Fabrication of Coated Polycaprolactone Scaffolds and Their Effects on Murine Embryonic Stem Cells

2005 ◽  
Vol 873 ◽  
Author(s):  
Michael H. Tollon ◽  
Takashi Hamazaki ◽  
Bradley J. Willenberg ◽  
Christopher Batich ◽  
Naohiro Terada
Keyword(s):  
Materials Science ◽  
Embryonic Stem ◽  
Science Research ◽  
Cell Types ◽  
Basement Membranes ◽  
Extracellular Matrices ◽  
Es Cell ◽  
Cellular Behaviors ◽  
Extracellular Materials ◽  
Mes Cells

AbstractIn the past decade, tissue engineering has become a great interest in materials science research. Embryonic stem (ES) cell transplantation has become one of the most researched therapies for restoring tissue and organ function. Many studies have investigated the use of porous biodegradable scaffolds to promote cell adhesion, growth, proliferation, differentiation, and to help steer the course of tissue development. Research has shown that extracellular matrices and the basement membranes affect various cell types and cellular behaviors. However, the effects of these materials on ES cell behavior are currently understudied and poorly understood.In this study, the synthetic biodegradable polymer polycaprolactone (PCL) was chosen to create an interconnected, fibrous foam structure. A phase separated scaffold method was developed and the product made was coated with various extracellular materials. When the phase separated PCL scaffolds were coated with Matrigel and gelatin solutions, murine ES (mES) cells attached, spread, and differentiated within the scaffolds. There was little growth on the uncoated material. Coating effects on mES cells were analyzed using flow cytometry, reverse-transcriptase polymerase chain reaction and scanning electron microscopy. It was found that coating the scaffold with different extracellular matrices affects mES cell morphology and differentiation. Matrigel coating causes expression of neural proteins and gelatin produces a hepatocyte-like cell.

scholarly journals Designer blood: creating hematopoietic lineages from embryonic stem cells

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1265-1275 ◽  
Author(s):  
Abby L. Olsen ◽  
David L. Stachura ◽  
Mitchell J. Weiss
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Basic Research ◽  
Cell Types ◽  
Patient Specific ◽  
Es Cell ◽  
Blood Disorders ◽  
Hematopoietic Stem

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases.

Development to term of mouse androgenetic aggregation chimeras

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1325-1333 ◽  
Author(s):  
J.R. Mann ◽  
C.L. Stewart
Keyword(s):  
Mouse Strain ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Considerable Improvement ◽  
Es Cell ◽  
Partial Explanation ◽  
Developmental Potential ◽  
Skeletal Abnormalities ◽  
Embryonic Tissues

Diploid androgenetic eggs contain two sperm-derived genomes, and only rarely develop to the early somite stage. Also, previous studies have indicated that androgenetic eggs cannot be rescued in aggregation chimeras beyond embryonic stages. Paradoxically, in blastocyst injection chimeras made with androgenetic embryonic stem (ES) cells of the 129/Sv strain, we previously obtained considerable improvement in developmental potential. Although considerable death occurred in utero, overtly normal chimeric fetuses and occasional postnatal chimeras that developed skeletal abnormalities were observed. Consequently, we have re-evaluated the developmental potential of androgenetic aggregation chimeras utilizing androgenetic eggs of the 129/Sv strain, and of the BALB/c and CD-1 strains for comparison. Regardless of strain, androgenetic aggregation chimeras were generally more inviable than previously observed with androgenetic ES cell chimeras, and often the embryoproper was abnormal even when an androgenetic contribution was detected only in the extra-embryonic membranes. This is at least a partial explanation of the greater viability of androgenetic ES cell chimeras, as ES cells do not colonize significantly certain extra-embryonic tissues. Nevertheless, in the 129/Sv strain, occasional development of chimeras to term was obtained, and one chimera that survived postnatally developed identical skeletal abnormalities to those observed previously in androgenetic ES cell chimeras. This result demonstrates that at least one example of paternal imprinting is faithfully conserved in androgenetic ES cells. Also, the postnatal chimerism shows that androgenetic eggs can give rise to terminally differentiated cell types, and are therefore pluripotent. In contrast, only possibly one BALB/c and no CD-1 androgenetic aggregation chimeras developed to term. Therefore, the developmental potential of androgenetic aggregation chimeras is to some extent dependent on mouse strain.

scholarly journals T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal

2012 ◽  
Vol 302 (3) ◽  
pp. C494-C504 ◽  
Author(s):  
José A. Rodríguez-Gómez ◽  
Konstantín L. Levitsky ◽  
José López-Barneo
Keyword(s):  
Cell Cycle ◽  
Alkaline Phosphatase ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Mrna Levels ◽  
Es Cell ◽  
External Application ◽  
Self Renewal ◽  
Mouse Es Cells

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na+ currents as well as transient Ca2+ currents abolished by the external application of Ni2+. Biophysical and pharmacological data indicated that the Ca2+ current is predominantly mediated by T-type (Cav3.2) channels. The number of cells expressing T-type channels and Cav3.2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca2+ currents with Ni2+ induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Cav3.2) Ca2+ channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Cav3.2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca2+ entry mediated by Cav3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.

