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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.

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Expression of neural related proteins. Immunofluorescence for neural related proteins in undifferentiated hMSCs. (A) Enolase2, (B) MAP1b, (C) Nurr1 and (D) nestin. Staining revealed cytoplasmic distribution of Enolase2 and Nurr1, whereas the staining of MAP1b and nestin was cytoskeletal. Scale bar 100 μm. (E) Quantification of the percentage of stained cells from three different donors revealed following data: Enolase2 expression was found in 59% ± 27.1% of all cells, 66.7% ± 12.2% Map1b positive cells, and 46.3% ± 10.8% of the cells expressed Nurr1. Nestin expression was found to be present in all cells analyzed.
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Figure 4: Expression of neural related proteins. Immunofluorescence for neural related proteins in undifferentiated hMSCs. (A) Enolase2, (B) MAP1b, (C) Nurr1 and (D) nestin. Staining revealed cytoplasmic distribution of Enolase2 and Nurr1, whereas the staining of MAP1b and nestin was cytoskeletal. Scale bar 100 μm. (E) Quantification of the percentage of stained cells from three different donors revealed following data: Enolase2 expression was found in 59% ± 27.1% of all cells, 66.7% ± 12.2% Map1b positive cells, and 46.3% ± 10.8% of the cells expressed Nurr1. Nestin expression was found to be present in all cells analyzed.

Mentions: In addition to RT-PCR, a number of non-differentiated donor hMSC samples were chosen for immunocytochemical analysis. Staining of Enolase2 revealed a cytoplasmic distribution (Figure 4A). A cytoskeletal staining was observed with antibodies against MAP1b (Figure 4B) and nestin (Figure 4D). However, staining for Nurr1 was detected at different intensities and revealed a cytoplasmic distribution (Figure 4C). Quantification of immunocytochemical staining from three donors (Figure 4E) revealed that Enolase2 expression was found in 59 ± 27.1% of all cells, Map1b expression was found in 66.7 ± 12.2% of all cells, and Nurr1 expression was found in 46.3 ± 10.8% of the cells expressed. Nestin expression was found to be present in all cells of the three donors analyzed. Thus, this data demonstrates that both protein and mRNAs of a range of neurally-related markers is already expressed by non-differentiated hMSC.


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)

Expression of neural related proteins. Immunofluorescence for neural related proteins in undifferentiated hMSCs. (A) Enolase2, (B) MAP1b, (C) Nurr1 and (D) nestin. Staining revealed cytoplasmic distribution of Enolase2 and Nurr1, whereas the staining of MAP1b and nestin was cytoskeletal. Scale bar 100 μm. (E) Quantification of the percentage of stained cells from three different donors revealed following data: Enolase2 expression was found in 59% ± 27.1% of all cells, 66.7% ± 12.2% Map1b positive cells, and 46.3% ± 10.8% of the cells expressed Nurr1. Nestin expression was found to be present in all cells analyzed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Expression of neural related proteins. Immunofluorescence for neural related proteins in undifferentiated hMSCs. (A) Enolase2, (B) MAP1b, (C) Nurr1 and (D) nestin. Staining revealed cytoplasmic distribution of Enolase2 and Nurr1, whereas the staining of MAP1b and nestin was cytoskeletal. Scale bar 100 μm. (E) Quantification of the percentage of stained cells from three different donors revealed following data: Enolase2 expression was found in 59% ± 27.1% of all cells, 66.7% ± 12.2% Map1b positive cells, and 46.3% ± 10.8% of the cells expressed Nurr1. Nestin expression was found to be present in all cells analyzed.
Mentions: In addition to RT-PCR, a number of non-differentiated donor hMSC samples were chosen for immunocytochemical analysis. Staining of Enolase2 revealed a cytoplasmic distribution (Figure 4A). A cytoskeletal staining was observed with antibodies against MAP1b (Figure 4B) and nestin (Figure 4D). However, staining for Nurr1 was detected at different intensities and revealed a cytoplasmic distribution (Figure 4C). Quantification of immunocytochemical staining from three donors (Figure 4E) revealed that Enolase2 expression was found in 59 ± 27.1% of all cells, Map1b expression was found in 66.7 ± 12.2% of all cells, and Nurr1 expression was found in 46.3 ± 10.8% of the cells expressed. Nestin expression was found to be present in all cells of the three donors analyzed. Thus, this data demonstrates that both protein and mRNAs of a range of neurally-related markers is already expressed by non-differentiated hMSC.

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