; CD1d-restricted invariant natural killer T (iNKT) cells, for example, can either promote or inhibit Th1 and Th2 responses. Recently, a new subset of CD4+ T helper cells, called Th17, was identified that is implicated in mucosal immunity and autoimmune disorders. To investigate the influence of iNKT cells on the differentiation of naïve T cells we used an adoptive transfer model of traceable antigen-specific CD4+ T cells. Transferred naïve CD25−CD62L+ CD4+ T cells were primed by antigen immunization of the recipient mice, permitting their expansion and Th17 differentiation. This study establishes that in vivo activation of iNKT cells during T-cell priming impedes the commitment of naïve T cells to the Th17 lineage. In vivo cytokine neutralization experiments revealed a role for IL-4, IL-10, and IFN-γ in the iNKT-cell-mediated regulation of T-cell lineage development. Moreover, by comparing IL-17 production by antigen-experienced T cells from unmanipulated wild-type mice and iNKT-cell-deficient mice, we demonstrate an enhanced Th17 response in mice lacking iNKT cells. This invigorated Th17 response reverts to physiological levels when iNKT cells are introduced into Jα18−/− mice by adoptive transfer, indicating that iNKT cells control the Th17 compartment at steady state. We conclude that iNKT cells play an important role in limiting development of the Th17 lineage and suggest that iNKT cells provide a natural barrier against Th17 responses. PMID:19325124

Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

PubMed

Dorn, Tatjana; Kornherr, Jessica; Parrotta, Elvira I; Zawada, Dorota; Ayetey, Harold; Santamaria, Gianluca; Iop, Laura; Mastantuono, Elisa; Sinnecker, Daniel; Goedel, Alexander; Dirschinger, Ralf J; My, Ilaria; Laue, Svenja; Bozoglu, Tarik; Baarlink, Christian; Ziegler, Tilman; Graf, Elisabeth; Hinkel, Rabea; Cuda, Giovanni; Kääb, Stefan; Grace, Andrew A; Grosse, Robert; Kupatt, Christian; Meitinger, Thomas; Smith, Austin G; Laugwitz, Karl-Ludwig; Moretti, Alessandra

2018-06-15

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Sox2+ Stem Cells Contribute to All Epithelial Lineages of the Tooth via Sfrp5+ Progenitors

PubMed Central

Juuri, Emma; Saito, Kan; Ahtiainen, Laura; Seidel, Kerstin; Tummers, Mark; Hochedlinger, Konrad; Klein, Ophir D.; Thesleff, Irma; Michon, Frederic

2012-01-01

SUMMARY The continuously growing mouse incisor serves as a valuable model to study stem cell regulation during organ renewal. Epithelial stem cells are localized in the proximal end of the incisor in the labial cervical loop. Here, we show that the transcription factor Sox2 is a specific marker for these stem cells. Sox2+ cells became restricted to the labial cervical loop during tooth morphogenesis, and they contributed to the renewal of enamel-producing ameloblasts as well as all other epithelial cell lineages of the tooth. The early progeny of Sox2-positive stem cells transiently expressed the Wnt inhibitor Sfrp5. Sox2 expression was regulated by the tooth initiation marker FGF8 and specific miRNAs, suggesting a fine-tuning to maintain homeostasis of the dental epithelium. The identification of Sox2 as a marker for the dental epithelial stem cells will facilitate further studies on their lineage segregation and differentiation during tooth renewal. PMID:22819339

TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells.

PubMed

Tsagaratou, Ageliki; González-Avalos, Edahí; Rautio, Sini; Scott-Browne, James P; Togher, Susan; Pastor, William A; Rothenberg, Ellen V; Chavez, Lukas; Lähdesmäki, Harri; Rao, Anjana

2017-01-01

TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4 + CD8 + double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).

Inner ear progenitor cells can be generated in vitro from human bone marrow mesenchymal stem cells.

PubMed

Boddy, Sarah L; Chen, Wei; Romero-Guevara, Ricardo; Kottam, Lucksy; Bellantuono, Illaria; Rivolta, Marcelo N

2012-11-01

Mouse mesenchymal stem cells (MSCs) can generate sensory neurons and produce inner ear hair cell-like cells. An equivalent source from humans is highly desirable, given their potential application in patient-specific regenerative therapies for deafness. In this study, we explored the ability of human MSCs (hMSCs) to differentiate into otic lineages. hMSCs were exposed to culture media conditioned by human fetal auditory stem cells. Conditioned media induced the expression of otic progenitor markers PAX8, PAX2, GATA3 and SOX2. After 4 weeks, cells coexpressed ATOH1, MYO7A and POU4F3 (indicators of hair cell lineage) or neuronal markers NEUROG1, POU4F1 and NEFH. Inhibition of WNT signaling prevented differentiation into otic progenitors, while WNT activation partially phenocopied results seen with the conditioned media. This study demonstrates that hMSCs can be driven to express key genes found in the otic lineages and thereby promotes their status as candidates for regenerative therapies for deafness.

Conserved Genetic Pathways Controlling the Development of the Diffuse Endocrine System in Vertebrates and Drosophila

PubMed Central

Hartenstein, Volker; Takashima, Shigeo; Adams, Katrina

2014-01-01

The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type. PMID:20005229

Cosmopolitan Species As Models for Ecophysiological Responses to Global Change: The Common Reed Phragmites australis.

PubMed

Eller, Franziska; Skálová, Hana; Caplan, Joshua S; Bhattarai, Ganesh P; Burger, Melissa K; Cronin, James T; Guo, Wen-Yong; Guo, Xiao; Hazelton, Eric L G; Kettenring, Karin M; Lambertini, Carla; McCormick, Melissa K; Meyerson, Laura A; Mozdzer, Thomas J; Pyšek, Petr; Sorrell, Brian K; Whigham, Dennis F; Brix, Hans

2017-01-01

Phragmites australis is a cosmopolitan grass and often the dominant species in the ecosystems it inhabits. Due to high intraspecific diversity and phenotypic plasticity, P. australis has an extensive ecological amplitude and a great capacity to acclimate to adverse environmental conditions; it can therefore offer valuable insights into plant responses to global change. Here we review the ecology and ecophysiology of prominent P. australis lineages and their responses to multiple forms of global change. Key findings of our review are that: (1) P. australis lineages are well-adapted to regions of their phylogeographic origin and therefore respond differently to changes in climatic conditions such as temperature or atmospheric CO 2 ; (2) each lineage consists of populations that may occur in geographically different habitats and contain multiple genotypes; (3) the phenotypic plasticity of functional and fitness-related traits of a genotype determine the responses to global change factors; (4) genotypes with high plasticity to environmental drivers may acclimate or even vastly expand their ranges, genotypes of medium plasticity must acclimate or experience range-shifts, and those with low plasticity may face local extinction; (5) responses to ancillary types of global change, like shifting levels of soil salinity, flooding, and drought, are not consistent within lineages and depend on adaptation of individual genotypes. These patterns suggest that the diverse lineages of P. australis will undergo intense selective pressure in the face of global change such that the distributions and interactions of co-occurring lineages, as well as those of genotypes within-lineages, are very likely to be altered. We propose that the strong latitudinal clines within and between P. australis lineages can be a useful tool for predicting plant responses to climate change in general and present a conceptual framework for using P. australis lineages to predict plant responses to global change and its consequences.

Comparative characterization of stromal vascular cells derived from three types of vascular wall and adipose tissue.

PubMed

Yang, Santsun; Eto, Hitomi; Kato, Harunosuke; Doi, Kentaro; Kuno, Shinichiro; Kinoshita, Kahori; Ma, Hsu; Tsai, Chi-Han; Chou, Wan-Ting; Yoshimura, Kotaro

2013-12-01

Multipotent stem/progenitor cells localize perivascularly in many organs and vessel walls. These tissue-resident stem/progenitor cells differentiate into vascular endothelial cells, pericytes, and other mesenchymal lineages, and participate in physiological maintenance and repair of vasculatures. In this study, we characterized stromal vascular cells obtained through the explant culture method from three different vessel walls in humans: arterial wall (ART; >500 μm in diameter), venous wall (VN; >500 μm in diameter), and small vessels in adipose tissue (SV; arterioles and venules, <100 μm in diameter). These were examined for functionality and compared with adipose-derived stem/stromal cells (ASCs). All stromal vascular cells of different origins presented fibroblast-like morphology and we could not visually discriminate one population from another. Flow cytometry showed that the cultured population heterogeneously expressed a variety of surface antigens associated with stem/progenitor cells, but CD105 was expressed by most cells in all groups, suggesting that the cells generally shared the characteristics of mesenchymal stem cells. Our histological and flow cytometric data suggested that the main population of vessel wall-derived stromal vascular cells were CD34(+)/CD31(-) and came from the tunica adventitia and areola tissue surrounding the adventitia. CD271 (p75NTR) was expressed by the vasa vasorum in the VN adventitia and by a limited population in the adventitia of SV. All three populations differentiated into multiple lineages as did ASCs. ART cells induced the largest quantity of calcium formation in the osteogenic medium, whereas ASCs showed the greatest adipogenic differentiation. SV and VN stromal cells had greater potency for network formation than did ART stromal cells. In conclusion, the three stromal vascular populations exhibited differential functional properties. Our results have clinical implications for vascular diseases such as arterial wall calcification and possible applications to regenerative therapies involving each vessel wall-resident stromal population.

A post-transcriptional mechanism pacing expression of neural genes with precursor cell differentiation status

PubMed Central

Dai, Weijun; Li, Wencheng; Hoque, Mainul; Li, Zhuyun; Tian, Bin; Makeyev, Eugene V.

2015-01-01

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3′-untranslated regions (3′ UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36), an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly, TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand, inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons. PMID:26144867

Evolutionary Convergence of Cell-Specific Gene Expression in Independent Lineages of C4 Grasses1[W][OPEN

PubMed Central

John, Christopher R.; Smith-Unna, Richard D.; Woodfield, Helen; Covshoff, Sarah; Hibberd, Julian M.