Human Embryonic Stem Cell-Derived Mesenchymal Stroma Cells (hES-MSC) Share Basic Properties with Bone Marrow MSC, They Have Potent Stroma Support Capacities but Lack Immune-Modulatory Function. Implications for off-the Shelf ES-MSC Therapies.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3858-3858 ◽  
Author(s):  
Ou Li ◽  
Ariane Tormin ◽  
Jan Claas Brune ◽  
Berit Sundberg ◽  
Johan Hyllner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Cell Types ◽  
Es Cell ◽  
Nsg Mice ◽  
Mesenchymal Stroma ◽  
Mesenchymal Stroma Cells

Abstract Abstract 3858 Mesenchymal stroma cells (MSC) have a high potential for novel cell therapy approaches in clinical transplantation due to their intriguing properties, e.g. high proliferation and differentiation capacity, stromal support and immune-modulation. Commonly, bone marrow-derived MSC (BM-MSC) are used for clinical MSC cell therapies. However, BM-derived MSC have a restricted proliferative capacity and cultured BM-MSC are heterogeneous and thus difficult to standardize. Human embryonic stem cell-derived mesenchymal stroma cells (hES-MSC) have recently been developed and might represent an alternative and unlimited source of hMSCs. We therefore aimed to characterize human ES-cell-derived MSC, i.e. the hES-MSC line hES-MP002.5 (Cellartis) and compare its properties with normal human bone marrow (BM) derived MSC. We found that hES-MP cells have lower yet reasonable CFU-F capacity when compared with BM-MSC (6+3 vs 25+1 CFU-F per 100 cells). hES-MP cells showed similar immunophenotypic properties compared with BM-MSC (flow cytometry): Both cell types were positive for CD105, CD73, CD166, HLA Class I, CD44, CD146 and CD90, and cells were negative for surface markers such as CD45, CD34, CD14, CD31, CD19, and HLA-DR. hES-MP, like BM-MSC, could be differentiated into adipocytes, osteoblasts and chondrocytes upon induction in vitro. In order to test whether MSC were capable of homing to the bone marrow after intravenous injection, hES-MP and BM-MSC were markerd with GFP, and sorted GFP-positive cells were injected intravenously into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. GFP-positive cells were not detected in the bone marrow 24 hours after injection, neither when hES-MP cells were injected, nor - and as expected - when cultured BM-MSC were used. Intra-femoral transplantation into NSG mice using GFP expressing hES-MP and BM-MSC on the other hand demonstrated successful long-term engraftment (8 weeks) for both cell types. Morphology and intra-femoral localization of hES-MP were similar compared to BM-MSC. LTC-IC and co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP, like BM-MSC, possess potent stroma support function both in vitro and in vivo. However, hES-MP showed no or only little activity in mixed lymphocyte cultures and PHA lymphocyte stimulation assays. In summary, our data demonstrate that MSC derived from hES cells have biological properties and potent stroma functions similar to conventional BM-MSC. Thus, ES-cell derived MSC might be an attractive and reliable alternative and unlimited source for obtaining MSC for clinical cell therapy. However, hES-MP probably have no or only little immuno-modulative capacity, which may limit their potential clinical use. Disclosures: Hyllner: Cellartis AB: Employment.

The WNT receptor FZD7 contributes to self-renewal signaling of human embryonic stem cells

2008 ◽  
Vol 389 (7) ◽  
Author(s):  
Kai Melchior ◽  
Jonathan Weiß ◽  
Holm Zaehres ◽  
Yong-mi Kim ◽  
Carolyn Lutzko ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Marker Genes ◽  
Specific Marker ◽  
Mrna Levels ◽  
Es Cell ◽  
Specific Expression ◽  
Self Renewal ◽  
Human Es Cells

Abstract A number of recent studies identified nuclear factors that together have the unique ability to induce pluripotency in differentiated cell types. However, little is known about the factors that are needed to maintain human embryonic stem (ES) cells in an undifferentiated state. In a search for such requirements, we performed a comprehensive meta-analysis of publicly available SAGE and microarray data. The rationale for this analysis was to identify genes that are exclusively expressed in human ES cell lines compared to 30 differentiated tissue types. The WNT receptor FZD7 was found among the genes with an ES cell-specific expression profile in both SAGE and microarray analyses. Subsequent validation by quantitative RT-PCR and flow cytometry confirmed that FZD7 mRNA levels in human ES cells are up to 200-fold higher compared to differentiated cell types. ShRNA-mediated knockdown of FZD7 in human ES cells induced dramatic changes in the morphology of ES cell colonies, perturbation of expression levels of germ layer-specific marker genes, and a rapid loss of expression of the ES cell-specific transcription factor OCT4. These findings identify the WNT receptor FZD7 as a novel ES cell-specific surface antigen with a likely important role in the maintenance of ES cell self-renewal capacity.

scholarly journals Permissive and restricted virus infection of murine embryonic stem cells

