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Adult human brain neural progenitor cells (NPCs) and fibroblast-like cells have similar properties in vitro but only NPCs differentiate into neurons.

Park TI, Monzo H, Mee EW, Bergin PS, Teoh HH, Montgomery JM, Faull RL, Curtis MA, Dragunow M - PLoS ONE (2012)

Bottom Line: This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures.These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations.This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting AhNPC-based experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand.

ABSTRACT
The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC) culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP) of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia), and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs). These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5-6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting AhNPC-based experiments.

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The FbCs isolated from biopsy specimens show NPC-like properties under NPC culture conditions but failed to differentiate into functional neurons.(A) Bright-field (BF) image showing the distinct morphology of the FbCs. They exhibited a flat and diffuse cytoplasm and were generally phase-dark. These cells showed characteristics of fibroblasts, as they lack the expression of neuronal βIII-tubulin (B), but had high levels of collagen synthesizing enzyme prolyl-4-hydroxylase (P4H; D) and Vimentin (F). However, they also expressed stem cell-like markers such as Nestin (C) and Sox-2 (E). Furthermore, when cultured under neurosphere-forming conditions, FbCs also formed spheres (G) that could be passaged for at least 2 – 3 passages. When FbCs were cultured in NPC culture conditions, their morphology changed to phase-bright polar shaped cells (H) and started expressing high levels of βIII-tubulin (I) and up-regulated their expression of Nestin (J). In agreement with immunostaining results, quantitative image analysis revealed NPC conditions gradually increased the percentage of FbCs expressing Nestin and βIII-tubulin at a detectable level (K). When compared to the control astrocytic culture condition, these differences were significant (P<0.01). Q-RT-PCR data also showed βIII-tubulin mRNA levels increased by over 10 fold by 14 days of exposure to NPC conditions (L; P<0.01). Due to the relatively high basal levels of Nestin in the control cells, the relative increase in Nestin was only 1.5 fold, but this still reached significance (L; P<0.01). Finally, western blots validated many of the above observations, as in control conditions, they showed βIII-tubulin at near un-detectable levels, but increased significantly during the first 3 days in NPC media and continued to increase till day 7 (M). Sox-2 and Vimentin were highly expressed in control and NPC media-induced FbCs (M). These results were consistently observed in all the cases tested (n = 5). Scale: 100 µm.
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pone-0037742-g004: The FbCs isolated from biopsy specimens show NPC-like properties under NPC culture conditions but failed to differentiate into functional neurons.(A) Bright-field (BF) image showing the distinct morphology of the FbCs. They exhibited a flat and diffuse cytoplasm and were generally phase-dark. These cells showed characteristics of fibroblasts, as they lack the expression of neuronal βIII-tubulin (B), but had high levels of collagen synthesizing enzyme prolyl-4-hydroxylase (P4H; D) and Vimentin (F). However, they also expressed stem cell-like markers such as Nestin (C) and Sox-2 (E). Furthermore, when cultured under neurosphere-forming conditions, FbCs also formed spheres (G) that could be passaged for at least 2 – 3 passages. When FbCs were cultured in NPC culture conditions, their morphology changed to phase-bright polar shaped cells (H) and started expressing high levels of βIII-tubulin (I) and up-regulated their expression of Nestin (J). In agreement with immunostaining results, quantitative image analysis revealed NPC conditions gradually increased the percentage of FbCs expressing Nestin and βIII-tubulin at a detectable level (K). When compared to the control astrocytic culture condition, these differences were significant (P<0.01). Q-RT-PCR data also showed βIII-tubulin mRNA levels increased by over 10 fold by 14 days of exposure to NPC conditions (L; P<0.01). Due to the relatively high basal levels of Nestin in the control cells, the relative increase in Nestin was only 1.5 fold, but this still reached significance (L; P<0.01). Finally, western blots validated many of the above observations, as in control conditions, they showed βIII-tubulin at near un-detectable levels, but increased significantly during the first 3 days in NPC media and continued to increase till day 7 (M). Sox-2 and Vimentin were highly expressed in control and NPC media-induced FbCs (M). These results were consistently observed in all the cases tested (n = 5). Scale: 100 µm.

