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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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NPCs differentiated on PDL/Laminin-coated coverslips give rise to cells showing neurophysiological properties.(A-C) Bright field image of a differentiated NPC during patch-clamp recordings and the electrophysiological responses to multiple and single current steps respectively (left to right). This cell had a RMP of −65 mV and fired an immature action potential (AP) in response to depolarizing current. The insert in (C) was the stimulus protocol. (D–F) AhNPC differentiated for 5 days. This cell did not express βIII-tubulin or GFAP, but had clear neurite processes (D1–4). It exhibited a small depolarization and a small inward current in response to depolarizing stimuli (E–F). (G–L) AhNPCs recorded after 3–4 weeks of differentiation. (G–I) Recording from a βIII-tubulin positive, GFAP-negative cell (D3–D4) that fired an immature APs (H), and exhibited small, fast-inactivating inward and slow-inactivating outward currents (I). (J1–4) An ‘asteron’ cell expressing both GFAP and βIII-tubulin. These cells failed to elicit any active voltage or current responses to depolarizing stimuli (K-L). (M-N) The stimulus protocols for current clamp (M) and voltage clamp (N). All traces are representative recordings from 5 different biopsy cases that were conducted from either passage 2 or 3 cells.
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pone-0037742-g002: NPCs differentiated on PDL/Laminin-coated coverslips give rise to cells showing neurophysiological properties.(A-C) Bright field image of a differentiated NPC during patch-clamp recordings and the electrophysiological responses to multiple and single current steps respectively (left to right). This cell had a RMP of −65 mV and fired an immature action potential (AP) in response to depolarizing current. The insert in (C) was the stimulus protocol. (D–F) AhNPC differentiated for 5 days. This cell did not express βIII-tubulin or GFAP, but had clear neurite processes (D1–4). It exhibited a small depolarization and a small inward current in response to depolarizing stimuli (E–F). (G–L) AhNPCs recorded after 3–4 weeks of differentiation. (G–I) Recording from a βIII-tubulin positive, GFAP-negative cell (D3–D4) that fired an immature APs (H), and exhibited small, fast-inactivating inward and slow-inactivating outward currents (I). (J1–4) An ‘asteron’ cell expressing both GFAP and βIII-tubulin. These cells failed to elicit any active voltage or current responses to depolarizing stimuli (K-L). (M-N) The stimulus protocols for current clamp (M) and voltage clamp (N). All traces are representative recordings from 5 different biopsy cases that were conducted from either passage 2 or 3 cells.

Mentions: The differentiated AhNPCs with neurite-bearing morphology from 5 separate cases were subjected to electrophysiological studies to confirm their functional differentiation. During the first week of differentiation, neuron-like cells exhibited a depolarized resting membrane potential (RMP) and a high membrane resistance (Rm) of −45 mV±3.2 mV and 654±210 MΩ (n = 7), respectively. When tested for active membrane properties, 2 of the 7 cells responded with a small depolarization (Figure 2 D–F). Continued differentiation (3–4 weeks) resulted in RMP and Rm values sitting at near physiological levels of −65±2.1 mV and 195±50 MΩ (n = 10), respectively. Active membrane properties also matured with 3 out of the 10 cells (30%) firing an immature action potential (iAP) in response to the depolarizing stimuli (represented by Figure 2 A–C & G-I). These APs were accompanied by a relatively fast inactivating inward and a slow inactivating outward current. The same AP firing cells exhibited βIII-tubulin staining (Figure 2 G1–4). The majority of the fiber-bearing cells elicited little or no active membrane responses to depolarizing currents or voltages (Figure 2 J–L) and generally had a hyperpolarized RMP and a lower Rm. When separately grouped into iAP firing and non-firing cells, the iAP firing cells had an average RMP and Rm of −61.7±3.0 mV and 333±100 MΩ, respectively, while the non-firing cells showed glial-like properties with a RMP of −67.5±1.6 mV and Rm of 118±13 MΩ (P<0.05). The cells that expressed both GFAP and βIII-tubulin (Figure 2 J–L) did not fire an iAP but exhibited a slight inward current under large depolarizing stimuli. These results demonstrated that our AhNPC cultures gave rise to at least 2 brain cell populations in vitro, neurons and astrocytes.