2014-01-01

Leaves of almost all C4 lineages separate the reactions of photosynthesis into the mesophyll (M) and bundle sheath (BS). The extent to which messenger RNA profiles of M and BS cells from independent C4 lineages resemble each other is not known. To address this, we conducted deep sequencing of RNA isolated from the M and BS of Setaria viridis and compared these data with publicly available information from maize (Zea mays). This revealed a high correlation (r = 0.89) between the relative abundance of transcripts encoding proteins of the core C4 pathway in M and BS cells in these species, indicating significant convergence in transcript accumulation in these evolutionarily independent C4 lineages. We also found that the vast majority of genes encoding proteins of the C4 cycle in S. viridis are syntenic to homologs used by maize. In both lineages, 122 and 212 homologous transcription factors were preferentially expressed in the M and BS, respectively. Sixteen shared regulators of chloroplast biogenesis were identified, 14 of which were syntenic homologs in maize and S. viridis. In sorghum (Sorghum bicolor), a third C4 grass, we found that 82% of these trans-factors were also differentially expressed in either M or BS cells. Taken together, these data provide, to our knowledge, the first quantification of convergence in transcript abundance in the M and BS cells from independent lineages of C4 grasses. Furthermore, the repeated recruitment of syntenic homologs from large gene families strongly implies that parallel evolution of both structural genes and trans-factors underpins the polyphyletic evolution of this highly complex trait in the monocotyledons. PMID:24676859

Cell-type-specific expression of NFIX in the developing and adult cerebellum.

PubMed

Fraser, James; Essebier, Alexandra; Gronostajski, Richard M; Boden, Mikael; Wainwright, Brandon J; Harvey, Tracey J; Piper, Michael

2017-07-01

Transcription factors from the nuclear factor one (NFI) family have been shown to play a central role in regulating neural progenitor cell differentiation within the embryonic and post-natal brain. NFIA and NFIB, for instance, promote the differentiation and functional maturation of granule neurons within the cerebellum. Mice lacking Nfix exhibit delays in the development of neuronal and glial lineages within the cerebellum, but the cell-type-specific expression of this transcription factor remains undefined. Here, we examined the expression of NFIX, together with various cell-type-specific markers, within the developing and adult cerebellum using both chromogenic immunohistochemistry and co-immunofluorescence labelling and confocal microscopy. In embryos, NFIX was expressed by progenitor cells within the rhombic lip and ventricular zone. After birth, progenitor cells within the external granule layer, as well as migrating and mature granule neurons, expressed NFIX. Within the adult cerebellum, NFIX displayed a broad expression profile, and was evident within granule cells, Bergmann glia, and interneurons, but not within Purkinje neurons. Furthermore, transcriptomic profiling of cerebellar granule neuron progenitor cells showed that multiple splice variants of Nfix are expressed within this germinal zone of the post-natal brain. Collectively, these data suggest that NFIX plays a role in regulating progenitor cell biology within the embryonic and post-natal cerebellum, as well as an ongoing role within multiple neuronal and glial populations within the adult cerebellum.

Histone Methylation and microRNA-dependent Regulation of Epigenetic Activities in Neural Progenitor Self-Renewal and Differentiation.

PubMed

Cacci, Emanuele; Negri, Rodolfo; Biagioni, Stefano; Lupo, Giuseppe

2017-01-01

Neural stem/progenitor cell (NSPC) self-renewal and differentiation in the developing and the adult brain are controlled by extra-cellular signals and by the inherent competence of NSPCs to produce appropriate responses. Stage-dependent responsiveness of NSPCs to extrinsic cues is orchestrated at the epigenetic level. Epigenetic mechanisms such as DNA methylation, histone modifications and non-coding RNA-mediated regulation control crucial aspects of NSPC development and function, and are also implicated in pathological conditions. While their roles in the regulation of stem cell fate have been largely explored in pluripotent stem cell models, the epigenetic signature of NSPCs is also key to determine their multipotency as well as their progressive bias towards specific differentiation outcomes. Here we review recent developments in this field, focusing on the roles of histone methylation marks and the protein complexes controlling their deposition in NSPCs of the developing cerebral cortex and the adult subventricular zone. In this context, we describe how bivalent promoters, carrying antagonistic epigenetic modifications, feature during multiple steps of neural development, from neural lineage specification to neuronal differentiation. Furthermore, we discuss the emerging cross-talk between epigenetic regulators and microRNAs, and how the interplay between these different layers of regulation can finely tune the expression of genes controlling NSPC maintenance and differentiation. In particular, we highlight recent advances in the identification of astrocyte-enriched microRNAs and their function in cell fate choices of NSPCs differentiating towards glial lineages.

Controlling Differentiation of Stem Cells for Developing Personalized Organ-on-Chip Platforms.

PubMed

Geraili, Armin; Jafari, Parya; Hassani, Mohsen Sheikh; Araghi, Behnaz Heidary; Mohammadi, Mohammad Hossein; Ghafari, Amir Mohammad; Tamrin, Sara Hasanpour; Modarres, Hassan Pezeshgi; Kolahchi, Ahmad Rezaei; Ahadian, Samad; Sanati-Nezhad, Amir

2018-01-01

Organ-on-chip (OOC) platforms have attracted attentions of pharmaceutical companies as powerful tools for screening of existing drugs and development of new drug candidates. OOCs have primarily used human cell lines or primary cells to develop biomimetic tissue models. However, the ability of human stem cells in unlimited self-renewal and differentiation into multiple lineages has made them attractive for OOCs. The microfluidic technology has enabled precise control of stem cell differentiation using soluble factors, biophysical cues, and electromagnetic signals. This study discusses different tissue- and organ-on-chip platforms (i.e., skin, brain, blood-brain barrier, bone marrow, heart, liver, lung, tumor, and vascular), with an emphasis on the critical role of stem cells in the synthesis of complex tissues. This study further recaps the design, fabrication, high-throughput performance, and improved functionality of stem-cell-based OOCs, technical challenges, obstacles against implementing their potential applications, and future perspectives related to different experimental platforms. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita

2010-03-12

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activitymore » in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.« less

SBR-Blood: systems biology repository for hematopoietic cells.

PubMed

Lichtenberg, Jens; Heuston, Elisabeth F; Mishra, Tejaswini; Keller, Cheryl A; Hardison, Ross C; Bodine, David M

2016-01-04

Extensive research into hematopoiesis (the development of blood cells) over several decades has generated large sets of expression and epigenetic profiles in multiple human and mouse blood cell types. However, there is no single location to analyze how gene regulatory processes lead to different mature blood cells. We have developed a new database framework called hematopoietic Systems Biology Repository (SBR-Blood), available online at http://sbrblood.nhgri.nih.gov, which allows user-initiated analyses for cell type correlations or gene-specific behavior during differentiation using publicly available datasets for array- and sequencing-based platforms from mouse hematopoietic cells. SBR-Blood organizes information by both cell identity and by hematopoietic lineage. The validity and usability of SBR-Blood has been established through the reproduction of workflows relevant to expression data, DNA methylation, histone modifications and transcription factor occupancy profiles. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Different roles of TGF-β in the multi-lineage differentiation of stem cells

PubMed Central

Wang, Ming-Ke; Sun, Hui-Qin; Xiang, Ying-Chun; Jiang, Fan; Su, Yong-Ping; Zou, Zhong-Min

2012-01-01

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells. Transforming growth factor β (TGF-β) family is a superfamily of growth factors, including TGF-β1, TGF-β2 and TGF-β3, bone morphogenetic proteins, activin/inhibin, and some other cytokines such as nodal, which plays very important roles in regulating a wide variety of biological processes, such as cell growth, differentiation, cell death. TGF-β, a pleiotropic cytokine, has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells, through the Smad pathway, non-Smad pathways including mitogen-activated protein kinase pathways, phosphatidylinositol-3-kinase/AKT pathways and Rho-like GTPase signaling pathways, and their cross-talks. For instance, it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells, immature cardiomyocytes, chondrocytes, neurocytes, hepatic stellate cells, Th17 cells, and dendritic cells. However, TGF-β inhibits the differentiation of stem cells into myotubes, adipocytes, endothelial cells, and natural killer cells. Additionally, TGF-β can provide competence for early stages of osteoblastic differentiation, but at late stages TGF-β acts as an inhibitor. The three mammalian isoforms (TGF-β1, 2 and 3) have distinct but overlapping effects on hematopoiesis. Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology, and will facilitate both basic research and clinical applications of stem cells. In this article, we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells. PMID:22993659

ADAM10 Regulates Notch Function in Intestinal Stem Cells of Mice

PubMed Central

Tsai, Yu-Hwai; VanDussen, Kelli L.; Sawey, Eric T.; Wade, Alex W.; Kasper, Chelsea; Rakshit, Sabita; Bhatt, Riha G.; Stoeck, Alex; Maillard, Ivan; Crawford, Howard C.; Samuelson, Linda C.; Dempsey, Peter J.

2014-01-01

BACKGROUND & AIMS ADAM10 is a cell surface sheddase that regulates physiological processes including Notch signaling. ADAM10 is expressed in all intestinal epithelial cell types but the requirement for ADAM10 signaling in crypt homeostasis is not well defined. METHODS We analyzed intestinal tissues from mice with constitutive (Vil-Cre;Adam10f/f mice) and conditional (Vil-CreER;Adam10f/f and Lgr5-CreER;Adam10f/f mice) deletion of ADAM10. We performed cell lineage tracing experiments in mice that expressed a gain-of-function allele of Notch in the intestine (Rosa26NICD) or mice with intestine-specific disruption of Notch (Rosa26DN-MAML), to examine the effects of ADAM10 deletion on cell fate specification and intestinal stem cell maintenance. RESULTS Loss of ADAM10 from developing and adult intestine caused lethality associated with altered intestinal morphology, reduced progenitor cell proliferation, and increased secretory cell differentiation. ADAM10 deletion led to the replacement of intestinal cell progenitors with 2 distinct, post-mitotic, secretory cell lineages: intermediate-like (Paneth/goblet) and enteroendocrine cells. Based on analysis of Rosa26NICD and Rosa26DN-MAML mice, we determined that ADAM10 controls these cell fate decisions by regulating Notch signaling. Cell lineage tracing experiments showed that ADAM10 is required for survival of Lgr5+ crypt-based columnar cells. Our findings indicate that Notch-activated stem cells have a competitive advantage for occupation of the stem cell niche. CONCLUSIONS ADAM10 acts in a cell autonomous manner within the intestinal crypt compartment to regulate Notch signaling. This process is required for progenitor cell lineage specification and crypt-based columnar cell maintenance. PMID:25038433

Native Human Monoclonal Antibodies with Potent Cross-Lineage Neutralization of Influenza B Viruses

PubMed Central

Vigil, Adam; Estélles, Angeles; Kauvar, Lawrence M.; Johnson, Scott K.