Mentions: To elucidate whether the differences in morphology and the stronger expression of Nestin was due to the NPC culture conditions, FbCs cultured in serum-containing culture conditions from the temporal cortex [21] were cultured in our serum-free NPC proliferation media. NPC culture conditions resulted in the FbCs showing markedly different characteristics to those cultured in serum containing conditions. Immunostaining revealed that the NPC marker Nestin, although expressed at detectable levels in FbCs cultured in serum containing media, further increased during the 14 days of NPC media induction (Figure 4 C & J). This increase was quantified using image analysis and q-RT-PCR and showed a 1.5 to 2 fold change in expression (P<0.01; Figure 4 L). Furthermore, βIII-tubulin, which was not present at immunologically detectable levels in FbCs cultured in serum containing media (Figure 4 B), increased significantly during the 2 weeks in NPC media (Figure 4 I) with image analysis and q-RT-PCR data indicating a larger than 10 fold increase in expression (P<0.01; Figure 4 K–L). Western blot results were consistent with the above (Figure 4 M). NPC media-exposed FbCs also changed their morphology to resemble cells those from the AhNPC cultures with bipolar processes and phase-bright somas (Figure 4 H). Furthermore, when the FbCs were cultured in NS forming conditions (i.e. plated onto non-adhesive surfaces at clonal densities in NPC media), a large number of cells formed primary spheres (Figure 4 G) that were capable of forming secondary spheres from their dissociated cells. These results were confirmed in all of 5 trialed independent cases and demonstrated the close phenotypic resemblance between the FbCs and the cells present in our AhNPC cultures. However, like the cells found during the later passages (>6) of our AhNPC cultures, the FbC-derived spheres were not capable of undergoing full neuronal or astrocytic differentiation, as they gave rise to cells expressing βIII-tubulin but not MAP2 or GFAP. They also failed to give rise to cells exhibiting neurophysiological properties with average RMP of the cells sitting at –6.5±6 mV and Rm 500±300 MΩ (n = 5), and failed to elicit any form of active membrane properties (data not shown). Table 1 summarizes these findings, which indicate that the majority of the mitotic cells present in the later passages of a primary AhNPC cultures are likely to be the FbCs and not NPCs.


Adult human brain neural progenitor cells (NPCs) and fibroblast-like cells have similar properties in vitro but only NPCs differentiate into neurons.

Park TI, Monzo H, Mee EW, Bergin PS, Teoh HH, Montgomery JM, Faull RL, Curtis MA, Dragunow M - PLoS ONE (2012)

The FbCs isolated from biopsy specimens show NPC-like properties under NPC culture conditions but failed to differentiate into functional neurons.(A) Bright-field (BF) image showing the distinct morphology of the FbCs. They exhibited a flat and diffuse cytoplasm and were generally phase-dark. These cells showed characteristics of fibroblasts, as they lack the expression of neuronal βIII-tubulin (B), but had high levels of collagen synthesizing enzyme prolyl-4-hydroxylase (P4H; D) and Vimentin (F). However, they also expressed stem cell-like markers such as Nestin (C) and Sox-2 (E). Furthermore, when cultured under neurosphere-forming conditions, FbCs also formed spheres (G) that could be passaged for at least 2 – 3 passages. When FbCs were cultured in NPC culture conditions, their morphology changed to phase-bright polar shaped cells (H) and started expressing high levels of βIII-tubulin (I) and up-regulated their expression of Nestin (J). In agreement with immunostaining results, quantitative image analysis revealed NPC conditions gradually increased the percentage of FbCs expressing Nestin and βIII-tubulin at a detectable level (K). When compared to the control astrocytic culture condition, these differences were significant (P<0.01). Q-RT-PCR data also showed βIII-tubulin mRNA levels increased by over 10 fold by 14 days of exposure to NPC conditions (L; P<0.01). Due to the relatively high basal levels of Nestin in the control cells, the relative increase in Nestin was only 1.5 fold, but this still reached significance (L; P<0.01). Finally, western blots validated many of the above observations, as in control conditions, they showed βIII-tubulin at near un-detectable levels, but increased significantly during the first 3 days in NPC media and continued to increase till day 7 (M). Sox-2 and Vimentin were highly expressed in control and NPC media-induced FbCs (M). These results were consistently observed in all the cases tested (n = 5). Scale: 100 µm.
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Related In: Results  -  Collection