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)

NPCs differentiated on PDL/Laminin-coated coverslips give rise to cells showing neurophysiological properties.(A-C) Bright field image of a differentiated NPC during patch-clamp recordings and the electrophysiological responses to multiple and single current steps respectively (left to right). This cell had a RMP of −65 mV and fired an immature action potential (AP) in response to depolarizing current. The insert in (C) was the stimulus protocol. (D–F) AhNPC differentiated for 5 days. This cell did not express βIII-tubulin or GFAP, but had clear neurite processes (D1–4). It exhibited a small depolarization and a small inward current in response to depolarizing stimuli (E–F). (G–L) AhNPCs recorded after 3–4 weeks of differentiation. (G–I) Recording from a βIII-tubulin positive, GFAP-negative cell (D3–D4) that fired an immature APs (H), and exhibited small, fast-inactivating inward and slow-inactivating outward currents (I). (J1–4) An ‘asteron’ cell expressing both GFAP and βIII-tubulin. These cells failed to elicit any active voltage or current responses to depolarizing stimuli (K-L). (M-N) The stimulus protocols for current clamp (M) and voltage clamp (N). All traces are representative recordings from 5 different biopsy cases that were conducted from either passage 2 or 3 cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366988&req=5

pone-0037742-g002: NPCs differentiated on PDL/Laminin-coated coverslips give rise to cells showing neurophysiological properties.(A-C) Bright field image of a differentiated NPC during patch-clamp recordings and the electrophysiological responses to multiple and single current steps respectively (left to right). This cell had a RMP of −65 mV and fired an immature action potential (AP) in response to depolarizing current. The insert in (C) was the stimulus protocol. (D–F) AhNPC differentiated for 5 days. This cell did not express βIII-tubulin or GFAP, but had clear neurite processes (D1–4). It exhibited a small depolarization and a small inward current in response to depolarizing stimuli (E–F). (G–L) AhNPCs recorded after 3–4 weeks of differentiation. (G–I) Recording from a βIII-tubulin positive, GFAP-negative cell (D3–D4) that fired an immature APs (H), and exhibited small, fast-inactivating inward and slow-inactivating outward currents (I). (J1–4) An ‘asteron’ cell expressing both GFAP and βIII-tubulin. These cells failed to elicit any active voltage or current responses to depolarizing stimuli (K-L). (M-N) The stimulus protocols for current clamp (M) and voltage clamp (N). All traces are representative recordings from 5 different biopsy cases that were conducted from either passage 2 or 3 cells.
Mentions: The differentiated AhNPCs with neurite-bearing morphology from 5 separate cases were subjected to electrophysiological studies to confirm their functional differentiation. During the first week of differentiation, neuron-like cells exhibited a depolarized resting membrane potential (RMP) and a high membrane resistance (Rm) of −45 mV±3.2 mV and 654±210 MΩ (n = 7), respectively. When tested for active membrane properties, 2 of the 7 cells responded with a small depolarization (Figure 2 D–F). Continued differentiation (3–4 weeks) resulted in RMP and Rm values sitting at near physiological levels of −65±2.1 mV and 195±50 MΩ (n = 10), respectively. Active membrane properties also matured with 3 out of the 10 cells (30%) firing an immature action potential (iAP) in response to the depolarizing stimuli (represented by Figure 2 A–C & G-I). These APs were accompanied by a relatively fast inactivating inward and a slow inactivating outward current. The same AP firing cells exhibited βIII-tubulin staining (Figure 2 G1–4). The majority of the fiber-bearing cells elicited little or no active membrane responses to depolarizing currents or voltages (Figure 2 J–L) and generally had a hyperpolarized RMP and a lower Rm. When separately grouped into iAP firing and non-firing cells, the iAP firing cells had an average RMP and Rm of −61.7±3.0 mV and 333±100 MΩ, respectively, while the non-firing cells showed glial-like properties with a RMP of −67.5±1.6 mV and Rm of 118±13 MΩ (P<0.05). The cells that expressed both GFAP and βIII-tubulin (Figure 2 J–L) did not fire an iAP but exhibited a slight inward current under large depolarizing stimuli. These results demonstrated that our AhNPC cultures gave rise to at least 2 brain cell populations in vitro, neurons and astrocytes.

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