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Development of a human extracellular matrix for applications related with stem cells and tissue engineering.

Escobedo-Lucea C, Ayuso-Sacido A, Xiong C, Prado-López S, del Pino MS, Melguizo D, Bellver-Estellés C, Gonzalez-Granero S, Valero ML, Moreno R, Burks DJ, Stojkovic M - Stem Cell Rev (2012)

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Affiliation: Comparative Neurobiology Unit, Instituto Cavanilles, University of Valencia- RETICS, 46980, Valencia, Spain. esluma@uv.es

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The presence of characteristic ECM adhesion proteins before and after the treatment was confirmed by immunostaining (Fig.  2b) using antibodies against fibronectin (Fig.  2), collagen (Fig.  2) and laminin (Fig.  2)... Collectively, the results of these studies demonstrate that the hffECM retains a structure similar to the ECM of intact HFF, suggesting that the lysis conditions do not damage significantly the organization of the matrix... In contrast, colonies plated on hffECM with TERS1 conditioned media retained the morphology of undifferentiated colonies with defined borders and a compact sphere, similar to hESC maintained on feeders (Fig.  4a, b)... Telomerase activity was similar between H9 cells at the onset of the experiment (passage 31) and those maintained for 21 additional passages (total of 52 passages) on either hffECM or HFF (Fig.  4d)... Additionally, the hESC maintained on hffECM continued to express markers of pluripotency at levels equivalent or superior to those detected in hESC grown on feeder layers (Fig.  4e)... To determine the compatibility of the hffECM with the differentiation of hESC by chemically-defined protocols, H9 colonies were cultured on this matrix under conditions known to generate human adipocytes... Indeed, our hffECM was capable of maintaining hESC in the undifferentiated state without inducing chromosomal aberrations during a significant amount of time (21 passages)... One observation regarding the hffECM that we initially interpreted as a limitation was the slight reduction in the number of colonies when hESC were continuously passaged on hffECM as compared with those grown on HFF... Our proteomics data reveal that the majority of the ECM partners which interact with integrins are retained in the hffECM (Fig.  3b), providing at least one molecular explanation for the capacity of this matrix to support growth and pluripotency of hESC during an extended period of time (21 passages)... With the aim of extrapolating in vitro experiments to in vivo processes, Zaman et al., 2006 compared cell migration parameters in 2D with Matrigel and observed clear differences between these supports... When we compared the migration of U87 cells on Matrigel versus hffECM as 3D supports, we observed similar patterns, although the speed of migration was superior on hffECM... Our hffECM supports the maintenance and differentiation of hESC and based on our data, can be applied to other types of stem cells which require a physical support... The taxonomy was set to Homo sapiens... The Paragon algorithm used by ProteinPilot does not require the manual setting of any of these parameters.

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Identification of protein components of the hffECM. After removal of HFF by lysis, the remaining hffECM was eluted from culture plates and subjected to proteomic analysis. a STRING analysis of the proteins contained in the cellular component terms of gene ontology that are significantly enriched in the hffECM samples. The two principal clusters correspond mainly to proteins of the extracellular matrix (blue) or cellular surface components (green). Note that the components form tight networks with several inter-cluster connections. b Protein components of the human ECM-receptor interaction pathway of the KEGG database. The proteins of the hffECM identified by mass spectrometry are in indicated in red. Laminin, in blue, was detected by immnunocytochemical analysis of the hffECM. Most of the extracellular matrix partners of the pathway have been identified
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Fig3: Identification of protein components of the hffECM. After removal of HFF by lysis, the remaining hffECM was eluted from culture plates and subjected to proteomic analysis. a STRING analysis of the proteins contained in the cellular component terms of gene ontology that are significantly enriched in the hffECM samples. The two principal clusters correspond mainly to proteins of the extracellular matrix (blue) or cellular surface components (green). Note that the components form tight networks with several inter-cluster connections. b Protein components of the human ECM-receptor interaction pathway of the KEGG database. The proteins of the hffECM identified by mass spectrometry are in indicated in red. Laminin, in blue, was detected by immnunocytochemical analysis of the hffECM. Most of the extracellular matrix partners of the pathway have been identified

Mentions: Experimental design for the obtention, analysis and applications of hffECM. Human foreskin fibroblasts (passage 11 to 18) were cultured during 7–9 days before inactivation with mitomycin C. At this stage, cultures were treated overnight at 4°C with lysis buffer to remove cells and the resulting hffECM was subjected to one of the following procedures: (a-1) Assessment of hECM protein integrity and characterization. Electron microscopy analysis was performed to evaluate the integrity and composition of hECM after the treatment and to establish the optimal period for obtention (see Fig. 2a). Immunocytochemistry (ICQ) was used to confirm visually the presence of characteristic ECM components (see Fig. 2b). (a-2) Identification of ECM proteins by proteomic analysis (Fig. 3). (a-3) Application of hffECM to biological processes. To test the functional capacity of the hffECM, we designed and performed different experiments related with pluripotency (Fig. 4a-e) and differentiation of hESC towards the three germ layers (Fig. 5 and Supplementary Fig. 1a-f) as well as the migration of adults cells (Fig. 6 a-c and Supplementary videos 1–3)


Development of a human extracellular matrix for applications related with stem cells and tissue engineering.

