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IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells.

Huat TJ, Khan AA, Pati S, Mustafa Z, Abdullah JM, Jaafar H - BMC Neurosci (2014)

Bottom Line: To unravel the roles and effects of different growth factors in the differentiation of MSCs into neural lineages, we have differentiated MSCs into neural lineages using different combinations of growth factors.The NPCs derived from IGF-1 treatment also resulted in a better yield after the terminal differentiation into neurons and glial cells than that of the other treated groups.Our results suggested that IGF-1 has a crucial role in the differentiation of MSCs into neuronal lineage by enhancing the proliferation and reducing the apoptosis in the NPCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medical Sciences, Universiti Sains Malaysia, Jalan Hospital Universiti Sains Malaysia, 16150 Kubang Kerian, Kota Bharu, Kelantan, Malaysia. teejonghuat@gmail.com.

ABSTRACT

Background: There has been increasing interest recently in the plasticity of mesenchymal stem cells (MSCs) and their potential to differentiate into neural lineages. To unravel the roles and effects of different growth factors in the differentiation of MSCs into neural lineages, we have differentiated MSCs into neural lineages using different combinations of growth factors. Based on previous studies of the roles of insulin-like growth factor 1 (IGF-1) in neural stem cell isolation in the laboratory, we hypothesized that IGF-1 can enhance proliferation and reduce apoptosis in neural progenitor-like cells (NPCs) during differentiation of MSCs into NCPs.We induced MSCs differentiation under four different combinations of growth factors: (A) EGF + bFGF, (B) EGF + bFGF + IGF-1, (C) EGF + bFGF + LIF, (D) EGF + bFGF + BDNF, and (E) without growth factors, as a negative control. The neurospheres formed were characterized by immunofluorescence staining against nestin, and the expression was measured by flow cytometry. Cell proliferation and apoptosis were also studied by MTS and Annexin V assay, respectively, at three different time intervals (24 hr, 3 days, and 5 days). The neurospheres formed in the four groups were then terminally differentiated into neuron and glial cells.

Results: The four derived NPCs showed a significantly higher expression of nestin than was shown by the negative control. Among the groups treated with growth factors, NPCs treated with IGF-1 showed the highest expression of nestin. Furthermore, NPCs derived using IGF-1 exhibited the highest cell proliferation and cell survival among the treated groups. The NPCs derived from IGF-1 treatment also resulted in a better yield after the terminal differentiation into neurons and glial cells than that of the other treated groups.

Conclusions: Our results suggested that IGF-1 has a crucial role in the differentiation of MSCs into neuronal lineage by enhancing the proliferation and reducing the apoptosis in the NPCs. This information will be beneficial in the long run for improving both cell-based and cell-free therapy for neurodegenerative diseases.

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Apoptosis assay. Immunostaining revealed that transdifferentiation is associated with apoptotic activities. (A) SYTOX Blue nuclei staining, (B) FITC-conjugated Annexin V, (C) propidium iodide (PI), and (D) overlay image. All images are viewed at 40× magnification under a confocal microscope.
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Fig6: Apoptosis assay. Immunostaining revealed that transdifferentiation is associated with apoptotic activities. (A) SYTOX Blue nuclei staining, (B) FITC-conjugated Annexin V, (C) propidium iodide (PI), and (D) overlay image. All images are viewed at 40× magnification under a confocal microscope.

Mentions: To understand the role of the combination of growth factors on the survival of NPCs in vitro, we also studied the apoptotic activity of NPCs at three time intervals (24 hr, 72 hr, and 120 hr) using the Annexin V kit. Immunofluorescence staining of NPCs revealed that the differentiation process induces cellular apoptosis (Figure 6). Apoptotic activities such as necrosis and early and late apoptosis were quantified by flow cytometry. The group treated with IGF-1 maintained the highest percentage of live cells at day 3 post induction, 48.6 ± 19.5% (n = 3; Figure 7A). Only 9.3 ± 7.6%, 0.5 ± 0.7%, and 1.3 ± 1.4% (n = 3) of cells in Group B underwent necrosis at day 1, day 3 and day 5 respectively (Figure 7B). Furthermore, only 3.7 ± 3.3% of cells in Group B underwent early apoptosis. The number increases to 24.6 ± 15% on day 3 and decreases to 13 ± 11.5% on day 5 (Figure 7C). A total of 28.1 ± 15.05% of cells from Group B underwent late apoptosis, decreasing to 26.3 ± 14.3% on day 3 and further decreasing to 15.6 ± 6.7% on day 5 (Figure 7D). Taken together, the early and late apoptosis graphs suggest that the group treated with IGF-1 has better survival efficiency than the rest of the groups.Figure 6


IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells.

