In vitro differentiation of reprogrammed murine somatic cells into hepatic precursor cells

2008 ◽  
Vol 389 (7) ◽  
Author(s):  
Tobias Cantz ◽  
Martina Bleidißel ◽  
Martin Stehling ◽  
Hans R. Schöler
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Es Cells ◽  
Marrow Cell ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Precursor Cells ◽  
Differentiation Potential ◽  
Teratoma Formation ◽  
Transplantation Experiments

Abstract Recently, a new approach to reprogram somatic cells into pluripotent stem cells was shown by fusion of somatic cells with embryonic stem (ES) cells, which results in a tetraploid karyotype. Normal hepatocytes are often polyploid, so we decided to investigate the differentiation potential of fusion hybrids into hepatic cells. We chose toxic milk mice (a model of Wilson's disease) and performed initial transplantation experiments using this potential cell therapy approach. Mononuclear bone marrow cells from Rosa26 mice were fused with OG2 (Oct4-GFP transgenic) ES cells. Unfused ES cells were eliminated by selection with G418 for OG2-Rosa26 hybrids and fusion-derived colonies could be subcloned. Using an endodermal differentiation protocol, hepatic precursor cells could be generated. After FACS depletion of contaminating Oct4-GFP-positive cells, the hepatic precursor cells were transplanted into immunosuppressed toxic milk mice by intrasplenic injection. However, five out of eight mice showed teratoma formation within 3–6 weeks after transplantation in the spleen and liver. In conclusion, a hepatic precursor cell type was achieved from mononuclear bone marrow cell-ES cell hybrids and preliminary transplantation experiments confirmed engraftment, but also showed teratoma formation, which needs to be excluded by using more stringent purification strategies.

225 TERATOMA FORMATION BY BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 229
Author(s):  
M. L. Lim ◽  
I. Vassiliev ◽  
P. J. Verma
Keyword(s):  
Growth Factor ◽  
Es Cells ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Scid Mice ◽  
Bovine Embryos ◽  
Differentiation Potential ◽  
Teratoma Formation

Teratoma formation is commonly used as a model for examining the in vivo differentiation potential of embryonic stem cells. We wanted to investigate the teratoma-forming ability of bovine ES cells; however, there are no reports of teratoma-forming ability of bovine pluripotent cells including pre-implantation embryos. In vivo-produced bovine embryos at stages earlier than Day 14 failed to develop teratomas when transplanted into one of the kidneys of immuno-deficient mice (Anderson et al. 1996 Anim. Reprod. Sci. 45, 231–240), and this prompted questions about the ability of bovine embryos to form teratomas. Bovine oocytes were cultured for 20 to 22 h after aspiration at 39�C (5% CO2/95% air) in TCM-199-bicarbonate medium supplemented with GlutaMax6" (Invitrogen Australia Pty Ltd., Mount Waverley, Victoria, Australia), penicillin/streptomycin, β-mercaptoethanol, 17β-estradiol, fetal calf serum, LH, follicle stimulating hormone, basic fibroblast growth factor, epidermal growth factor, glycine, and l-cysteine. Oocytes were fertilized with IVF media (Cook Australia, Brisbane, Queensland, Australia) and kept for 7 days at 39�C in 5% CO2/95% air to generate blastocysts. The zona pellucida of Day 7 blastocysts was enzymatically removed, and one or two zona-free embryos were injected into each testis of 5-week-old immunodeficient (SCID) mice (CB-17/ICR-Prkdcscid strain; Walter and Eliza Hall Institute, Melbourne, Australia). Eight weeks post-injection, teratomas partially expelled from testes were identified. Histological analysis has confirmed the derivatives of all 3 germ layers in teratomas. In conclusion, we report that Day 7 in vitro-produced embryos can form teratomas when injected into testes of SCID mice.

scholarly journals CONTRIBUTION OF BONE MARROW CELLS AND LACK OF EXPRESSION OF THYMOCYTES IN GENETIC CONTROLS OF IMMUNE RESPONSES FOR TWO IMMUNOPOTENT REGIONS WITHIN POLY-(PHE,GLU)-POLY-PRO--POLY-LYS IN INBRED MOUSE STRAINS

1971 ◽  
Vol 134 (1) ◽  
pp. 141-161 ◽  
Author(s):  
Edna Mozes ◽  
G. M. Shearer
Keyword(s):  
Bone Marrow ◽  
Immune Responses ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Genetic Defect ◽  
Precursor Cells ◽  
Immunocompetent Cells ◽  
Mouse Strains ◽  
Limiting Dilution ◽  
Marrow Cells