Escobedo-Lucea C, Ayuso-Sacido A, Xiong C, Prado-López S, del Pino MS, Melguizo D, Bellver-Estellés C, Gonzalez-Granero S, Valero ML, Moreno R, Burks DJ, Stojkovic M - Stem Cell Rev (2012)

Identification of protein components of the hffECM. After removal of HFF by lysis, the remaining hffECM was eluted from culture plates and subjected to proteomic analysis. a STRING analysis of the proteins contained in the cellular component terms of gene ontology that are significantly enriched in the hffECM samples. The two principal clusters correspond mainly to proteins of the extracellular matrix (blue) or cellular surface components (green). Note that the components form tight networks with several inter-cluster connections. b Protein components of the human ECM-receptor interaction pathway of the KEGG database. The proteins of the hffECM identified by mass spectrometry are in indicated in red. Laminin, in blue, was detected by immnunocytochemical analysis of the hffECM. Most of the extracellular matrix partners of the pathway have been identified
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285767&req=5

Fig3: Identification of protein components of the hffECM. After removal of HFF by lysis, the remaining hffECM was eluted from culture plates and subjected to proteomic analysis. a STRING analysis of the proteins contained in the cellular component terms of gene ontology that are significantly enriched in the hffECM samples. The two principal clusters correspond mainly to proteins of the extracellular matrix (blue) or cellular surface components (green). Note that the components form tight networks with several inter-cluster connections. b Protein components of the human ECM-receptor interaction pathway of the KEGG database. The proteins of the hffECM identified by mass spectrometry are in indicated in red. Laminin, in blue, was detected by immnunocytochemical analysis of the hffECM. Most of the extracellular matrix partners of the pathway have been identified
Mentions: Experimental design for the obtention, analysis and applications of hffECM. Human foreskin fibroblasts (passage 11 to 18) were cultured during 7–9 days before inactivation with mitomycin C. At this stage, cultures were treated overnight at 4°C with lysis buffer to remove cells and the resulting hffECM was subjected to one of the following procedures: (a-1) Assessment of hECM protein integrity and characterization. Electron microscopy analysis was performed to evaluate the integrity and composition of hECM after the treatment and to establish the optimal period for obtention (see Fig. 2a). Immunocytochemistry (ICQ) was used to confirm visually the presence of characteristic ECM components (see Fig. 2b). (a-2) Identification of ECM proteins by proteomic analysis (Fig. 3). (a-3) Application of hffECM to biological processes. To test the functional capacity of the hffECM, we designed and performed different experiments related with pluripotency (Fig. 4a-e) and differentiation of hESC towards the three germ layers (Fig. 5 and Supplementary Fig. 1a-f) as well as the migration of adults cells (Fig. 6 a-c and Supplementary videos 1–3)

View Article: PubMed Central - PubMed

Affiliation: Comparative Neurobiology Unit, Instituto Cavanilles, University of Valencia- RETICS, 46980, Valencia, Spain. esluma@uv.es

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

The presence of characteristic ECM adhesion proteins before and after the treatment was confirmed by immunostaining (Fig.  2b) using antibodies against fibronectin (Fig.  2), collagen (Fig.  2) and laminin (Fig.  2)... Collectively, the results of these studies demonstrate that the hffECM retains a structure similar to the ECM of intact HFF, suggesting that the lysis conditions do not damage significantly the organization of the matrix... In contrast, colonies plated on hffECM with TERS1 conditioned media retained the morphology of undifferentiated colonies with defined borders and a compact sphere, similar to hESC maintained on feeders (Fig.  4a, b)... Telomerase activity was similar between H9 cells at the onset of the experiment (passage 31) and those maintained for 21 additional passages (total of 52 passages) on either hffECM or HFF (Fig.  4d)... Additionally, the hESC maintained on hffECM continued to express markers of pluripotency at levels equivalent or superior to those detected in hESC grown on feeder layers (Fig.  4e)... To determine the compatibility of the hffECM with the differentiation of hESC by chemically-defined protocols, H9 colonies were cultured on this matrix under conditions known to generate human adipocytes... Indeed, our hffECM was capable of maintaining hESC in the undifferentiated state without inducing chromosomal aberrations during a significant amount of time (21 passages)... One observation regarding the hffECM that we initially interpreted as a limitation was the slight reduction in the number of colonies when hESC were continuously passaged on hffECM as compared with those grown on HFF... Our proteomics data reveal that the majority of the ECM partners which interact with integrins are retained in the hffECM (Fig.  3b), providing at least one molecular explanation for the capacity of this matrix to support growth and pluripotency of hESC during an extended period of time (21 passages)... With the aim of extrapolating in vitro experiments to in vivo processes, Zaman et al., 2006 compared cell migration parameters in 2D with Matrigel and observed clear differences between these supports... When we compared the migration of U87 cells on Matrigel versus hffECM as 3D supports, we observed similar patterns, although the speed of migration was superior on hffECM... Our hffECM supports the maintenance and differentiation of hESC and based on our data, can be applied to other types of stem cells which require a physical support... The taxonomy was set to Homo sapiens... The Paragon algorithm used by ProteinPilot does not require the manual setting of any of these parameters.

Show MeSH
Related in: MedlinePlus