Huat TJ, Khan AA, Pati S, Mustafa Z, Abdullah JM, Jaafar H - BMC Neurosci (2014)

Apoptosis assay. Immunostaining revealed that transdifferentiation is associated with apoptotic activities. (A) SYTOX Blue nuclei staining, (B) FITC-conjugated Annexin V, (C) propidium iodide (PI), and (D) overlay image. All images are viewed at 40× magnification under a confocal microscope.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4117972&req=5

Fig6: Apoptosis assay. Immunostaining revealed that transdifferentiation is associated with apoptotic activities. (A) SYTOX Blue nuclei staining, (B) FITC-conjugated Annexin V, (C) propidium iodide (PI), and (D) overlay image. All images are viewed at 40× magnification under a confocal microscope.
Mentions: To understand the role of the combination of growth factors on the survival of NPCs in vitro, we also studied the apoptotic activity of NPCs at three time intervals (24 hr, 72 hr, and 120 hr) using the Annexin V kit. Immunofluorescence staining of NPCs revealed that the differentiation process induces cellular apoptosis (Figure 6). Apoptotic activities such as necrosis and early and late apoptosis were quantified by flow cytometry. The group treated with IGF-1 maintained the highest percentage of live cells at day 3 post induction, 48.6 ± 19.5% (n = 3; Figure 7A). Only 9.3 ± 7.6%, 0.5 ± 0.7%, and 1.3 ± 1.4% (n = 3) of cells in Group B underwent necrosis at day 1, day 3 and day 5 respectively (Figure 7B). Furthermore, only 3.7 ± 3.3% of cells in Group B underwent early apoptosis. The number increases to 24.6 ± 15% on day 3 and decreases to 13 ± 11.5% on day 5 (Figure 7C). A total of 28.1 ± 15.05% of cells from Group B underwent late apoptosis, decreasing to 26.3 ± 14.3% on day 3 and further decreasing to 15.6 ± 6.7% on day 5 (Figure 7D). Taken together, the early and late apoptosis graphs suggest that the group treated with IGF-1 has better survival efficiency than the rest of the groups.Figure 6

Bottom Line: To unravel the roles and effects of different growth factors in the differentiation of MSCs into neural lineages, we have differentiated MSCs into neural lineages using different combinations of growth factors.The NPCs derived from IGF-1 treatment also resulted in a better yield after the terminal differentiation into neurons and glial cells than that of the other treated groups.Our results suggested that IGF-1 has a crucial role in the differentiation of MSCs into neuronal lineage by enhancing the proliferation and reducing the apoptosis in the NPCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medical Sciences, Universiti Sains Malaysia, Jalan Hospital Universiti Sains Malaysia, 16150 Kubang Kerian, Kota Bharu, Kelantan, Malaysia. teejonghuat@gmail.com.

ABSTRACT

Background: There has been increasing interest recently in the plasticity of mesenchymal stem cells (MSCs) and their potential to differentiate into neural lineages. To unravel the roles and effects of different growth factors in the differentiation of MSCs into neural lineages, we have differentiated MSCs into neural lineages using different combinations of growth factors. Based on previous studies of the roles of insulin-like growth factor 1 (IGF-1) in neural stem cell isolation in the laboratory, we hypothesized that IGF-1 can enhance proliferation and reduce apoptosis in neural progenitor-like cells (NPCs) during differentiation of MSCs into NCPs.We induced MSCs differentiation under four different combinations of growth factors: (A) EGF + bFGF, (B) EGF + bFGF + IGF-1, (C) EGF + bFGF + LIF, (D) EGF + bFGF + BDNF, and (E) without growth factors, as a negative control. The neurospheres formed were characterized by immunofluorescence staining against nestin, and the expression was measured by flow cytometry. Cell proliferation and apoptosis were also studied by MTS and Annexin V assay, respectively, at three different time intervals (24 hr, 3 days, and 5 days). The neurospheres formed in the four groups were then terminally differentiated into neuron and glial cells.

Results: The four derived NPCs showed a significantly higher expression of nestin than was shown by the negative control. Among the groups treated with growth factors, NPCs treated with IGF-1 showed the highest expression of nestin. Furthermore, NPCs derived using IGF-1 exhibited the highest cell proliferation and cell survival among the treated groups. The NPCs derived from IGF-1 treatment also resulted in a better yield after the terminal differentiation into neurons and glial cells than that of the other treated groups.

Conclusions: Our results suggested that IGF-1 has a crucial role in the differentiation of MSCs into neuronal lineage by enhancing the proliferation and reducing the apoptosis in the NPCs. This information will be beneficial in the long run for improving both cell-based and cell-free therapy for neurodegenerative diseases.

Show MeSH
Related in: MedlinePlus