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Functional fingerprinting of human mesenchymal stem cells using high-throughput RNAi screening.

Erdmann G, Suchanek M, Horn P, Graf F, Volz C, Horn T, Zhang X, Wagner W, Ho AD, Boutros M - Genome Med (2015)

Bottom Line: We profiled primary human MSCs against human fibroblasts.Network analysis showed a kinome fingerprint that differs from human primary fibroblasts as well as fibroblast cell lines.In conclusion, this study shows that high-throughput screening in primary human MSCs can be reliably used for kinome fingerprinting.

View Article: PubMed Central - PubMed

Affiliation: German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics and Heidelberg University, Department of Cell and Molecular Biology, Medical Faculty Mannheim, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany.

ABSTRACT
Mesenchymal stem cells (MSCs) are promising candidates for cellular therapies ranging from tissue repair in regenerative medicine to immunomodulation in graft versus host disease after allogeneic transplantation or in autoimmune diseases. Nonetheless, progress has been hampered by their enormous phenotypic as well as functional heterogeneity and the lack of uniform standards and guidelines for quality control. In this study, we describe a method to perform cellular phenotyping by high-throughput RNA interference in primary human bone marrow MSCs. We have shown that despite heterogeneity of MSC populations, robust functional assays can be established that are suitable for high-throughput and high-content screening. We profiled primary human MSCs against human fibroblasts. Network analysis showed a kinome fingerprint that differs from human primary fibroblasts as well as fibroblast cell lines. In conclusion, this study shows that high-throughput screening in primary human MSCs can be reliably used for kinome fingerprinting.

No MeSH data available.


Related in: MedlinePlus

PIK3C2A and WEE1 silencing alters the cell cycle profile of primary MSCs. Cytometric analysis of MSCs 96 h after siRNA treatment confirms viability phenotypes of selected candidates. a Representative fluorescence flow cytometry analysis of different MSC donors. MSCs were stained with 200 μg/ml of propidium iodide (PI), 0.1 %, phycoerythrin (PE), 0.1 %, sodium azide, 0.1 % Triton-X100 and 10 μg/ml RNAses for 2–4 h at 4 °C. Single cells were analyzed for fragmented DNA and dead cells were gated to quantify viable cells (PE-A). The percentage of viable cells was calculated from three biological replicates and average viability was plotted. b Representative histograms of MSCs analyzed with FlowJo 887 for sub G1, S and G2 peaks (PI-A). PIK3C2A and WEE1 show a shift in DNA content (blue) compared with the negative control (red). Quantification of cell cycle percentages shows that PIK3C2A and WEE1 silencing led to a significant (p ≤ 0.01) increase in S-phase and G2 peaks, respectively. Average of three biological replicates is shown and significant cell cycle changes were calculated using unpaired two tailed student’s T-test
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Fig4: PIK3C2A and WEE1 silencing alters the cell cycle profile of primary MSCs. Cytometric analysis of MSCs 96 h after siRNA treatment confirms viability phenotypes of selected candidates. a Representative fluorescence flow cytometry analysis of different MSC donors. MSCs were stained with 200 μg/ml of propidium iodide (PI), 0.1 %, phycoerythrin (PE), 0.1 %, sodium azide, 0.1 % Triton-X100 and 10 μg/ml RNAses for 2–4 h at 4 °C. Single cells were analyzed for fragmented DNA and dead cells were gated to quantify viable cells (PE-A). The percentage of viable cells was calculated from three biological replicates and average viability was plotted. b Representative histograms of MSCs analyzed with FlowJo 887 for sub G1, S and G2 peaks (PI-A). PIK3C2A and WEE1 show a shift in DNA content (blue) compared with the negative control (red). Quantification of cell cycle percentages shows that PIK3C2A and WEE1 silencing led to a significant (p ≤ 0.01) increase in S-phase and G2 peaks, respectively. Average of three biological replicates is shown and significant cell cycle changes were calculated using unpaired two tailed student’s T-test

Mentions: Nuclear intensity and ATP content are two parameters that have been frequently used to determine cell viability. To assess whether the decrease in cell viability and nuclear intensity observed after RNAi treatment (Fig. 3) was caused by lower cell proliferation, decreased metabolism or apoptosis, the total percentage of dead cells was measured by fluorescence flow cytometry 96 h post-transfection. All tested candidates showed a significant decrease in cell number 96 h post-RNAi treatment (Fig. 4). Both PIK3CA2 and MAP3K9 were associated with weaker reduction in cell viability compared with the CTG-based re-tests (Fig. 3). Conversely, the phenotypes for ABL1 and WEE1 were more pronounced in the fluorescence flow cytometry analysis (Fig. 4). Cell cycle profiling revealed an increase in G2/M DNA content in ABL1 (26 %) and WEE1 (38 %) RNAi treated samples compared with 12 % in the control (Fig. 4). Although we observed an increase in the G2/M DNA content after both ABL1 and WEE1 ablation, only the increase in WEE1 proved statistically significant (p ≤ 0.05). In the case of PIK3CA2, 9 % of cells were scored to be in S-phase, which is significantly more compared with the 2 % measured in the control. These experiments further supported that the screening experiments have identified valid candidates. Several candidates showed severe cell cycle alterations in primary MSCs that led to the observed growth and viability effects.Fig. 4


Functional fingerprinting of human mesenchymal stem cells using high-throughput RNAi screening.