2018-01-01

ABSTRACT Although antibodies that effectively neutralize a broad set of influenza viruses exist in the human antibody repertoire, they are rare. We used a single-cell screening technology to identify rare monoclonal antibodies (MAbs) that recognized a broad set of influenza B viruses (IBV). The screen yielded 23 MAbs with diverse germ line origins that recognized hemagglutinins (HAs) derived from influenza strains of both the Yamagata and Victoria lineages of IBV. Of the 23 MAbs, 3 exhibited low expression in a transient-transfection system, 4 were neutralizers that bound to the HA head region, 11 were stalk-binding nonneutralizers, and 5 were stalk-binding neutralizers, with 4 of these 5 having unique antibody sequences. Of these four unique stalk-binding neutralizing MAbs, all were broadly reactive and neutralizing against a panel of multiple strains spanning both IBV lineages as well as highly effective in treating lethal IBV infections in mice at both 24 and 72 h postinfection. The MAbs in this group were thermostable and bound different epitopes in the highly conserved HA stalk region. These characteristics suggest that these MAbs are suitable for consideration as candidates for clinical studies to address their effectiveness in the treatment of IBV-infected patients. PMID:29507069

Liver-enriched transcription factors are critical for the expression of hepatocyte marker genes in mES-derived hepatocyte-lineage cells.

PubMed

Kheolamai, Pakpoom; Dickson, Alan J

2009-04-23

Induction of stem cell differentiation toward functional hepatocytes is hampered by lack of knowledge of the hepatocyte differentiation processes. The overall objective of this project is to characterize key stages in the hepatocyte differentiation process. We established a mouse embryonic stem (mES) cell culture system which exhibited changes in gene expression profiles similar to those observed in the development of endodermal and hepatocyte-lineage cells previously described in the normal mouse embryo. Transgenic mES cells were established that permitted isolation of enriched hepatocyte-lineage populations. This approach has isolated mES-derived hepatocyte-lineage cells that express several markers of mature hepatocytes including albumin, glucose-6-phosphatase, tyrosine aminotransferase, cytochrome P450-3a, phosphoenolpyruvate carboxykinase and tryptophan 2,3-dioxygenase. In addition, our results show that the up-regulation of the expression levels of hepatocyte nuclear factor-3alpha, -4alpha, -6, and CCAAT-enhancer binding protein-beta might be critical for passage into late-stage differentiation towards functional hepatocytes. These data present important steps for definition of regulatory phenomena that direct specific cell fate determination. The mES cell culture system generated in this study provides a model for studying transition between stages of the hepatocyte development and has significant potential value for studying the molecular basis of hepatocyte differentiation in vitro.

Lineage-Specific Restraint of Pituitary Gonadotroph Cell Adenoma Growth

PubMed Central

Chesnokova, Vera; Zonis, Svetlana; Zhou, Cuiqi; Ben-Shlomo, Anat; Wawrowsky, Kolja; Toledano, Yoel; Tong, Yunguang; Kovacs, Kalman; Scheithauer, Bernd; Melmed, Shlomo

2011-01-01

Although pituitary adenomas are usually benign, unique trophic mechanisms restraining cell proliferation are unclear. As GH-secreting adenomas are associated with p53/p21-dependent senescence, we tested mechanisms constraining non-functioning pituitary adenoma growth. Thirty six gonadotroph-derived non-functioning pituitary adenomas all exhibited DNA damage, but undetectable p21 expression. However, these adenomas all expressed p16, and >90% abundantly expressed cytoplasmic clusterin associated with induction of the Cdk inhibitor p15 in 70% of gonadotroph and in 26% of somatotroph lineage adenomas (p = 0.006). Murine LβT2 and αT3 gonadotroph pituitary cells, and αGSU.PTTG transgenic mice with targeted gonadotroph cell adenomas also abundantly expressed clusterin and exhibited features of oncogene-induced senescence as evidenced by C/EBPβ and C/EBPδ induction. In turn, C/EBPs activated the clusterin promoter ∼5 fold, and elevated clusterin subsequently elicited p15 and p16 expression, acting to arrest murine gonadotroph cell proliferation. In contrast, specific clusterin suppression by RNAis enhanced gonadotroph proliferation. FOXL2, a tissue-specific gonadotroph lineage factor, also induced the clusterin promoter ∼3 fold in αT3 pituitary cells. As nine of 12 pituitary carcinomas were devoid of clusterin expression, this protein may limit proliferation of benign adenomatous pituitary cells. These results point to lineage-specific pathways restricting uncontrolled murine and human pituitary gonadotroph adenoma cell growth. PMID:21464964

Systems and Trans-System Level Analysis Identifies Conserved Iron Deficiency Responses in the Plant Lineage[W][OA

PubMed Central

Urzica, Eugen I.; Casero, David; Yamasaki, Hiroaki; Hsieh, Scott I.; Adler, Lital N.; Karpowicz, Steven J.; Blaby-Haas, Crysten E.; Clarke, Steven G.; Loo, Joseph A.; Pellegrini, Matteo; Merchant, Sabeeha S.

2012-01-01

We surveyed the iron nutrition-responsive transcriptome of Chlamydomonas reinhardtii using RNA-Seq methodology. Presumed primary targets were identified in comparisons between visually asymptomatic iron-deficient versus iron-replete cells. This includes the known components of high-affinity iron uptake as well as candidates for distributive iron transport in C. reinhardtii. Comparison of growth-inhibited iron-limited versus iron-replete cells revealed changes in the expression of genes in chloroplastic oxidative stress response pathways, among hundreds of other genes. The output from the transcriptome was validated at multiple levels: by quantitative RT-PCR for assessing the data analysis pipeline, by quantitative proteomics for assessing the impact of changes in RNA abundance on the proteome, and by cross-species comparison for identifying conserved or universal response pathways. In addition, we assessed the functional importance of three target genes, VITAMIN C 2 (VTC2), MONODEHYDROASCORBATE REDUCTASE 1 (MDAR1), and CONSERVED IN THE GREEN LINEAGE AND DIATOMS 27 (CGLD27), by biochemistry or reverse genetics. VTC2 and MDAR1, which are key enzymes in de novo ascorbate synthesis and ascorbate recycling, respectively, are likely responsible for the 10-fold increase in ascorbate content of iron-limited cells. CGLD27/At5g67370 is a highly conserved, presumed chloroplast-localized pioneer protein and is important for growth of Arabidopsis thaliana in low iron. PMID:23043051

A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates.

PubMed

Plouhinec, Jean-Louis; Medina-Ruiz, Sofía; Borday, Caroline; Bernard, Elsa; Vert, Jean-Philippe; Eisen, Michael B; Harland, Richard M; Monsoro-Burq, Anne H

2017-10-01

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.

A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates

PubMed Central

Borday, Caroline; Bernard, Elsa; Vert, Jean-Philippe; Eisen, Michael B.; Harland, Richard M.

2017-01-01

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research. PMID:29049289

Y-chromosome lineage determines cardiovascular organ T-cell infiltration in the stroke-prone spontaneously hypertensive rat.

PubMed

Khan, Shanzana I; Andrews, Karen L; Jackson, Kristy L; Memon, Basimah; Jefferis, Ann-Maree; Lee, Man K S; Diep, Henry; Wei, Zihui; Drummond, Grant R; Head, Geoffrey A; Jennings, Garry L; Murphy, Andrew J; Vinh, Antony; Sampson, Amanda K; Chin-Dusting, Jaye P F

2018-05-01

The essential role of the Y chromosome in male sex determination has largely overshadowed the possibility that it may exert other biologic roles. Here, we show that Y-chromosome lineage is a strong determinant of perivascular and renal T-cell infiltration in the stroke-prone spontaneously hypertensive rat, which, in turn, may influence vascular function and blood pressure (BP). We also show, for the first time to our knowledge, that augmented perivascular T-cell levels can directly instigate vascular dysfunction, and that the production of reactive oxygen species that stimulate cyclo-oxygenase underlies this. We thus provide strong evidence for the consideration of Y-chromosome lineage in the diagnosis and treatment of male hypertension, and point to the modulation of cardiovascular organ T-cell infiltration as a possible mechanism that underpins Y- chromosome regulation of BP.-Khan, S. I., Andrews, K. L., Jackson, K. L., Memon, B., Jefferis, A.-M., Lee, M. K. S., Diep, H., Wei, Z., Drummond, G. R., Head, G. A., Jennings, G. L., Murphy, A. J., Vinh, A., Sampson, A. K., Chin-Dusting, J. P. F. Y-chromosome lineage determines cardiovascular organ T-cell infiltration in the stroke-prone spontaneously hypertensive rat.

Multiple maternal origins and weak phylogeographic structure in domestic goats

PubMed Central

Luikart, Gordon; Gielly, Ludovic; Excoffier, Laurent; Vigne, Jean-Denis; Bouvet, Jean; Taberlet, Pierre

2001-01-01

Domestic animals have played a key role in human history. Despite their importance, however, the origins of most domestic species remain poorly understood. We assessed the phylogenetic history and population structure of domestic goats by sequencing a hypervariable segment (481 bp) of the mtDNA control region from 406 goats representing 88 breeds distributed across the Old World. Phylogeographic analysis revealed three highly divergent goat lineages (estimated divergence >200,000 years ago), with one lineage occurring only in eastern and southern Asia. A remarkably similar pattern exists in cattle, sheep, and pigs. These results, combined with recent archaeological findings, suggest that goats and other farm animals have multiple maternal origins with a possible center of origin in Asia, as well as in the Fertile Crescent. The pattern of goat mtDNA diversity suggests that all three lineages have undergone population expansions, but that the expansion was relatively recent for two of the lineages (including the Asian lineage). Goat populations are surprisingly less genetically structured than cattle populations. In goats only ≈10% of the mtDNA variation is partitioned among continents. In cattle the amount is ≥50%. This weak structuring suggests extensive intercontinental transportation of goats and has intriguing implications about the importance of goats in historical human migrations and commerce. PMID:11344314

Apoptotic cell-induced AhR activity is required for immunological tolerance and suppression of systemic lupus erythematosus in mice and humans.