Previous cellular studies on the genetic regulation of immunological responsiveness for two immunopotent regions within the branched chain synthetic polypeptide (Phe, G)-Pro--L demonstrated a direct correlation between the number of detectable immunocompetent splenic precursor cells and the response patterns of SJL, DBA/1, and F1 mice (21). In order to establish the cellular origin(s) of the genetic defect, the present study first demonstrated that thymus and bone marrow cell cooperation was required for (Phe, G)- and Pro--L-specific immune responses. Secondly, limiting dilution experiments, in which several graded and limiting inocula of marrow cells were mixed with a non-limiting number of 108 thymocytes and injected into irradiated, syngeneic recipients, indicated that the low responsiveness of the SJL and DBA/1 strains to the (Phe, G) and Pro--L specificities, respectively, could be attributed to a reduced number of precursor cells found in bone marrow. About five times more marrow precursors were detected in SJL mice for Pro--L than for (Phe, G), whereas about five times as many precursor cells were estimated for (Phe, G) as for Pro--L in the DBA/1 strain. These differences are similar to those obtained using spleen cells from unimmunized SJL and DBA/1 donors (21), and indicate that these genetically determined variations in responsiveness can be accounted for by differences in the frequencies of monospecific populations of immunocompetent cells present in bone marrow. In contrast, limiting dilution transfers of thymocytes or thymus-derived cells with an excess of syngeneic marrow cells resulted in equally frequent (Phe, G) and Pro--L responses for both SJL ad DBA/1 strains. This finding in conjunction with the observation that the generation of (Phe, G)- and Pro--L-specific responses were associated in individual recipients injected with limiting inocula of thymocytes indicated that a single population of thymocytes was stimulated by (Phe,G)-Pro--L. Therefore, it is improbable that the thymic population of immunocompetent cells contributes to expression of these genetically controlled defects.

scholarly journals Role of variable region gene expression and environmental selection in determining the antiphosphorylcholine B cell repertoire.

1983 ◽  
Vol 158 (6) ◽  
pp. 1948-1961 ◽  
Author(s):  
N R Klinman ◽  
M R Stone
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Variable Region ◽  
Precursor Cells ◽  
Cell Repertoire ◽  
Region Gene ◽  
B Cell Repertoire

To evaluate the role of environmental selective processes, as opposed to variable region gene expression, in the determination of B cell repertoire expression, we have assessed the phosphorylcholine (PC)-specific repertoire of precursor cells that remain in bone marrow cell populations after the removal of surface immunoglobulin (sIg)-bearing cells. Such cells are assumed to represent a stage in B cell maturation before the expression of sIg, and thus at a time when they have not as yet interfaced with environmental influences that operate through sIg receptors such as antigenic stimulation, tolerance, or antiidiotypic regulation. The repertoire as expressed in these cells, therefore, should reflect the readout of immunoglobulin variable region genes as they are expressed in progenitors to B cells. The results of these studies indicate that, as in mature primary B cell pools of BALB/c mice, the majority of PC-responsive sIg- bone marrow cells are of the T15 clonotype. Thus, environmental selective mechanisms would not appear to be required for the high frequency of B cells of the T15 idiotype in the primary B cell repertoire of BALB/c mice. Analysis of the sIg- bone marrow cells in (CBA/N X BALB/c)F1 male mice demonstrated that the deficit of PC-responsive mature B cells, which is a characteristic of this murine strain, must occur after receptor expression, since a normal frequency of PC-responsive and T15-expressing cells is present in their sIg- bone marrow population. Finally, these same mice were used to obtain bone marrow cell preparations from individual leg bones, so as to permit an analysis of the occurrence of T15+ and T15- clonotypes within individual bone marrow populations. The findings from these studies indicate that T15+ B cells occur as a high frequency event within bone marrow generative cell pools. Furthermore, bone marrow populations that are positive for PC-responsive precursor cells often display multiple copies of such precursor cells that are exclusively either T15+ or T15-. This finding indicates that clonal expansion of cells within the B cell lineage apparently occurs before immunoglobulin receptor acquisition.

