Zhang SC, Wernig M, Duncan ID, Brustle O, and Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19: 1129-1133, 2001.
Summary: This paper describes how human embryonic stem (ES) cells could be used in neural transplantation therapies. Because ES cells could differentiate into various somatic cells, they have the potential of becoming specific donor cells used in transplantation therapies. For example, in the presence of fibroblast growth factor 2 (FGF-2) in vitro, ES cells could differentiate into neural tube-like structures that eventually become neurons and astrocytes. Studies performed on mouse ES cells have shown that they are capable of in vitro differentiation into CNS related cells such as neurons and glial cells. As a matter of fact, after transplantation into the mouse CNS, these ES-derived cells integrated well with the host tissue and even yielded functional improvements. This paper proposes a method to extend these findings and apply them to human ES cells.
The first step in isolating transplantable neural precursors from human ES cells was to initiate the differentiation process by growing human ES cell colonies as embryoid bodies (EBs). Then the EBs were cultured in the presence of FGF-2. After several days, most of the cells displayed “rosette formations” that resemble early neural tube development in humans. Zhang et. al subcultured these “rosettes” and discovered that they could generate all three major cell types of the CNS: astrocytes, oligodendrocytes, and neurons. Furthermore, upon implanting the neural precursors into the lateral ventricles of newborn mice the transplanted cells migrated to various regions of the host brain, forming clusters. Individual mice analyzed after eight weeks still showed the precursor cells. To specifically locate where these grafted cells were located in the host brain, DNA in situ hybridization with a human-specific probe was used. Gray matter areas that exhibited donor cell incorporation included the hippocampus, thalamus, hypothalamus, olfactory bulb, septum, striatum, and midbrain. White matter areas that exhibited donor cell incorporation included the internal capsule, hippocampal fiber, and corpus callosum. Immunostaining with specific antibodies indicated that the incorporate cells differentiated into neurons, glial cells, and astrocytes. Oligodendrocytes, however, could not be detected in vivo with antibodies to myelin proteins.
Significance: The study of in vitro differentiation of human ES cells could provide great insight into the early development stages of the human nervous system from a cellular and molecular level. Since this study discovered that the ES cells only formed neural tube-like structures in vitro, this phenomenon could be used to study human neural tube formation under extremely controlled conditions. This could lead to new discoveries regarding how the nervous system forms during early stages of development. Also, this study shows that human neural precursor cells could be generated in vitro and possibly used in nervous system repair. As shown with the mice, the donor cells incorporated nicely into the host brain without forming teratomata. In the future, this discovery could potentially lead to successful transplantation therapies for neurological diseases or brain cancer. Another application could be for the regeneration of lost neurons, which do not replicate on their own, in the adult human brain. Although more rigorous testing in nonhuman primates need to be done before any actual clinical applications, there is a promising future in using human ES cells as a source for neurological repair.
4 comments:
This is an interesting subject, the ability of FGF-2 to stimulate neural cell growth is promising. I have a few questions though, so don’t feel left out with no comments, Dien. What substrate are the cells cultured on or in, if any? For implantation, is it important to have neural cells with in-tact dendrites, as taking cells off of such substrates might reduce functionality of the neural cells.
Just a curiosity question: how functional are neural cells that are cultured in-vitro upon implantation? The reason I ask this is because when culturing the cells, they have no real electrical stimulation, so how do they incorporate with functional portions of the human brain? I envision in-vitro cultured neural cells to be like animals raised in captivity; most that are re-released into the wild have a strong affinity to get eaten soon thereafter because they’ve never had to deal with any real danger.
Ben, you bring up some very good points. From what I understood from the original paper, it appears that ES cells were only tested in vitro to see if they are capable of differentiating into neural precursors. I believe the ES cells were implanted in the mouse before they differentiated completely. As for the culture substrate, I'm not quite sure what they used. However, I'd imagine that the culture environment contains many factors found in the CNS.
This is an interesting area of research regarding studies and observations of neuronal cells. FGF-2 appears to allow ES cells to differentiate into CNS cells, and the studies in mice where the cells integrated and allowed for improved function seemed rather interesting. I was wondering what type of functional improvements did implantation of these cells in mice yielded and how these were quantified. Also, in growing neurons in-vitro, did the researchers mention if they used any specific scaffold onto which these cells were growing?
Nikit, I'm not sure if the authors included the functional improvements in the mice after implantation. However, I believe other studies out there have shown that these implants have the ability to dramatically improve motor skills in mice with damages in their neural cord. Also, like I said previously, I'm not sure what scaffold the researchers used to grow the ES cells in vitro.
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