PubMed

Shinde, Rahul; Hezaveh, Kebria; Halaby, Marie Jo; Kloetgen, Andreas; Chakravarthy, Ankur; da Silva Medina, Tiago; Deol, Reema; Manion, Kieran P; Baglaenko, Yuriy; Eldh, Maria; Lamorte, Sara; Wallace, Drew; Chodisetti, Sathi Babu; Ravishankar, Buvana; Liu, Haiyun; Chaudhary, Kapil; Munn, David H; Tsirigos, Aristotelis; Madaio, Michael; Gabrielsson, Susanne; Touma, Zahi; Wither, Joan; De Carvalho, Daniel D; McGaha, Tracy L

2018-06-01

The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.

E3 ubiquitin ligase Cbl-b in innate and adaptive immunity

PubMed Central

Liu, Qingjun; Zhou, Hong; Langdon, Wallace Y; Zhang, Jian

2014-01-01

Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING finger E3 ubiquitin-protein ligase, has been demonstrated to play a crucial role in establishing the threshold for T-cell activation and controlling peripheral T-cell tolerance via multiple mechanisms. Accumulating evidence suggests that Cbl-b also regulates innate immune responses and plays an important role in host defense to pathogens. Understanding the signaling pathways regulated by Cbl-b in innate and adaptive immune cells is therefore essential for efficient manipulation of Cbl-b in emerging immunotherapies for human disorders such as autoimmune diseases, allergic inflammation, infections, and cancer. In this article, we review the latest developments in the molecular structural basis of Cbl-b function, the regulation of Cbl-b expression, the signaling mechanisms of Cbl-b in immune cells, as well as the biological function of Cbl-b in physiological and pathological immune responses in animal models and human diseases. PMID:24875217

Single-cell genomics of multiple uncultured stramenopiles reveals underestimated functional diversity across oceans.

PubMed

Seeleuthner, Yoann; Mondy, Samuel; Lombard, Vincent; Carradec, Quentin; Pelletier, Eric; Wessner, Marc; Leconte, Jade; Mangot, Jean-François; Poulain, Julie; Labadie, Karine; Logares, Ramiro; Sunagawa, Shinichi; de Berardinis, Véronique; Salanoubat, Marcel; Dimier, Céline; Kandels-Lewis, Stefanie; Picheral, Marc; Searson, Sarah; Pesant, Stephane; Poulton, Nicole; Stepanauskas, Ramunas; Bork, Peer; Bowler, Chris; Hingamp, Pascal; Sullivan, Matthew B; Iudicone, Daniele; Massana, Ramon; Aury, Jean-Marc; Henrissat, Bernard; Karsenti, Eric; Jaillon, Olivier; Sieracki, Mike; de Vargas, Colomban; Wincker, Patrick

2018-01-22

Single-celled eukaryotes (protists) are critical players in global biogeochemical cycling of nutrients and energy in the oceans. While their roles as primary producers and grazers are well appreciated, other aspects of their life histories remain obscure due to challenges in culturing and sequencing their natural diversity. Here, we exploit single-cell genomics and metagenomics data from the circumglobal Tara Oceans expedition to analyze the genome content and apparent oceanic distribution of seven prevalent lineages of uncultured heterotrophic stramenopiles. Based on the available data, each sequenced genome or genotype appears to have a specific oceanic distribution, principally correlated with water temperature and depth. The genome content provides hypotheses for specialization in terms of cell motility, food spectra, and trophic stages, including the potential impact on their lifestyles of horizontal gene transfer from prokaryotes. Our results support the idea that prominent heterotrophic marine protists perform diverse functions in ocean ecology.

Neural Stem Cells: Implications for the Conventional Radiotherapy of Central Nervous System Malignancies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barani, Igor J.; Benedict, Stanley H.; Lin, Peck-Sun

Advances in basic neuroscience related to neural stem cells and their malignant counterparts are challenging traditional models of central nervous system tumorigenesis and intrinsic brain repair. Neurogenesis persists into adulthood predominantly in two neurogenic centers: subventricular zone and subgranular zone. Subventricular zone is situated adjacent to lateral ventricles and subgranular zone is confined to the dentate gyrus of the hippocampus. Neural stem cells not only self-renew and differentiate along multiple lineages in these regions, but also contribute to intrinsic brain plasticity and repair. Ionizing radiation can depopulate these exquisitely sensitive regions directly or impair in situ neurogenesis by indirect, dose-dependentmore » and inflammation-mediated mechanisms, even at doses <2 Gy. This review discusses the fundamental neural stem cell concepts within the framework of cumulative clinical experience with the treatment of central nervous system malignancies using conventional radiotherapy.« less

Reporter-Based Isolation of Developmental Myogenic Progenitors

PubMed Central

Kheir, Eyemen; Cusella, Gabriella; Messina, Graziella; Cossu, Giulio; Biressi, Stefano

2018-01-01

The formation and activity of mammalian tissues entail finely regulated processes, involving the concerted organization and interaction of multiple cell types. In recent years the prospective isolation of distinct progenitor and stem cell populations has become a powerful tool in the hands of developmental biologists and has rendered the investigation of their intrinsic properties possible. In this protocol, we describe how to purify progenitors with different lineage history and degree of differentiation from embryonic and fetal skeletal muscle by fluorescence-activated cell sorting (FACS). The approach takes advantage of a panel of murine strains expressing fluorescent reporter genes specifically in the myogenic progenitors. We provide a detailed description of the dissection procedures and of the enzymatic dissociation required to maximize the yield of mononucleated cells for subsequent FACS-based purification. The procedure takes ~6–7 h to complete and allows for the isolation and the subsequent molecular and phenotypic characterization of developmental myogenic progenitors. PMID:29674978

Transplantation of motoneurons derived from MASH1-transfected mouse ES cells reconstitutes neural networks and improves motor function in hemiplegic mice.

PubMed

Ikeda, Ritsuko; Kurokawa, Manae S; Chiba, Shunmei; Yoshikawa, Hideshi; Hashimoto, Takuo; Tadokoro, Mamoru; Suzuki, Noboru

2004-10-01

Mouse embryonic stem (ES) cells were transfected with a MASH1 expression vector and G418-resistant cells were selected. The MASH1-transfected cells became neuron-like appearance and expressed betaIIItubulin and panNCAM. Glial fibrillary acidic protein (GFAP) and galactocerebroside (GalC)-expressing cells were rarely detected. Half of the neural cells differentiated into the Islet1+ motoneuron lineage. Thus, we obtained motoneuron lineage-enriched neuronal cells by transfection of ES cells with MASH1. A hemiplegic model of mice was developed by cryogenic injury of the motor cortex, and motoneuron lineage-enriched neuronal cells were transplanted underneath the injured motor cortex neighboring the periventricular region. The motor function of the recipients was assessed by a beam walking and rotarod tests, whereby the results gradually improved, but little improvement was observed in vehicle injected control mice. We found that the grafted cells not only remained close to the implantation site, but also exhibited substantial migration, penetrating into the damaged lesion in a directed manner up to the cortical region. Grafted neuronal cells that had migrated into the cortex were elongated axon-positive for neurofilament middle chain (NFM). Synaptophysin immunostaining showed a positive staining pattern around the graft, suggesting that the transplanted neurons interacted with the recipient neurons to form a neural network. Our study suggests that the motoneuron lineage can be induced from ES cells, and grafted cells adapt to the host environment and can reconstitute a neural network to improve motor function of a paralyzed limb.

Circulating Hematopoietic Stem and Progenitor Cells in Aging Atomic Bomb Survivors.

PubMed

Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Kajimura, Junko; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Young, Lauren F; Shieh, Jae-Hung; Moore, Malcolm A; van den Brink, Marcel R M; Kusunoki, Yoichiro

2016-01-01

It is not yet known whether hematopoietic stem and progenitor cells (HSPCs) are compromised in the aging population of atomic bomb (A-bomb) survivors after their exposure nearly 70 years ago. To address this, we evaluated age- and radiation-related changes in different subtypes of circulating HSPCs among the CD34-positive/lineage marker-negative (CD34(+)Lin(-)) cell population in 231 Hiroshima A-bomb survivors. We enumerated functional HSPC subtypes, including: cobblestone area-forming cells; long-term culture-initiating cells; erythroid burst-forming units; granulocyte and macrophage colony-forming units; and T-cell and natural killer cell progenitors using cell culture. We obtained the count of each HSPC subtype per unit volume of blood and the proportion of each HSPC subtype in CD34(+)Lin(-) cells to represent the lineage commitment trend. Multivariate analyses, using sex, age and radiation dose as variables, showed significantly decreased counts with age in the total CD34(+)Lin(-) cell population and all HSPC subtypes. As for the proportion, only T-cell progenitors decreased significantly with age, suggesting that the commitment to the T-cell lineage in HSPCs continuously declines with age throughout the lifetime. However, neither the CD34(+)Lin(-) cell population, nor HSPC subtypes showed significant radiation-induced dose-dependent changes in counts or proportions. Moreover, the correlations of the proportions among HSPC subtypes in the survivors properly revealed the hierarchy of lineage commitments. Taken together, our findings suggest that many years after exposure to radiation and with advancing age, the number and function of HSPCs in living survivors as a whole may have recovered to normal levels.

Circulating Hematopoietic Stem and Progenitor Cells in Aging Atomic Bomb Survivors

PubMed Central

Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Kajimura, Junko; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Young, Lauren F.; Shieh, Jae-Hung; Moore, Malcolm A.; van den Brink, Marcel R. M.; Kusunoki, Yoichiro

2016-01-01

It is not yet known whether hematopoietic stem and progenitor cells (HSPCs) are compromised in the aging population of atomic bomb (A-bomb) survivors after their exposure nearly 70 years ago. To address this, we evaluated age- and radiation-related changes in different subtypes of circulating HSPCs among the CD34-positive/lineage marker-negative (CD34+Lin− ) cell population in 231 Hiroshima A-bomb survivors. We enumerated functional HSPC subtypes, including: cobblestone area-forming cells; long-term culture-initiating cells; erythroid burst-forming units; granulocyte and macrophage colony-forming units; and T-cell and natural killer cell progenitors using cell culture. We obtained the count of each HSPC subtype per unit volume of blood and the proportion of each HSPC subtype in CD34+Lin− cells to represent the lineage commitment trend. Multivariate analyses, using sex, age and radiation dose as variables, showed significantly decreased counts with age in the total CD34+Lin− cell population and all HSPC subtypes. As for the proportion, only T-cell progenitors decreased significantly with age, suggesting that the commitment to the T-cell lineage in HSPCs continuously declines with age throughout the lifetime. However, neither the CD34+Lin− cell population, nor HSPC subtypes showed significant radiation-induced dose-dependent changes in counts or proportions. Moreover, the correlations of the proportions among HSPC subtypes in the survivors properly revealed the hierarchy of lineage commitments. Taken together, our findings suggest that many years after exposure to radiation and with advancing age, the number and function of HSPCs in living survivors as a whole may have recovered to normal levels. PMID:26720799

Mosaic Expression of Thyroid Hormone Regulatory Genes Defines Cell Type-Specific Dependency in the Developing Chicken Cerebellum.