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pone-0037742-g004: The FbCs isolated from biopsy specimens show NPC-like properties under NPC culture conditions but failed to differentiate into functional neurons.(A) Bright-field (BF) image showing the distinct morphology of the FbCs. They exhibited a flat and diffuse cytoplasm and were generally phase-dark. These cells showed characteristics of fibroblasts, as they lack the expression of neuronal βIII-tubulin (B), but had high levels of collagen synthesizing enzyme prolyl-4-hydroxylase (P4H; D) and Vimentin (F). However, they also expressed stem cell-like markers such as Nestin (C) and Sox-2 (E). Furthermore, when cultured under neurosphere-forming conditions, FbCs also formed spheres (G) that could be passaged for at least 2 – 3 passages. When FbCs were cultured in NPC culture conditions, their morphology changed to phase-bright polar shaped cells (H) and started expressing high levels of βIII-tubulin (I) and up-regulated their expression of Nestin (J). In agreement with immunostaining results, quantitative image analysis revealed NPC conditions gradually increased the percentage of FbCs expressing Nestin and βIII-tubulin at a detectable level (K). When compared to the control astrocytic culture condition, these differences were significant (P<0.01). Q-RT-PCR data also showed βIII-tubulin mRNA levels increased by over 10 fold by 14 days of exposure to NPC conditions (L; P<0.01). Due to the relatively high basal levels of Nestin in the control cells, the relative increase in Nestin was only 1.5 fold, but this still reached significance (L; P<0.01). Finally, western blots validated many of the above observations, as in control conditions, they showed βIII-tubulin at near un-detectable levels, but increased significantly during the first 3 days in NPC media and continued to increase till day 7 (M). Sox-2 and Vimentin were highly expressed in control and NPC media-induced FbCs (M). These results were consistently observed in all the cases tested (n = 5). Scale: 100 µm.
Mentions: To elucidate whether the differences in morphology and the stronger expression of Nestin was due to the NPC culture conditions, FbCs cultured in serum-containing culture conditions from the temporal cortex [21] were cultured in our serum-free NPC proliferation media. NPC culture conditions resulted in the FbCs showing markedly different characteristics to those cultured in serum containing conditions. Immunostaining revealed that the NPC marker Nestin, although expressed at detectable levels in FbCs cultured in serum containing media, further increased during the 14 days of NPC media induction (Figure 4 C & J). This increase was quantified using image analysis and q-RT-PCR and showed a 1.5 to 2 fold change in expression (P<0.01; Figure 4 L). Furthermore, βIII-tubulin, which was not present at immunologically detectable levels in FbCs cultured in serum containing media (Figure 4 B), increased significantly during the 2 weeks in NPC media (Figure 4 I) with image analysis and q-RT-PCR data indicating a larger than 10 fold increase in expression (P<0.01; Figure 4 K–L). Western blot results were consistent with the above (Figure 4 M). NPC media-exposed FbCs also changed their morphology to resemble cells those from the AhNPC cultures with bipolar processes and phase-bright somas (Figure 4 H). Furthermore, when the FbCs were cultured in NS forming conditions (i.e. plated onto non-adhesive surfaces at clonal densities in NPC media), a large number of cells formed primary spheres (Figure 4 G) that were capable of forming secondary spheres from their dissociated cells. These results were confirmed in all of 5 trialed independent cases and demonstrated the close phenotypic resemblance between the FbCs and the cells present in our AhNPC cultures. However, like the cells found during the later passages (>6) of our AhNPC cultures, the FbC-derived spheres were not capable of undergoing full neuronal or astrocytic differentiation, as they gave rise to cells expressing βIII-tubulin but not MAP2 or GFAP. They also failed to give rise to cells exhibiting neurophysiological properties with average RMP of the cells sitting at –6.5±6 mV and Rm 500±300 MΩ (n = 5), and failed to elicit any form of active membrane properties (data not shown). Table 1 summarizes these findings, which indicate that the majority of the mitotic cells present in the later passages of a primary AhNPC cultures are likely to be the FbCs and not NPCs.

Bottom Line: This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures.These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations.This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting AhNPC-based experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand.

ABSTRACT
The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC) culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP) of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia), and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs). These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5-6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting AhNPC-based experiments.

Show MeSH
Related in: MedlinePlus