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Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture

BACKGROUND: Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but wit...

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Autores principales: Peters, Rowayda, Wolf, Monika J., van den Broek, Maries, Nuvolone, Mario, Dannenmann, Stefanie, Stieger, Bruno, Rapold, Reto, Konrad, Daniel, Rubin, Arnold, Bertino, Joseph R., Aguzzi, Adriano, Heikenwalder, Mathias, Knuth, Alexander K.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012708/
https://www.ncbi.nlm.nih.gov/pubmed/21209896
http://dx.doi.org/10.1371/journal.pone.0015689
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author Peters, Rowayda
Wolf, Monika J.
van den Broek, Maries
Nuvolone, Mario
Dannenmann, Stefanie
Stieger, Bruno
Rapold, Reto
Konrad, Daniel
Rubin, Arnold
Bertino, Joseph R.
Aguzzi, Adriano
Heikenwalder, Mathias
Knuth, Alexander K.
author_facet Peters, Rowayda
Wolf, Monika J.
van den Broek, Maries
Nuvolone, Mario
Dannenmann, Stefanie
Stieger, Bruno
Rapold, Reto
Konrad, Daniel
Rubin, Arnold
Bertino, Joseph R.
Aguzzi, Adriano
Heikenwalder, Mathias
Knuth, Alexander K.
author_sort Peters, Rowayda
collection PubMed
description BACKGROUND: Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC. METHODS AND FINDINGS: UCB-derived mononuclear cells (MNC) or selected CD34(+) cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34(+) selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin(+), CD133(+) and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages. Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies. CONCLUSIONS: This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice.
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spelling pubmed-30127082011-01-05 Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture Peters, Rowayda Wolf, Monika J. van den Broek, Maries Nuvolone, Mario Dannenmann, Stefanie Stieger, Bruno Rapold, Reto Konrad, Daniel Rubin, Arnold Bertino, Joseph R. Aguzzi, Adriano Heikenwalder, Mathias Knuth, Alexander K. PLoS One Research Article BACKGROUND: Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC. METHODS AND FINDINGS: UCB-derived mononuclear cells (MNC) or selected CD34(+) cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34(+) selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin(+), CD133(+) and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages. Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies. CONCLUSIONS: This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice. Public Library of Science 2010-12-30 /pmc/articles/PMC3012708/ /pubmed/21209896 http://dx.doi.org/10.1371/journal.pone.0015689 Text en Peters et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Peters, Rowayda
Wolf, Monika J.
van den Broek, Maries
Nuvolone, Mario
Dannenmann, Stefanie
Stieger, Bruno
Rapold, Reto
Konrad, Daniel
Rubin, Arnold
Bertino, Joseph R.
Aguzzi, Adriano
Heikenwalder, Mathias
Knuth, Alexander K.
Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture
title Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture
title_full Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture
title_fullStr Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture
title_full_unstemmed Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture
title_short Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture
title_sort efficient generation of multipotent mesenchymal stem cells from umbilical cord blood in stroma-free liquid culture
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012708/
https://www.ncbi.nlm.nih.gov/pubmed/21209896
http://dx.doi.org/10.1371/journal.pone.0015689
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