PubMed

Delbaere, Joke; Van Herck, Stijn L J; Bourgeois, Nele M A; Vancamp, Pieter; Yang, Shuo; Wingate, Richard J T; Darras, Veerle M

2016-12-01

The cerebellum is a morphologically unique brain structure that requires thyroid hormones (THs) for the correct coordination of key cellular events driving its development. Unravelling the interplay between the multiple factors that can regulate intracellular TH levels is a key step to understanding their role in the regulation of these cellular processes. We therefore investigated the regional/cell-specific expression pattern of TH transporters and deiodinases in the cerebellum using the chicken embryo as a model. In situ hybridisation revealed expression of the TH transporters monocarboxylate transporter 8 (MCT8) and 10 (MCT10), L-type amino acid transporter 1 (LAT1) and organic anion transporting polypeptide 1C1 (OATP1C1) as well as the inactivating type 3 deiodinase (D3) in the fourth ventricle choroid plexus, suggesting a possible contribution of the resulting proteins to TH exchange and subsequent inactivation of excess hormone at the blood-cerebrospinal fluid barrier. Exclusive expression of LAT1 and the activating type 2 deiodinase (D2) mRNA was found at the level of the blood-brain barrier, suggesting a concerted function for LAT1 and D2 in the direct access of active T 3 to the developing cerebellum via the capillary endothelial cells. The presence of MCT8 mRNA in Purkinje cells and cerebellar nuclei during the first 2 weeks of embryonic development points to a potential role of this transporter in the uptake of T 3 in central neurons. At later stages, together with MCT10, detection of MCT8 signal in close association with the Purkinje cell dendritic tree suggests a role of both transporters in TH signalling during Purkinje cell synaptogenesis. MCT10 was also expressed in late-born cells in the rhombic lip lineage with a clear hybridisation signal in the outer external granular layer, indicating a potential role for MCT10 in the proliferation of granule cell precursors. By contrast, expression of D3 in the first-born rhombic lip-derived population may serve as a buffering mechanism against high T 3 levels during early embryonic development, a hypothesis supported by the pattern of expression of a fluorescent TH reporter in this lineage. Overall, this study builds a picture of the TH dependency in multiple cerebellar cell types starting from early embryonic development.

Acute leukemia coexpressing myeloid, B- and T-lineage associated markers: multiparameter analysis of criteria defining lineage commitment and maturational stage in a case of undifferentiated leukemia.

PubMed

Meckenstock, G; Heyll, A; Schneider, E M; Hildebrandt, B; Runde, V; Aul, C; Bartram, C R; Ludwig, W D; Schneider, W

1995-02-01

Coexpression of myeloid, B-, and T-lineage associated markers was found in a patient with morphologically and cytochemically undifferentiated acute leukemia. Surface marker analysis using two-color immunofluorescence staining characterized blast cells to express CD34, CD38, CD117, and class II antigens, coexpressing TdT, CD4, CD7, CD13, CD19, and CD33. Cytoplasmic expression of myeloperoxidase, CD3, and CD22 could not be demonstrated. Monosomy for chromosome 7 was found by cytogenetic analysis. The absence of clonal rearrangements of immunoglobulin or T-cell receptor genes was shown by Southern blot analysis. Using a 3H-thymidine incorporation assay, DNA synthesis of leukemic blasts could be stimulated by IL-3, IL-6 and G-CSF in vitro. The present case did not offer specific criteria of lineage commitment. Corresponding to an equivalent counterpart in normal hematopoiesis, the involved cell population may reflect an early, most immature developmental stage within a multipotent progenitor cell compartment.

Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo

PubMed Central

Posfai, Eszter; Petropoulos, Sophie; de Barros, Flavia Regina Oliveira; Schell, John Paul; Jurisica, Igor; Sandberg, Rickard; Lanner, Fredrik; Rossant, Janet

2017-01-01

The segregation of the trophectoderm (TE) from the inner cell mass (ICM) in the mouse blastocyst is determined by position-dependent Hippo signaling. However, the window of responsiveness to Hippo signaling, the exact timing of lineage commitment and the overall relationship between cell commitment and global gene expression changes are still unclear. Single-cell RNA sequencing during lineage segregation revealed that the TE transcriptional profile stabilizes earlier than the ICM and prior to blastocyst formation. Using quantitative Cdx2-eGFP expression as a readout of Hippo signaling activity, we assessed the experimental potential of individual blastomeres based on their level of Cdx2-eGFP expression and correlated potential with gene expression dynamics. We find that TE specification and commitment coincide and occur at the time of transcriptional stabilization, whereas ICM cells still retain the ability to regenerate TE up to the early blastocyst stage. Plasticity of both lineages is coincident with their window of sensitivity to Hippo signaling. DOI: http://dx.doi.org/10.7554/eLife.22906.001 PMID:28226240

Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-Thrombospondin-1 axis

PubMed Central

Lee, Joo-Hyeon; Bhang, Dong Ha; Beede, Alexander; Huang, Tian Lian; Stripp, Barry R.; Bloch, Kenneth D.; Wagers, Amy J.; Tseng, Yu-Hua; Ryeom, Sandra; Kim, Carla F.

2014-01-01

SUMMARY Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal three-dimensional (3D) co-cultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell co-cultures. Gain and loss of function experiments showed BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of Thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1-null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide new tools to understand the mechanisms of respiratory diseases at the single cell level. PMID:24485453

A review of adipocyte lineage cells and dermal papilla cells in hair follicle regeneration

PubMed Central

Zhang, Peipei; Kling, Russell E; Ravuri, Sudheer K; Kokai, Lauren E; Rubin, J Peter; Chai, Jia-ke

2014-01-01

Alopecia is an exceedingly prevalent problem effecting men and women of all ages. The standard of care for alopecia involves either transplanting existing hair follicles to bald areas or attempting to stimulate existing follicles with topical and/or oral medication. Yet, these treatment options are fraught with problems of cost, side effects, and, most importantly, inadequate long-term hair coverage. Innovative cell-based therapies have focused on the dermal papilla cell as a way to grow new hair in previously bald areas. However, despite this attention, many obstacles exist, including retention of dermal papilla inducing ability and maintenance of dermal papilla productivity after several passages of culture. The use of adipocyte lineage cells, including adipose-derived stem cells, has shown promise as a cell-based solution to regulate hair regeneration and may help in maintaining or increasing dermal papilla cells inducing hair ability. In this review, we highlight recent advances in the understanding of the cellular contribution and regulation of dermal papilla cells and summarize adipocyte lineage cells in hair regeneration. PMID:25383178

The bone marrow niche, stem cells, and leukemia: impact of drugs, chemicals, and the environment

PubMed Central

Greim, Helmut; Kaden, Debra A.; Larson, Richard A.; Palermo, Christine M.; Rice, Jerry M.; Ross, David; Snyder, Robert

2014-01-01

Hematopoietic stem cells (HSCs) are a unique population of somatic stem cells that can both self-renew for long-term reconstitution of HSCs and differentiate into hematopoietic progenitor cells, which in turn give rise, in a hierarchical manner, to the entire myeloid and lymphoid lineages. The differentiation and maturation of these lineages occurs in the bone marrow niche, a microenvironment that regulates self-renewal, survival, differentiation, and proliferation, with interactions among signaling pathways in the HSCs and the niche required to establish and maintain homeostasis. The accumulation of genetic mutations and cytogenetic abnormalities within cells of the partially differentiated myeloid lineage, particularly as a result of exposure to benzene or cytotoxic anticancer drugs, can give rise to malignancies like acute myeloid leukemia and myelodysplastic syndrome. Better understanding of the mechanisms driving these malignancies and susceptibility factors, both within hematopoietic progenitor cells and cells within the bone marrow niche, may lead to the development of strategies for prevention of occupational and cancer therapy–induced disease. PMID:24495159

[Cell lineage tracing of regenerating cells after partial pancreatectomy using pseudo-type retrovirus].

PubMed

Zhang, Lixin; Ju, Xiaofang; Wang, Fa; Guo, Zhiwei; Piao, Shanhua; Teng, Chunbo

2008-04-01

Pancreas is an important mixed gland having both endocrine and exocrine functions, and has been proven regeneration after injury. To explore the cell lineage tracing methods in pancreas in vivo and the regenerate cells source, we used pseudo-type retrovirus to transfect adult mouse pancreas which had been partially pancreatectomized by rubbing the kerf using a cotton stick saturated with retrovirus suspension then injecting 100 microL retrovirus suspension into pancreas, injecting 100 microL retrovirus by caudal vein, or interperitoneally injecting retrovirus respectively. The results showed that the method of rubbing the kerf then injection of retrovirus suspension into pancreas could more effectively mark the pancreatic cells than the caudal vein injection and the intraperitoneal injection did in vivo. Furthermore, this study also found that some acinus cells could accept injury stimulus signals to regenerate through resuming mitosis after pancreatic injury. This study establishes a cell lineage tracing method in pancreas in vivo using retrovirus and offers a clue for gene therapy of pancreatic diseases using retrovirus vectors.

Phylogenetic Analysis of Klebsiella pneumoniae from Hospitalized Children, Pakistan.

PubMed

Ejaz, Hasan; Wang, Nancy; Wilksch, Jonathan J; Page, Andrew J; Cao, Hanwei; Gujaran, Shruti; Keane, Jacqueline A; Lithgow, Trevor; Ul-Haq, Ikram; Dougan, Gordon; Strugnell, Richard A; Heinz, Eva

2017-11-01

Klebsiella pneumoniae shows increasing emergence of multidrug-resistant lineages, including strains resistant to all available antimicrobial drugs. We conducted whole-genome sequencing of 178 highly drug-resistant isolates from a tertiary hospital in Lahore, Pakistan. Phylogenetic analyses to place these isolates into global context demonstrate the expansion of multiple independent lineages, including K. quasipneumoniae.

