Differentiation of Human Embryonic Stem Cells to Regional Specific Neural Precursors in Chemically Defined Medium Conditions
Slaven Erceg, Sergio Lainez, Mohammad Ronaghi, Petra Stojkovic, Maria Amparo Perez-Arago, Victoria Moreno-Manzano, Ruben Moreno-Palanques, Rosa Planells-Cases, Miodrag Stojkovic.
Introduction
Human embryonic stem cells (hESC) are pluripotent cells that can be propagated in vitro and theoretically provide a source of precursor cell that can be differentiated into any cell type. Thus derivation of neural progenitors from hESC provides a mean to study the central and peripheral nervous system, and for potential cell therapy applications to treat diseases.
Currently, hESC differentiation towards neural lineages include presence of stromal cell lines and/or conditional medium includes a multi-step process which involves formation of embryoid bodies (EBs), risking pathogen cross-transfer or contamination with non-neural cells. Countermeasures such as developing feeder free conditions for growth and controlled generation of neural progenitors in feeder and animal-free conditions avoid the formation of EBs. They devised their protocol which includes usage of animal-free components of ECM and chemically defined medium. Furthermore, the protocol allows controlled differentiation towards a specific region by exposing the rosettes to a signaling factor.
Summary
Human ESC were cultured on human foreskin fibroblasts and ES medium (enriched natural seawater medium) was changed every second day. For controlled differentiation, hESC were passaged to plates in PBS in the chemically defined medium. After 24 hours the cells attached and exhibited typical hESC morphology and after day two there were signs of neural differentiation and even further morphological development such as
After day 7, the medium was replaced with GRM medium along with basic fibroblast growth factor (bFGF). Rapid growth of rosettes occurred and at day 28 immuncytochemical analysis showed that cells were positive for numerous neural progenitor markers.
Figure 3: Differentation of hESC to neural progenitors Day 28
Figure 4:
D: Oligodendrocyte and astrocytes expression profile
E: Changes in gene expression of main pluripotency markers and general neural markers
To identify the population types obtained they first determined the expression of rostral-caudal (anterior-posterior) CNS markers using RT-PCR. They analyzed transcription factors involved in dopaminergic differentiation (largely rostral and midbrain markers) and found that those of the rostral markers were upregulated while midbrain cells had weak expression. The neural progenitors were then examined for caudal characteristics using class I and class II, homeodomein proteins, all caudal markers. They found high expression but little to no differentiation towards caudal cells, suggesting that using bFGF differentiated towards rostral cells as well as activation of caudal markers only. They tried to alter this rostral-caudal relationship by exposing the cells to RA (a well known caudalizing signal). Using the same protocol and methods, the found that the rostral and midbrain markers were strongly suppressed, that the cells acquired a spinal positional identity, and the caudal markers were strongly expressed or remain unchanged. Interestingly, the cells differentiated into a motorneurons upon this RA treatment.
Figure 5
E: RT-PCR analysis of rostral markers of hESC derived neural progenitors with or without RA
F and G: RT-PCR analysis of spinal markers
Figure 6: Neuronal excitability and study of voltage dependent channels
A: Action potentials evoked by depolarizing current steps. Both cases show that the spikes were fully blocked with TTX (red)
B: Early inward current suggests presence of voltage dependent sodium channels and the second outward component is consistent with K channels
Discussion
The paper demonstrated that neural progenitors can be generated in a one step approach without forming EBs and showed that there are advantages including non spontaneous differentiation of hESC and early markers for neural cells. They didn't however discuss how the method can lead differentiation towards more specific neuronal cells and they deterred from using a feeder layer for culturing but they maintained they undifferentiated hESC on a feeder layer before starting their study which seemed contradictory. I should point out that their actual experiment didn't use those cells until they were transferred to a feeder-free culture. Although they succeeded differentiating their cells to general regions of the brain, it didn't seem very specific and may be hard to evaluate.
8 comments:
In response to your discussion, it does seem difficult to evaluate the validity of these methods.
For instance, although the cells overall tested positive for various neural progenitor markers, it is unclear as to what fraction of the cells were differentiated (ie. what the efficiency of this method actually was).
I agree on your point that they kept the hESCs on a feeder layer but later claimed to differentiate them via chemically-defined media, especially since it is unclear what state the hESCs were in just prior to directing differentiation. Did they culture the hESCs on other types of feeder layers? Additionally, the paper does not seem to include much genetic analysis to demonstrate that DNA modifications towards neural lineages were made. Do you think there would have been substantial DNA modification differences between hESCs and neural precursors?
But is a high fraction of differentiated cells necessary? Once the cells have a differentiated can't they be separated and just cultured normally.
It seems unlikeley that having them on a feeder layer alone would be enough to promote them to grow towards neural precursor cells, but I do agree that they should of at least run some of the tests on the "pre-diffirentiated" cells. It could serve as a negative control to show that no differentiation had already occurred.
I was really curious as to what specifically in the chemically defined media was contributing to the differentiation of these cells and how the authors came up with the media they used.
There is a medium switch at day 7 which isn't really explained either.
Also, since the authors are pushing for this feeder-free method to be used instead of standard methods, a direct comparison between the two would have been helpful (i.e. comparing differentiation markers at different time points). If this method is as good or better than previous ones in getting neural differentiation, then it seems like people would adopt this easier method.
The clinical application of stem cells is really hampered by the concerns of animal-derived materials contaminating the cells. To maintain the stem-like state, the authors use a human fibroblast feeder layer instead.
As previous comments mentioned, something like a methylation profile to see gene regulation would have been a nice inclusion to the study.
It would be interesting to see if these neural progenitors could be further differentiated into cells with phenotypes closer to adult cells. Efficiency does matter since the progenitors will have limited self-renewal capacity.
The ability of hESC to differentiate into neuronal cells can aid research efforts. In the paper, various markers for neuronal cells were tested and shown to be present in the differentiated cells. However, were more intensive studies done to further characterize these differentiated cells? Moreover, was re-differentiation or change to other cell types exhibited once the cells were not subject to the medium conditions?
It seems like the authors did a good job of characterizing these differentiated cells as neural-like, but I wonder how functional they are in vivo . The patch clamp analysis suggests that they function in vitro , but there's no practical application unless they can work in organisms.
It'd be interesting to see what would happen if they attempt to inject these differentiated cells in a mouse. Would they proliferate at all?
I thought this was an interesting paper...Did they compare levels of expression to actual neuronal cells when they performed RT-PCR (I was not sure if D4 was a control or not)? Also, did they provide any explanation as to how the addition of RA contributed to the differentiation the cells into motorneurons?
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