Erdmann G, Suchanek M, Horn P, Graf F, Volz C, Horn T, Zhang X, Wagner W, Ho AD, Boutros M - Genome Med (2015)

PIK3C2A and WEE1 silencing alters the cell cycle profile of primary MSCs. Cytometric analysis of MSCs 96 h after siRNA treatment confirms viability phenotypes of selected candidates. a Representative fluorescence flow cytometry analysis of different MSC donors. MSCs were stained with 200 μg/ml of propidium iodide (PI), 0.1 %, phycoerythrin (PE), 0.1 %, sodium azide, 0.1 % Triton-X100 and 10 μg/ml RNAses for 2–4 h at 4 °C. Single cells were analyzed for fragmented DNA and dead cells were gated to quantify viable cells (PE-A). The percentage of viable cells was calculated from three biological replicates and average viability was plotted. b Representative histograms of MSCs analyzed with FlowJo 887 for sub G1, S and G2 peaks (PI-A). PIK3C2A and WEE1 show a shift in DNA content (blue) compared with the negative control (red). Quantification of cell cycle percentages shows that PIK3C2A and WEE1 silencing led to a significant (p ≤ 0.01) increase in S-phase and G2 peaks, respectively. Average of three biological replicates is shown and significant cell cycle changes were calculated using unpaired two tailed student’s T-test
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4481116&req=5

Fig4: PIK3C2A and WEE1 silencing alters the cell cycle profile of primary MSCs. Cytometric analysis of MSCs 96 h after siRNA treatment confirms viability phenotypes of selected candidates. a Representative fluorescence flow cytometry analysis of different MSC donors. MSCs were stained with 200 μg/ml of propidium iodide (PI), 0.1 %, phycoerythrin (PE), 0.1 %, sodium azide, 0.1 % Triton-X100 and 10 μg/ml RNAses for 2–4 h at 4 °C. Single cells were analyzed for fragmented DNA and dead cells were gated to quantify viable cells (PE-A). The percentage of viable cells was calculated from three biological replicates and average viability was plotted. b Representative histograms of MSCs analyzed with FlowJo 887 for sub G1, S and G2 peaks (PI-A). PIK3C2A and WEE1 show a shift in DNA content (blue) compared with the negative control (red). Quantification of cell cycle percentages shows that PIK3C2A and WEE1 silencing led to a significant (p ≤ 0.01) increase in S-phase and G2 peaks, respectively. Average of three biological replicates is shown and significant cell cycle changes were calculated using unpaired two tailed student’s T-test
Mentions: Nuclear intensity and ATP content are two parameters that have been frequently used to determine cell viability. To assess whether the decrease in cell viability and nuclear intensity observed after RNAi treatment (Fig. 3) was caused by lower cell proliferation, decreased metabolism or apoptosis, the total percentage of dead cells was measured by fluorescence flow cytometry 96 h post-transfection. All tested candidates showed a significant decrease in cell number 96 h post-RNAi treatment (Fig. 4). Both PIK3CA2 and MAP3K9 were associated with weaker reduction in cell viability compared with the CTG-based re-tests (Fig. 3). Conversely, the phenotypes for ABL1 and WEE1 were more pronounced in the fluorescence flow cytometry analysis (Fig. 4). Cell cycle profiling revealed an increase in G2/M DNA content in ABL1 (26 %) and WEE1 (38 %) RNAi treated samples compared with 12 % in the control (Fig. 4). Although we observed an increase in the G2/M DNA content after both ABL1 and WEE1 ablation, only the increase in WEE1 proved statistically significant (p ≤ 0.05). In the case of PIK3CA2, 9 % of cells were scored to be in S-phase, which is significantly more compared with the 2 % measured in the control. These experiments further supported that the screening experiments have identified valid candidates. Several candidates showed severe cell cycle alterations in primary MSCs that led to the observed growth and viability effects.Fig. 4

Bottom Line: We profiled primary human MSCs against human fibroblasts.Network analysis showed a kinome fingerprint that differs from human primary fibroblasts as well as fibroblast cell lines.In conclusion, this study shows that high-throughput screening in primary human MSCs can be reliably used for kinome fingerprinting.

View Article: PubMed Central - PubMed

Affiliation: German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics and Heidelberg University, Department of Cell and Molecular Biology, Medical Faculty Mannheim, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany.

ABSTRACT
Mesenchymal stem cells (MSCs) are promising candidates for cellular therapies ranging from tissue repair in regenerative medicine to immunomodulation in graft versus host disease after allogeneic transplantation or in autoimmune diseases. Nonetheless, progress has been hampered by their enormous phenotypic as well as functional heterogeneity and the lack of uniform standards and guidelines for quality control. In this study, we describe a method to perform cellular phenotyping by high-throughput RNA interference in primary human bone marrow MSCs. We have shown that despite heterogeneity of MSC populations, robust functional assays can be established that are suitable for high-throughput and high-content screening. We profiled primary human MSCs against human fibroblasts. Network analysis showed a kinome fingerprint that differs from human primary fibroblasts as well as fibroblast cell lines. In conclusion, this study shows that high-throughput screening in primary human MSCs can be reliably used for kinome fingerprinting.

No MeSH data available.


Related in: MedlinePlus