Plasticity in Olfactory Epithelium: Is It a Sniffer or Shape Shifter?

PubMed

Konkimalla, Arvind; Tata, Purushothama Rao

2017-12-07

Precise lineage trajectories and the cellular sources that contribute to regeneration after injury are largely unknown in many tissues. In this issue of Cell Stem Cell, Gadye et al. (2017) and Lin et al. (2017) show that olfactory epithelial cells transit through unique and unfamiliar paths of differentiation and undergo lineage reversion, respectively, during regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Lineage-tracking of stem cell differentiation: a neutral model of hematopoiesis in rhesus macaque

NASA Astrophysics Data System (ADS)

Chou, Tom

How a potentially diverse population of hematopoietic stem cells (HSCs) differentiates and proliferates to supply more than 1011 mature blood cells every day in humans remains a key biological question. We investigated this process by quantitatively analyzing the clonal structure of peripheral blood that is generated by a population of transplanted lentivirus-marked HSCs in myeloablated rhesus macaques. Each transplanted HSC generates a clonal lineage of cells in the peripheral blood that is then detected and quantified through deep sequencing of the viral vector integration sites (VIS) common within each lineage. This approach allowed us to observe, over a period of 4-12 years, hundreds of distinct clonal lineages. Surprisingly, while the distinct clone sizes varied by three orders of magnitude, we found that collectively, they form a steady-state clone size-distribution with a distinctive shape. Our concise model shows that slow HSC differentiation followed by fast progenitor growth is responsible for the observed broad clone size-distribution. Although all cells are assumed to be statistically identical, analogous to a neutral theory for the different clone lineages, our mathematical approach captures the intrinsic variability in the times to HSC differentiation after transplantation. Steady-state solutions of our model show that the predicted clone size-distribution is sensitive to only two combinations of parameters. By fitting the measured clone size-distributions to our mechanistic model, we estimate both the effective HSC differentiation rate and the number of active HSCs. NSF and NIH.

TLX activates MASH1 for induction of neuronal lineage commitment of adult hippocampal neuroprogenitors.

PubMed

Elmi, Muna; Matsumoto, Yoshiki; Zeng, Zhao-jun; Lakshminarasimhan, Pavithra; Yang, Weiwen; Uemura, Akiyoshi; Nishikawa, Shin-ichi; Moshiri, Alicia; Tajima, Nobuyoshi; Agren, Hans; Funa, Keiko

2010-10-01

The orphan nuclear receptor TLX has been proposed to act as a repressor of cell cycle inhibitors to maintain the neural stem cells in an undifferentiated state, and prevents commitment into astrocyte lineages. However, little is known about the mechanism of TLX in neuronal lineage commitment and differentiation. A majority of adult rat hippocampus-derived progenitors (AHPs) cultured in the presence of FGF express a high level of TLX and a fraction of these cells also express the proneural gene MASH1. Upon FGF withdrawal, TLX rapidly decreased, while MASH1 was intensely expressed within 1h, decreasing gradually to disappear at 24h. Adenoviral transduction of TLX in AHP cells in the absence of FGF transiently increased cell proliferation, however, later resulted in neuronal differentiation by inducing MASH1, Neurogenin1, DCX, and MAP2ab. Furthermore, TLX directly targets and activates the MASH1 promoter through interaction with Sp1, recruiting co-activators whereas dismissing the co-repressor HDAC4. Conversely, silencing of TLX in AHPs decreased beta-III tubulin and DCX expression and promoted glial differentiation. Our results thus suggest that TLX not only acts as a repressor of cell cycle and glial differentiation but also activates neuronal lineage commitment in AHPs. Copyright 2010 Elsevier Inc. All rights reserved.

FoxO and Stress Responses in the Cnidarian Hydra vulgaris

PubMed Central

Bridge, Diane; Theofiles, Alexander G.; Holler, Rebecca L.; Marcinkevicius, Emily; Steele, Robert E.; Martínez, Daniel E.

2010-01-01

Background In the face of changing environmental conditions, the mechanisms underlying stress responses in diverse organisms are of increasing interest. In vertebrates, Drosophila, and Caenorhabditis elegans, FoxO transcription factors mediate cellular responses to stress, including oxidative stress and dietary restriction. Although FoxO genes have been identified in early-arising animal lineages including sponges and cnidarians, little is known about their roles in these organisms. Methods/Principal Findings We have examined the regulation of FoxO activity in members of the well-studied cnidarian genus Hydra. We find that Hydra FoxO is expressed at high levels in cells of the interstitial lineage, a cell lineage that includes multipotent stem cells that give rise to neurons, stinging cells, secretory cells and gametes. Using transgenic Hydra that express a FoxO-GFP fusion protein in cells of the interstitial lineage, we have determined that heat shock causes localization of the fusion protein to the nucleus. Our results also provide evidence that, as in bilaterian animals, Hydra FoxO activity is regulated by both Akt and JNK kinases. Conclusions These findings imply that basic mechanisms of FoxO regulation arose before the evolution of bilaterians and raise the possibility that FoxO is involved in stress responses of other cnidarian species, including corals. PMID:20657733

FoxO and stress responses in the cnidarian Hydra vulgaris.

PubMed

Bridge, Diane; Theofiles, Alexander G; Holler, Rebecca L; Marcinkevicius, Emily; Steele, Robert E; Martínez, Daniel E

2010-07-21

In the face of changing environmental conditions, the mechanisms underlying stress responses in diverse organisms are of increasing interest. In vertebrates, Drosophila, and Caenorhabditis elegans, FoxO transcription factors mediate cellular responses to stress, including oxidative stress and dietary restriction. Although FoxO genes have been identified in early-arising animal lineages including sponges and cnidarians, little is known about their roles in these organisms. We have examined the regulation of FoxO activity in members of the well-studied cnidarian genus Hydra. We find that Hydra FoxO is expressed at high levels in cells of the interstitial lineage, a cell lineage that includes multipotent stem cells that give rise to neurons, stinging cells, secretory cells and gametes. Using transgenic Hydra that express a FoxO-GFP fusion protein in cells of the interstitial lineage, we have determined that heat shock causes localization of the fusion protein to the nucleus. Our results also provide evidence that, as in bilaterian animals, Hydra FoxO activity is regulated by both Akt and JNK kinases. These findings imply that basic mechanisms of FoxO regulation arose before the evolution of bilaterians and raise the possibility that FoxO is involved in stress responses of other cnidarian species, including corals.

DNA methylation dynamics during in vivo differentiation of blood and skin stem cells

PubMed Central

Bock, Christoph; Beerman, Isabel; Lien, Wen-Hui; Smith, Zachary D.; Gu, Hongcang; Boyle, Patrick; Gnirke, Andreas; Fuchs, Elaine; Rossi, Derrick J.; Meissner, Alexander

2012-01-01

DNA methylation is a mechanism of epigenetic regulation that is common to all vertebrates. Functional studies underscore its relevance for tissue homeostasis, but the global dynamics of DNA methylation during in vivo differentiation remain underexplored. Here we report high-resolution DNA methylation maps of adult stem cell differentiation in mouse, focusing on 19 purified cell populations of the blood and skin lineages. DNA methylation changes were locus-specific and relatively modest in magnitude. They frequently overlapped with lineage-associated transcription factors and their binding sites, suggesting that DNA methylation may protect cells from aberrant transcription factor activation. DNA methylation and gene expression provided complementary information, and combining the two enabled us to infer the cellular differentiation hierarchy of the blood lineage directly from genomic data. In summary, these results demonstrate that in vivo differentiation of adult stem cells is associated with small but informative changes in the genomic distribution of DNA methylation. PMID:22841485

Fabrication of hydrogel based nanocomposite scaffold containing bioactive glass nanoparticles for myocardial tissue engineering.

PubMed

Barabadi, Zahra; Azami, Mahmoud; Sharifi, Esmaeel; Karimi, Roya; Lotfibakhshaiesh, Nasrin; Roozafzoon, Reza; Joghataei, Mohammad Taghi; Ai, Jafar

2016-12-01

Selecting suitable cell sources and angiogenesis induction are two important issues in myocardial tissue engineering. Human endometrial stromal cells (EnSCs) have been introduced as an abundant and easily available resource in regenerative medicine. Bioactive glass is an agent that induces angiogenesis and has been studied in some experiments. The aim of this study was to investigate in vitro differentiation capacity of endometrial stem cells into cardiomyocyte lineage and to evaluate capability of bioactive glass nanoparticles toward EnSCs differentiation into endothelial lineage and angiogenesis on hydrogel scaffold. Our findings suggests that endometrial stem cells could be programmed into cardiomyocyte linage and considered a suitable cell source for myocardial regeneration. This experiment also revealed that inclusion of bioactive glass nanoparticles in hydrogel scaffold could improve angiogenesis through differentiating EnSCs toward endothelial lineage and increasing level of vascular endothelial growth factor secretion. Copyright © 2016 Elsevier B.V. All rights reserved.

Green Algae and the Origins of Multicellularity in the Plant Kingdom

PubMed Central

Umen, James G.

2014-01-01

The green lineage of chlorophyte algae and streptophytes form a large and diverse clade with multiple independent transitions to produce multicellular and/or macroscopically complex organization. In this review, I focus on two of the best-studied multicellular groups of green algae: charophytes and volvocines. Charophyte algae are the closest relatives of land plants and encompass the transition from unicellularity to simple multicellularity. Many of the innovations present in land plants have their roots in the cell and developmental biology of charophyte algae. Volvocine algae evolved an independent route to multicellularity that is captured by a graded series of increasing cell-type specialization and developmental complexity. The study of volvocine algae has provided unprecedented insights into the innovations required to achieve multicellularity. PMID:25324214

Quantifying selective pressures driving bacterial evolution using lineage analysis

PubMed Central

Lambert, Guillaume; Kussell, Edo

2015-01-01

Organisms use a variety of strategies to adapt to their environments and maximize long-term growth potential, but quantitative characterization of the benefits conferred by the use of such strategies, as well as their impact on the whole population’s rate of growth, remains challenging. Here, we use a path-integral framework that describes how selection acts on lineages –i.e. the life-histories of individuals and their ancestors– to demonstrate that lineage-based measurements can be used to quantify the selective pressures acting on a population. We apply this analysis to E. coli bacteria exposed to cyclical treatments of carbenicillin, an antibiotic that interferes with cell-wall synthesis and affects cells in an age-dependent manner. While the extensive characterization of the life-history of thousands of cells is necessary to accurately extract the age-dependent selective pressures caused by carbenicillin, the same measurement can be recapitulated using lineage-based statistics of a single surviving cell. Population-wide evolutionary pressures can be extracted from the properties of the surviving lineages within a population, providing an alternative and efficient procedure to quantify the evolutionary forces acting on a population. Importantly, this approach is not limited to age-dependent selection, and the framework can be generalized to detect signatures of other trait-specific selection using lineage-based measurements. Our results establish a powerful way to study the evolutionary dynamics of life under selection, and may be broadly useful in elucidating selective pressures driving the emergence of antibiotic resistance and the evolution of survival strategies in biological systems. PMID:26213639

Biomaterial delivery of morphogens to mimic the natural healing cascade in bone.