2012 ◽  
Vol 93 (10) ◽  
pp. 2118-2130 ◽  
Author(s):  
Rachael Wash ◽  
Sabrina Calabressi ◽  
Stephanie Franz ◽  
Samantha J. Griffiths ◽  
David Goulding ◽  
...  
Keyword(s):  
Virus Infection ◽  
Embryonic Stem ◽  
Cell Types ◽  
Host Protein ◽  
Host Proteins ◽  
Transformed Cell Lines ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Mes Cells ◽  
Hsv 1

Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA ‘off-target’ effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host–pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host–virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host–virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence.

scholarly journals Nuclear Receptors in Regulation of Mouse ES Cell Pluripotency and Differentiation

PPAR Research ◽  
2007 ◽  
Vol 2007 ◽  
pp. 1-10 ◽  
Author(s):  
Eimear M. Mullen ◽  
Peili Gu ◽  
Austin J. Cooney
Keyword(s):  
Nuclear Receptors ◽  
Therapeutic Potential ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Cell Phenotype ◽  
Es Cell ◽  
Complex Signaling ◽  
Different Cell Types ◽  
Mouse Es Cell

Embryonic stem (ES) cells have great therapeutic potential because they are capable of indefinite self-renewal and have the potential to differentiate into over 200 different cell types that compose the human body. The switch from the pluripotent phenotype to a differentiated cell involves many complex signaling pathways including those involving LIF/Stat3 and the transcription factors Sox2, Nanog and Oct-4. Many nuclear receptors play an important role in the maintenance of pluripotence (ERRβ, SF-1, LRH-1, DAX-1) repression of the ES cell phenotype (RAR, RXR, GCNF) and also the differentiation of ES cells (PPARγ). Here we review the roles of the nuclear receptors involved in regulating these important processes in ES cells.

scholarly journals UTF1 is a chromatin-associated protein involved in ES cell differentiation

2007 ◽  
Vol 178 (6) ◽  
pp. 913-924 ◽  
Author(s):  
Vincent van den Boom ◽  
Susanne M. Kooistra ◽  
Marije Boesjes ◽  
Bart Geverts ◽  
Adriaan B. Houtsmuller ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Leucine Zipper ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryonic Cell ◽  
Es Cell ◽  
Self Renewal ◽  
Adult Cell ◽  
Core Histones

Embryonic stem (ES) cells are able to grow indefinitely (self-renewal) and have the potential to differentiate into all adult cell types (pluripotency). The regulatory network that controls pluripotency is well characterized, whereas the molecular basis for the transition from self-renewal to the differentiation of ES cells is much less understood, although dynamic epigenetic gene silencing and chromatin compaction are clearly implicated. In this study, we report that UTF1 (undifferentiated embryonic cell transcription factor 1) is involved in ES cell differentiation. Knockdown of UTF1 in ES and carcinoma cells resulted in a substantial delay or block in differentiation. Further analysis using fluorescence recovery after photobleaching assays, subnuclear fractionations, and reporter assays revealed that UTF1 is a stably chromatin-associated transcriptional repressor protein with a dynamic behavior similar to core histones. An N-terminal Myb/SANT domain and a C-terminal domain containing a putative leucine zipper are required for these properties of UTF1. These data demonstrate that UTF1 is a strongly chromatin-associated protein involved in the initiation of ES cell differentiation.

scholarly journals Human telomeres replicate using chromosome-specific, rather than universal, replication programs

2012 ◽  
Vol 197 (2) ◽  
pp. 253-266 ◽  
Author(s):  
William C. Drosopoulos ◽  
Settapong T. Kosiyatrakul ◽  
Zi Yan ◽  
Simone G. Calderano ◽  
Carl L. Schildkraut
Keyword(s):  
Single Molecule ◽  
Embryonic Stem ◽  
Cell Types ◽  
Es Cell ◽  
Site Location ◽  
Subtelomeric Heterochromatin ◽  
Basic Features ◽  
Different Cell Types ◽  
Single Molecule Analysis ◽  
Individual Human

Telomeric and adjacent subtelomeric heterochromatin pose significant challenges to the DNA replication machinery. Little is known about how replication progresses through these regions in human cells. Using single molecule analysis of replicated DNA (SMARD), we delineate the replication programs—i.e., origin distribution, termination site location, and fork rate and direction—of specific telomeres/subtelomeres of individual human chromosomes in two embryonic stem (ES) cell lines and two primary somatic cell types. We observe that replication can initiate within human telomere repeats but was most frequently accomplished by replisomes originating in the subtelomere. No major delay or pausing in fork progression was detected that might lead to telomere/subtelomere fragility. In addition, telomeres from different chromosomes from the same cell type displayed chromosome-specific replication programs rather than a universal program. Importantly, although there was some variation in the replication program of the same telomere in different cell types, the basic features of the program of a specific chromosome end appear to be conserved.

scholarly journals Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

1998 ◽  
Vol 142 (4) ◽  
pp. 1121-1133 ◽  
Author(s):  
Helen Priddle ◽  
Lance Hemmings ◽  
Susan Monkley ◽  
Alison Woods ◽  
Bipin Patel ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Es Cells ◽  
Embryonic Stem ◽  
Focal Adhesions ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Integrin Subunit ◽  
Β1 Integrin ◽  
Es Cell ◽  
Wild Type

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.

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