Enrichment of blood from embryonic stem cells in vitro

1998 ◽  
Vol 10 (8) ◽  
pp. 563 ◽  
Author(s):  
Andrew C. Perkins
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Embryonic Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Leukaemia Inhibitory Factor ◽  
Chimeric Mouse ◽  
Differentiation Potential

Murine embryonic stem (ES) cells are pluripotent. When injected into blastocysts they can give rise to every cell type of a derived chimeric mouse including germ cells. Embryonic stem cells also possess remarkable in vitro differentiation potential. When removed from stromal support and leukaemia inhibitory factor (LIF), ES cells differentiate into structures known as embryoid bodies (EBs), in which all three germ layers develop and interact. As ES cells from humans become available there is increasing interest in the potential for EBs to provide an unlimited supply of stem cells for somatic transplantation therapies. Realisation of this potential will require greater understanding of the molecular determinants of cell fate within EBs. Also, culture techniques for selective expansion of cell lineages of interest will reduce the risks associated with transplantation of EB-derived cells. In this paper the kinetics of expression of mRNA and protein for early mesoderm markers within EBs is reported. In addition, a three-step culture system incorporating co-cultivation on the bone marrow derived stromal cell line, MC3T3-G2/PA6 (PA6), is explored as a way to select for haematopoietic progenitor cells (HPCs) and against undifferentiated ES cells. A system like this could enhance purification of haematopoietic stem cells (HSCs) from ES cells for bone marrow transplantation.

scholarly journals CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4136-4142 ◽  
Author(s):  
I Kawashima ◽  
ED Zanjani ◽  
G Almaida-Porada ◽  
AW Flake ◽  
H Zeng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
In Utero ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Show Evidence ◽  
Marrow Cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

scholarly journals Epigenetic Regulation of Mesenchymal Stem Cells: A Focus on Osteogenic and Adipogenic Differentiation

2011 ◽  
Vol 2011 ◽  
pp. 1-18 ◽  
Author(s):  
Chad M. Teven ◽  
Xing Liu ◽  
Ning Hu ◽  
Ni Tang ◽  
Stephanie H. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Epigenetic Regulation ◽  
Adult Stem Cells ◽  
Es Cells ◽  
Adipogenic Differentiation ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiation Potential ◽  
Msc Differentiation

Stem cells are characterized by their capability to self-renew and terminally differentiate into multiple cell types. Somatic or adult stem cells have a finite self-renewal capacity and are lineage-restricted. The use of adult stem cells for therapeutic purposes has been a topic of recent interest given the ethical considerations associated with embryonic stem (ES) cells. Mesenchymal stem cells (MSCs) are adult stem cells that can differentiate into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. While various signaling molecules important to MSC differentiation have been identified, our complete understanding of this process is lacking. Recent investigations focused on the role of epigenetic regulation in lineage-specific differentiation of MSCs have shown that unique patterns of DNA methylation and histone modifications play an important role in the induction of MSC differentiation toward specific lineages. Nevertheless, MSC epigenetic profiles reflect a more restricted differentiation potential as compared to ES cells. Here we review the effect of epigenetic modifications on MSC multipotency and differentiation, with a focus on osteogenic and adipogenic differentiation. We also highlight clinical applications of MSC epigenetics and nuclear reprogramming.

Lidocaine depresses splenocyte immune functions following trauma-hemorrhage in mice

2006 ◽  
Vol 291 (5) ◽  
pp. C1049-C1055 ◽  
Author(s):  
Takashi Kawasaki ◽  
Mashkoor A. Choudhry ◽  
Martin G. Schwacha ◽  
Kirby I. Bland ◽  
Irshad H. Chaudry
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Immune Responses ◽  
Cytokine Production ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Sham Operation ◽  
Mapk Activation ◽  
Midline Laparotomy ◽  
Marrow Cells

Traumatic and/or surgical injury as well as hemorrhage induces profound suppression of cellular immunity. Although local anesthetics have been shown to impair immune responses, it remains unclear whether lidocaine affects lymphocyte functions following trauma-hemorrhage (T-H). We hypothesized that lidocaine will potentiate the suppression of lymphocyte functions after T-H. To test this, we randomly assigned male C3H/HeN (6–8 wk) mice to sham operation or T-H. T-H was induced by midline laparotomy and ∼90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4× shed blood volume in the form of Ringer lactate). Two hours later, the mice were killed and splenocytes and bone marrow cells were isolated. The effects of lidocaine on concanavalin A-stimulated splenocyte proliferation and cytokine production in both sham-operated and T-H mice were assessed. The effects of lidocaine on LPS-stimulated bone marrow cell proliferation and cytokine production were also assessed. The results indicate that T-H suppresses cell proliferation, Th1 cytokine production, and MAPK activation in splenocytes. In contrast, cell proliferation, cytokine production, and MAPK activation in bone marrow cells were significantly higher 2 h after T-H compared with shams. Lidocaine depressed immune responses in splenocytes; however, it had no effect in bone marrow cells in either sham or T-H mice. The enhanced immunosuppressive effects of lidocaine could contribute to the host's enhanced susceptibility to infection following T-H.