PubMed

Mehta, Manav; Schmidt-Bleek, Katharina; Duda, Georg N; Mooney, David J

2012-09-01

Complications in treatment of large bone defects using bone grafting still remain. Our understanding of the endogenous bone regeneration cascade has inspired the exploration of a wide variety of growth factors (GFs) in an effort to mimic the natural signaling that controls bone healing. Biomaterial-based delivery of single exogenous GFs has shown therapeutic efficacy, and this likely relates to its ability to recruit and promote replication of cells involved in tissue development and the healing process. However, as the natural bone healing cascade involves the action of multiple factors, each acting in a specific spatiotemporal pattern, strategies aiming to mimic the critical aspects of this process will likely benefit from the usage of multiple therapeutic agents. This article reviews the current status of approaches to deliver single GFs, as well as ongoing efforts to develop sophisticated delivery platforms to deliver multiple lineage-directing morphogens (multiple GFs) during bone healing. Copyright © 2012 Elsevier B.V. All rights reserved.

JMJD3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors

PubMed Central

Kochat, Veena; Equbal, Zaffar; Baligar, Prakash; Kumar, Vikash; Srivastava, Madhulika; Mukhopadhyay, Asok

2017-01-01

The strictly regulated unidirectional differentiation program in some somatic stem/progenitor cells has been found to be modified in the ectopic site (tissue) undergoing regeneration. In these cases, the lineage barrier is crossed by either heterotypic cell fusion or direct differentiation. Though studies have shown the role of coordinated genetic and epigenetic mechanisms in cellular development and differentiation, how the lineage fate of adult bone marrow progenitor cells (BMPCs) is reprogrammed during liver regeneration and whether this lineage switch is stably maintained are not clearly understood. In the present study, we wanted to decipher genetic and epigenetic mechanisms that involve in lineage reprogramming of BMPCs into hepatocyte-like cells. Here we report dynamic transcriptional change during cellular reprogramming of BMPCs to hepatocytes and dissect the epigenetic switch mechanism of BM cell-mediated liver regeneration after acute injury. Genome-wide gene expression analysis in BM-derived hepatocytes, isolated after 1 month and 5 months of transplantation, showed induction of hepatic transcriptional program and diminishing of donor signatures over the time. The transcriptional reprogramming of BM-derived cells was found to be the result of enrichment of activating marks (H3K4me3 and H3K9Ac) and loss of repressive marks (H3K27me3 and H3K9me3) at the promoters of hepatic transcription factors (HTFs). Further analyses showed that BMPCs possess bivalent histone marks (H3K4me3 and H3K27me3) at the promoters of crucial HTFs. H3K27 methylation dynamics at the HTFs was antagonistically regulated by EZH2 and JMJD3. Preliminary evidence suggests a role of JMJD3 in removal of H3K27me3 mark from promoters of HTFs, thus activating epigenetically poised hepatic genes in BMPCs prior to partial nuclear reprogramming. The importance of JMJD3 in reprogramming of BMPCs to hepatic phenotype was confirmed by inhibiting catalytic function of the enzyme using small molecule GSK-J4. Our results propose a potential role of JMJD3 in lineage conversion of BM cells into hepatic lineage. PMID:28328977

The effects of vitamin D binding protein-macrophage activating factor and colony-stimulating factor-1 on hematopoietic cells in normal and osteopetrotic rats.

PubMed

Benis, K A; Schneider, G B

1996-10-15

Osteopetrosis is a heterogeneous group of bone disorders characterized by the failure of osteoclasts to resorb bone and by several immunological defects including macrophage dysfunction. Two compounds, colony-stimulating factor-1 (CSF-1) and vitamin D-binding protein-macrophage activating factor (DBP-MAF) were used in the present study to evaluate their effects on the peritoneal population of cells and on cells within the bone marrow microenvironment in normal and incisors absent (ia) osteopetrotic rats. Previous studies in this laboratory have demonstrated that administration of DBP-MAF to newborn ia animals results in a substantial increase in bone marrow cavity size due to upregulated osteoclast function. To study the effects of these compounds on the macrophage/osteoclast precursors, DBP-MAF, CSF-1, and the combination of these compounds were given to newborn ia and normal littermate animals. Both the normal and mutant phenotypes responded similarly when treated with these compounds. Rats exhibited a profound shift toward the macrophage lineage from the neutrophil lineage when compared with vehicle-treated control animals after treatment with these compounds. In the in vivo peritoneal lavage study, animals received injections of CSF-1, DBP-MAF or DBP-MAF/CSF-1 over a 4-week period. The various types of cells in the peritoneal cavity were then enumerated. The in vitro study consisted of cells isolated from the bone marrow microenvironment and cultured on feeder layers of CSF-1, DBP-MAF, or DBP-MAF/CSF-1 for colony enumeration. The increase in macrophage numbers at the expense of neutrophil numbers could be seen in both the in vivo and in vitro experiments. The macrophage/osteoclast and neutrophil lineages have a common precursor, the granulocyte/macrophage colony-forming cell (GM-CFC). With the addition of CSF-1, the GM-CFC precursor may be induced into the macrophage/osteoclast lineage rather than the granulocyte lineage. This increased pool of cells in the macrophage/osteoclast lineage can be functionally upregulated with the subsequent addition of DBP-MAF to perform the activities of phagocytosis and bone resorption. The in vitro data also showed that DBP-MAF did not support colony development as in CSF-1 or the combination treatment. The recruitment and activation of cells into the macrophage/ osteoclast lineage may help to correct the bone and immune defects found in diseases demonstrating a significant lack of myeloid cells, as well as neutrophilia disorders and the disease, osteopetrosis.

Concise review: adult multipotent stromal cells and cancer: risk or benefit?

PubMed

Lazennec, Gwendal; Jorgensen, Christian

2008-06-01

This review focuses on the interaction between multipotent stromal cells (MSCs) and carcinoma and the possible use of MSCs in cell-based anticancer therapies. MSCs are present in multiple tissues and are defined as cells displaying the ability to differentiate in multiple lineages, including chondrocytes, osteoblasts, and adipocytes. Recent evidence also suggests that they could play a role in the progression of carcinogenesis and that MSCs could migrate toward primary tumors and metastatic sites. It is possible that MSCs could also be involved in the early stages of carcinogenesis through spontaneous transformation. In addition, it is thought that MSCs can modulate tumor growth and metastasis, although this issue remains controversial and not well understood. The immunosuppressive properties and proangiogenic properties of MSCs account, at least in part, for their effects on cancer development. On the other hand, cancer cells also have the ability to enhance MSC migration. This complex dialog between MSCs and cancer cells is certainly critical for the outcome of tumor development. Interestingly, several studies have shown that MSCs engineered to express antitumor factors could be an innovative choice as a cell-mediated gene therapy to counteract tumor growth. More evidence will be needed to understand how MSCs positively or negatively modulate carcinogenesis and to evaluate the safety of MSC use in cell-mediated gene strategies. Disclosure of potential conflicts of interest is found at the end of this article.

Concise review: adult multipotent stromal cells and cancer: risk or benefit?

PubMed Central

Lazennec, Gwendal; Jorgensen, Christian

2008-01-01

This review will focus on the interaction between multipotent stromal cells (MSCs) and carcinoma and the possible use of MSCs in cell-based anti-cancer therapies. MSCs are present in multiple tissues and are defined as cells displaying the ability to differentiate in multiple lineages including chondrocytes, osteoblasts and adipocytes. Recent evidence suggests also that they could play a role in the progression of carcinogenesis and that MSCs could migrate towards primary tumors and metastatic sites. It is possible that MSCs could be also involved in the early stages of carcinogenesis through spontaneous transformation. In addition, it is thought that MSCs can modulate tumor growth and metastasis, although this issue remains controversial and not well understood. The immuno-suppressive properties and pro-angiogenic properties of MSCs account, at least in part, for their effects on cancer development. On the other hand, cancer cells also have the ability to enhance MSC migration. This complex dialog between MSCs and cancer cells is certainly critical for the outcome of tumor development. Interestingly, several studies have shown that MSCs engineered to express anti-tumor factors could be an innovative choice as a cell-mediated gene therapy to counteract tumor growth. More evidence will be needed to understand how MSCs positively or negatively modulate carcinogenesis and to evaluate the safety of MSCs use in cell-mediated gene strategies. PMID:18388305

Rho-associated kinase inhibitors promote the cardiac differentiation of embryonic and induced pluripotent stem cells.

