Limits...
Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression.

Montzka K, Lassonczyk N, Tschöke B, Neuss S, Führmann T, Franzen R, Smeets R, Brook GA, Wöltje M - BMC Neurosci (2009)

Bottom Line: Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries.More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression.Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, RWTH Aachen University, Aachen, Germany. kmontzka@ukaachen.de

ABSTRACT

Background: In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors.

Results: The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression.

Conclusion: The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

Show MeSH

Related in: MedlinePlus

Surface marker expression of human MSCs. FACS analysis of the immunophenotypic surface profile for CD11b, CD19, CD34, CD45, CD73, CD90, CD105 and HLA-DR of isolated hMSCs. Red histograms represent the fluorescence from negative-control cells incubated with only secondary antibody; black histograms represent the counts of cells incubated with the relevant primary antibody. The logarithm on the X-axis (FL1-H channel) represents the intensity of the fluorescent signal and the number of cells is given on the Y-axis. HMSCs isolated in this study were positive for the markers CD73, CD90 and CD105, but negative for CD11b, CD19, CD34, CD45 and HLA-DR according to the criteria for MSCs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2655300&req=5

Figure 1: Surface marker expression of human MSCs. FACS analysis of the immunophenotypic surface profile for CD11b, CD19, CD34, CD45, CD73, CD90, CD105 and HLA-DR of isolated hMSCs. Red histograms represent the fluorescence from negative-control cells incubated with only secondary antibody; black histograms represent the counts of cells incubated with the relevant primary antibody. The logarithm on the X-axis (FL1-H channel) represents the intensity of the fluorescent signal and the number of cells is given on the Y-axis. HMSCs isolated in this study were positive for the markers CD73, CD90 and CD105, but negative for CD11b, CD19, CD34, CD45 and HLA-DR according to the criteria for MSCs.

Mentions: Fluorescent activated cell sorting (FACS) analysis demonstrated that the expanded, plastic adherent cells used in the present investigation were positive for the surface markers CD73, CD90 and CD105, but negative for CD11b, CD19, CD34, CD45 and HLA-DR (Figure 1). To demonstrate their multipotent potential, MSCs were differentiated to adipocytes, chondrocytes and osteocytes according to published protocols [24]. Lipid vacuoles in differentiated adipocytes were visualized with Oil Red O (Figure 2A). However, not all cells demonstrated the same degree of staining. Induction of chondrogenic differentiation was performed in cell pellets which developed a proteoglycan-rich extracellular matrix. Thin sections of these pellets were stained with Toluidine Blue (Figure 2B), demonstrating a metachromatic staining that was characteristic of cartilage matrix [25]. Osteogenic differentiation resulted in an immense production of mineral deposits that were stained with Alizarin-Red-S (Figure 2C). Thus, the cells used in this study fulfilled all criteria to be defined as MSCs.


Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression.

Montzka K, Lassonczyk N, Tschöke B, Neuss S, Führmann T, Franzen R, Smeets R, Brook GA, Wöltje M - BMC Neurosci (2009)

Surface marker expression of human MSCs. FACS analysis of the immunophenotypic surface profile for CD11b, CD19, CD34, CD45, CD73, CD90, CD105 and HLA-DR of isolated hMSCs. Red histograms represent the fluorescence from negative-control cells incubated with only secondary antibody; black histograms represent the counts of cells incubated with the relevant primary antibody. The logarithm on the X-axis (FL1-H channel) represents the intensity of the fluorescent signal and the number of cells is given on the Y-axis. HMSCs isolated in this study were positive for the markers CD73, CD90 and CD105, but negative for CD11b, CD19, CD34, CD45 and HLA-DR according to the criteria for MSCs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2655300&req=5

Figure 1: Surface marker expression of human MSCs. FACS analysis of the immunophenotypic surface profile for CD11b, CD19, CD34, CD45, CD73, CD90, CD105 and HLA-DR of isolated hMSCs. Red histograms represent the fluorescence from negative-control cells incubated with only secondary antibody; black histograms represent the counts of cells incubated with the relevant primary antibody. The logarithm on the X-axis (FL1-H channel) represents the intensity of the fluorescent signal and the number of cells is given on the Y-axis. HMSCs isolated in this study were positive for the markers CD73, CD90 and CD105, but negative for CD11b, CD19, CD34, CD45 and HLA-DR according to the criteria for MSCs.
Mentions: Fluorescent activated cell sorting (FACS) analysis demonstrated that the expanded, plastic adherent cells used in the present investigation were positive for the surface markers CD73, CD90 and CD105, but negative for CD11b, CD19, CD34, CD45 and HLA-DR (Figure 1). To demonstrate their multipotent potential, MSCs were differentiated to adipocytes, chondrocytes and osteocytes according to published protocols [24]. Lipid vacuoles in differentiated adipocytes were visualized with Oil Red O (Figure 2A). However, not all cells demonstrated the same degree of staining. Induction of chondrogenic differentiation was performed in cell pellets which developed a proteoglycan-rich extracellular matrix. Thin sections of these pellets were stained with Toluidine Blue (Figure 2B), demonstrating a metachromatic staining that was characteristic of cartilage matrix [25]. Osteogenic differentiation resulted in an immense production of mineral deposits that were stained with Alizarin-Red-S (Figure 2C). Thus, the cells used in this study fulfilled all criteria to be defined as MSCs.

Bottom Line: Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries.More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression.Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, RWTH Aachen University, Aachen, Germany. kmontzka@ukaachen.de

ABSTRACT

Background: In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors.

Results: The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression.

Conclusion: The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

Show MeSH
Related in: MedlinePlus