scholarly journals A Model for Sequestration of the Transmission Stages of Plasmodium falciparum: Adhesion of Gametocyte-Infected Erythrocytes to Human Bone Marrow Cells

2000 ◽  
Vol 68 (6) ◽  
pp. 3455-3462 ◽  
Author(s):  
Nicola J. Rogers ◽  
Belinda S. Hall ◽  
Jacktone Obiero ◽  
Geoffrey A. T. Targett ◽  
Colin J. Sutherland
Keyword(s):  
Bone Marrow ◽  
Plasmodium Falciparum ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Mature Stage ◽  
High Avidity ◽  
Necrosis Factor Alpha ◽  
Marrow Cells

ABSTRACT With the aim of developing an appropriate in vitro model of the sequestration of developing Plasmodium falciparumsexual-stage parasites, we have investigated the cytoadherence of gametocytes to human bone marrow cells of stromal and endothelial origin. Developing stage III and IV gametocytes, but not mature stage V gametocytes, adhere to bone marrow cells in significantly higher densities than do asexual-stage parasites, although these adhesion densities are severalfold lower than those encountered in classical CD36-dependent assays of P. falciparum cytoadherence. This implies that developing gametocytes undergo a transition from high-avidity, CD36-mediated adhesion during stages I and II to a lower-avidity adhesion during stages III and IV. We show that this adhesion is CD36 independent, fixation sensitive, stimulated by tumor necrosis factor alpha, and dependent on divalent cations and serum components. These data suggest that gametocytes and asexual parasites utilize distinct sets of receptors for adhesion during development in their respective sequestered niches. To identify receptors for gametocyte-specific adhesion of infected erythrocytes to bone marrow cells, we tested a large panel of antibodies for the ability to inhibit cytoadherence. Our results implicate ICAM-1, CD49c, CD166, and CD164 as candidate bone marrow cell receptors for gametocyte adhesion.

scholarly journals Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3932-3939 ◽  
Author(s):  
Yang Du ◽  
Nancy A. Jenkins ◽  
Neal G. Copeland
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Drug Targets ◽  
Insertional Mutagenesis ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Serial Passage ◽  
Mouse Bone Marrow ◽  
Cancer Genes ◽  
Differentiation Potential

Retroviruses can induce hematopoietic disease via insertional mutagenesis of cancer genes and provide valuable molecular tags for cancer gene discovery. Here we show that insertional mutagenesis can also identify genes that promote the immortalization of hematopoietic cells, which normally have only limited self-renewal. Transduction of mouse bone marrow cells with replication-incompetent murine stem cell virus (MSCV) expressing only neo, followed by serial passage in liquid culture containing stem cell factor (SCF) and interleukin-3 (IL-3), produced immortalized immature myeloid cell lines with neutrophil and macrophage differentiation potential in about 50% of the infected cultures. More than half of the lines have MSCV insertions at Evi1 or Prdm16. These loci encode transcription factor homologs and are validated human myeloid leukemia genes. Integrations are located in intron 1 or 2, where they promote expression of truncated proteins lacking the PRDI-BF1-RIZ1 homologous (PR) domain, similar to what is observed in human leukemias with EVI1 or PRDM16 mutations. Evi1 overexpression alone appears sufficient to immortalize immature myeloid cells and does not seem to require any other cooperating mutations. Genes identified by insertional mutagenesis by their nature could also be involved in immortalization of leukemic stem cells, and thus represent attractive drug targets for treating cancer.

scholarly journals Effects of neuraminidase on the regulation of erythropoiesis

Blood ◽  
1984 ◽  
Vol 63 (4) ◽  
pp. 784-788 ◽  
Author(s):  
VF LaRussa ◽  
F Sieber ◽  
LL Sensenbrenner ◽  
SJ Sharkis
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Colony Growth ◽  
Mouse Bone Marrow ◽  
Continuous Exposure ◽  
Low Concentrations ◽  
High Concentrations ◽  
Marrow Cells

Abstract In this article, we present evidence that sialic acid-containing surface components play a role in the regulation of erythropoiesis. A 1- hr exposure of mouse bone marrow cells to high concentrations of neuraminidase reduced erythroid colony formation. Coculture of 10(6) untreated thymocytes with neuraminidase-treated bone marrow cells restored erythroid colony growth. Neuraminidase-treated thymocytes retained their ability to suppress erythroid colony formation by untreated marrow cells, but lost their ability to enhance erythroid colony formation. Continuous exposure to low concentrations of neuraminidase enhanced erythroid bone marrow cell colony growth in response to a suboptimal dose of erythropoietin.

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