PubMed

Cheng, Ya-Ting; Yeih, Dong-Feng; Liang, Shu-Man; Chien, Chia-Ying; Yu, Yen-Ling; Ko, Bor-Sheng; Jan, Yee-Jee; Kuo, Cheng-Chin; Sung, Li-Ying; Shyue, Song-Kun; Chen, Ming-Fong; Yet, Shaw-Fang; Wu, Kenneth K; Liou, Jun-Yang

2015-12-15

Rho-associated kinase (ROCK) plays an important role in maintaining embryonic stem (ES) cell pluripotency. To determine whether ROCK is involved in ES cell differentiation into cardiac and hematopoietic lineages, we evaluated the effect of ROCK inhibitors, Y-27632 and fasudil on murine ES and induced pluripotent stem (iPS) cell differentiation. Gene expression levels were determined by real-time PCR, Western blot analysis and immunofluorescent confocal microscopy. Cell transplantation of induced differentiated cells were assessed in vivo in a mouse model (three groups, n=8/group) of acute myocardial infarction (MI). The cell engraftment was examined by immunohistochemical staining and the outcome was analyzed by echocardiography. Cells were cultured in hematopoietic differentiation medium in the presence or absence of ROCK inhibitor and colony formation as well as markers of ES, hematopoietic stem cells (HSC) and cells of cardiac lineages were analyzed. ROCK inhibition resulted in a drastic change in colony morphology accompanied by loss of hematopoietic markers (GATA-1, CD41 and β-Major) and expressed markers of cardiac lineages (GATA-4, Isl-1, Tbx-5, Tbx-20, MLC-2a, MLC-2v, α-MHC, cTnI and cTnT) in murine ES and iPS cells. Fasudil-induced cardiac progenitor (Mesp-1 expressing) cells were infused into a murine MI model. They engrafted into the peri-infarct and infarct regions and preserved left ventricular function. These findings provide new insights into the signaling required for ES cell differentiation into hematopoietic as well as cardiac lineages and suggest that ROCK inhibitors are useful in directing iPS cell differentiation into cardiac progenitor cells for cell therapy of cardiovascular diseases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Roles of CDX2 and EOMES in human induced trophoblast progenitor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ying, E-mail: ying.chen@hc.msu.edu; Wang, Kai; Gong, Yun Guo

Highlights: ► CDX2 and EOMES play critical roles in human induced trophoblast progenitors (iTP). ► iTP cells directly transformed from fibroblasts. ► Differentiation of iTP cells into extravillous trophoblasts and syncytiotrophoblasts. -- Abstract: Abnormal trophoblast lineage proliferation and differentiation in early pregnancy have been associated with the pathogenesis of placenta diseases of pregnancy. However, there is still a gap in understanding the molecular mechanisms of early placental development due to the limited primary trophoblast cultures and fidelity of immortalized trophoblast lines. Trophoblasts stem (TS) cells, an in vitro model of trophectoderm that can differentiate into syncytiotrophoblasts and extravillous trophoblasts, canmore » be an attractive tool for early pregnancy research. TS cells are well established in mouse but not in humans due to insufficient knowledge of which trophoblast lineage-specific transcription factors are involved in human trophectoderm (TE) proliferation and differentiation. Here, we applied induced pluripotent stem cell technique to investigate the human trophoblast lineage-specific transcription factors. We established human induced trophoblast progenitor (iTP) cells by direct reprogramming the fibroblasts with a pool of mouse trophoblast lineage-specific transcription factors consisting of CDX2, EOMES, and ELF5. The human iTP cells exhibit epithelial morphology and can be maintained in vitro for more than 2 months. Gene expression profile of these cells was tightly clustered with human trophectoderm but not with human neuron progenitor cells, mesenchymal stem cells, or endoderm cells. These cells are capable of differentiating into cells with an invasive capacity, suggesting extravillous trophoblasts. They also form multi-nucleated cells which secrete human chorionic gonadotropin and estradiol, consistent with a syncytiotrophoblast phenotype. Our results provide the evidence that transcription factors CDX2 and EOMES may play critical roles in human iTP cell generation.« less

Establishment of segment polarity in the ectoderm of the leech Helobdella

NASA Technical Reports Server (NTRS)

Seaver, E. C.; Shankland, M.

2001-01-01

The segmented ectoderm and mesoderm of the leech arise via a stereotyped cell lineage from embryonic stem cells called teloblasts. Each teloblast gives rise to a column of primary blast cell daughters, and the blast cells generate descendant clones that serve as the segmental repeats of their particular teloblast lineage. We have examined the mechanism by which the leech primary blast cell clones acquire segment polarity - i.e. a fixed sequence of positional values ordered along the anteroposterior axis of the segmental repeat. In the O and P teloblast lineages, the earliest divisions of the primary blast cell segregate anterior and posterior cell fates along the anteroposterior axis. Using a laser microbeam, we ablated single cells from both o and p blast cell clones at stages when the clone was two to four cells in length. The developmental fate of the remaining cells was characterized with rhodamine-dextran lineage tracer. Twelve different progeny cells were ablated, and in every case the ablation eliminated the normal descendants of the ablated cell while having little or no detectable effect on the developmental fate of the remaining cells. This included experiments in which we specifically ablated those blast cell progeny that are known to express the engrailed gene, or their lineal precursors. These findings confirm and extend a previous study by showing that the establishment of segment polarity in the leech ectoderm is largely independent of cell interactions conveyed along the anteroposterior axis. Both intercellular signaling and engrailed expression play an important role in the segment polarity specification of the Drosophila embryo, and our findings suggest that there may be little or no conservation of this developmental mechanism between those two organisms.

Effects of granulocyte-macrophage colony-stimulating factor and interleukin 6 on the growth of leukemic blasts in suspension culture.

PubMed

Tsao, C J; Cheng, T Y; Chang, S L; Su, W J; Tseng, J Y

1992-05-01

We examined the stimulatory effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL)-6 on the in vitro proliferation of leukemic blast cells from patients with acute leukemia. Bone marrow or peripheral blood leukemic blast cells were obtained from 21 patients, including 14 cases of acute myeloblastic leukemia (AML), four cases of acute lymphoblastic leukemia (ALL), two cases of acute undifferentiated leukemia, and one case of acute mixed-lineage leukemia. The proliferation of leukemic blast cells was evaluated by measuring the incorporation of 3H-thymidine into cells incubated with various concentrations of cytokines for 3 days. GM-CSF stimulated the DNA synthesis (with greater than 2.0 stimulation index) of blast cells in 9 of 14 (64%) AML cases, two cases of acute undifferentiated leukemia and one case of acute mixed-lineage leukemia. Only two cases of AML blasts responded to IL-6 to grow in the short-term suspension cultures. GM-CSF and IL-6 did not display a synergistic effect on the growth of leukemic cells. Moreover, GM-CSF and IL-6 did not stimulate the proliferation of ALL blast cells. Binding study also revealed the specific binding of GM-CSF on the blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia. Our results indicated that leukemic blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia possessed functional GM-CSF receptors.

Quantitative Detection of Low-Abundance Transcripts at Single-Cell Level in Human Epidermal Keratinocytes by Digital Droplet Reverse Transcription-Polymerase Chain Reaction.

PubMed

Auvré, Frédéric; Coutier, Julien; Martin, Michèle T; Fortunel, Nicolas O

2018-05-08

Genetic and epigenetic characterization of the large cellular diversity observed within tissues is essential to understanding the molecular networks that ensure the regulation of homeostasis, repair, and regeneration, but also pathophysiological processes. Skin is composed of multiple cell lineages and is therefore fully concerned by this complexity. Even within one particular lineage, such as epidermal keratinocytes, different immaturity statuses or differentiation stages are represented, which are still incompletely characterized. Accordingly, there is presently great demand for methods and technologies enabling molecular investigation at single-cell level. Also, most current methods used to analyze gene expression at RNA level, such as RT-qPCR, do not directly provide quantitative data, but rather comparative ratios between two conditions. A second important need in skin biology is thus to determine the number of RNA molecules in a given cell sample. Here, we describe a workflow that we have set up to meet these specific needs, by means of transcript quantification in cellular micro-samples using flow cytometry sorting and reverse transcription-digital droplet polymerase chain reaction. As a proof-of-principle, the workflow was tested for the detection of transcription factor transcripts expressed at low levels in keratinocyte precursor cells. A linear correlation was found between quantification values and keratinocyte input numbers in a low quantity range from 40 cells to 1 cell. Interpretable signals were repeatedly obtained from single-cell samples corresponding to estimated expression levels as low as 10-20 transcript copies per keratinocyte or less. The present workflow may have broad applications for the detection and quantification of low-abundance nucleic acid species in single cells, opening up perspectives for the study of cell-to-cell genetic and molecular heterogeneity. Interestingly, the process described here does not require internal references such as house-keeping gene expression, as it is initiated with defined cell numbers, precisely sorted by flow cytometry.

A regulatory sequence from the retinoid X receptor γ gene directs expression to horizontal cells and photoreceptors in the embryonic chicken retina.

PubMed

Blixt, Maria K E; Hallböök, Finn

2016-01-01

Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression-specific cell lineage tracing in the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors. A 208 bp gene regulatory sequence from the chicken retinoid X receptor γ gene (RXRγ208) was used to drive Cre expression. RXRγ is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac "donor" vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures. Cells that drove Cre expression from the regulatory RXRγ208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRγ208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with RXRγ gene expression in the early retina. In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the RXRγ-gene regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor.

Chromatin associated Sin3A is essential for male germ cell lineage in the mouse

PubMed Central

Pellegrino, Jessica; Castrillon, Diego H.; David, Gregory

2012-01-01

Spermatogenesis is a complex process that requires coordinated proliferation and differentiation of male germ cells. The molecular events that dictate this process are largely unknown, but are likely to involve highly regulated transcriptional control. In this study, we investigate the contribution of chromatin associated Sin3A in mouse germ cell lineage development. Genetic inactivation of Sin3A in the male germline leads to sterility that results from the early and penetrant apoptotic death observed in Sin3A-deleted germ cells, coincident with the reentry in mitosis. Sin3A-deleted testes exhibit a Sertoli-cell only phenotype, consistent with the absolute requirement for Sin3A in germ cells’ development and/or viability. Interestingly, transcripts analysis revealed that the expression program of Sertoli cells is altered upon inactivation of Sin3A in germ cells. These studies identified a central role for the mammalian Sin3-HDAC complex in the germ cell lineage, and point to an exquisite transcriptional crosstalk between germ cells and their niche to support fertility in mammals. PMID:22820070

Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells

PubMed Central

Li, Li; Grausam, Katie B.; Wang, Jun; Lun, Melody P.; Ohli, Jasmin; Lidov, Hart G. W.; Calicchio, Monica L.; Zeng, Erliang; Salisbury, Jeffrey L.; Wechsler-Reya, Robert J.; Lehtinen, Maria K.; Schüller, Ulrich; Zhao, Haotian

2016-01-01

Aberrant Notch signaling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly pediatric brain neoplasms. We developed animal models of CP tumours by inducing sustained expression of Notch1 that recapitulate properties of human CP tumours with aberrant NOTCH signaling. Whole transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate diffferentiation. A Shh-driven signaling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from mono-ciliated progenitors in the roof plate characterized by elevated Notch signaling. Abnormal SHH signaling and distinct ciliogenesis are detected in human CP tumours, suggesting SHH pathway and cilia differentiation as potential therapeutic avenues. PMID:26999738