Xenotransplantation of adult hippocampal neural progenitors into the developing zebrafish for assessment of stem cell plasticity

Adult stem cells are considered multipotent, restricted to differentiate into a few tissue-specific cell types. With the advent of technologies which can dedifferentiate and transdifferentiate cell types, assumptions about the process of cell fate determination must be reconsidered, including the role of extrinsic versus intrinsic factors. To determine the plasticity of adult neural progenitors, rat hippocampal progenitor cells were xenotransplanted into embryonic zebrafish. These animals allow for easy detection of transplanted cells due to their external development and transparency at early stages. Adult neural progenitors were observed throughout the zebrafish for the duration of the experiment (at least five days post-transplantation). While the majority of transplanted cells were observed in the central nervous system, a large percentage of cells were located in superficial tissues. However, approximately one-third of these cells retained neural morphology and expression of the neuronal marker, Class III β-tubulin, indicating that the transplanted adult neural progenitors did not adapt alternate fates. A very small subset of cells demonstrated unique, non-neural flattened morphology, suggesting that adult neural progenitors may exhibit plasticity in this model, though at a very low rate. These findings demonstrate that the developing zebrafish may be an efficient model to explore plasticity of a variety of adult stem cell types and the role of external factors on cell fate

Adult neural stem cell plasticity

Stem cells derived from adult tissues have long been considered multipotent, able to differentiate into a limited number of cell types found in their tissue of origin. Embryonic stem cells, in contrast, are pluripotent, which may differentiate into almost all cell types. With the ability to create induced pluripotent stem cells from somatic cells now available, the properties of multipotent stem cells are being re-evaluated. If adult cells may be reverted to pluripotent stem cells, can multipotent stem cells also be manipulated towards pluripotency? Advancements in biotechnology now allow for better methods to investigate stem cell plasticity, such as the relative influence of external versus intrinsic factors on cell fate. Recent studies indicate that adult neural stem cells (NSCs) demonstrate greater plasticity under certain conditions, resulting in the derivation of a variety of cell types including muscle, hematopoietic, and epithelial cells. This suggests that NSCs may provide a potential source of rare cell types for clinical application as an alternative to embryonic stem cells. Producing rare cell types from NSCs rather than embryonic stem cells avoids the ethical issues surrounding the use of this cell type. Further, NSCs may be an advantageous source compared to induced pluripotent stem cells, which are difficult to create, expensive, and time-consuming to develop. Adult NSCs have the ability to form neurons, astrocytes, and oligodendrocytes in vitro. More recently, evidence has arisen which indicates adult NSCs may have extended plasticity. Studies have demonstrated differentiation into cell types of all three germ layers through a variety of methods

Xenotransplantation of adult hippocampal neural progenitors into the developing zebrafish for assessment of stem cell plasticity.

Adult stem cells are considered multipotent, restricted to differentiate into a few tissue-specific cell types. With the advent of technologies which can dedifferentiate and transdifferentiate cell types, assumptions about the process of cell fate determination must be reconsidered, including the role of extrinsic versus intrinsic factors. To determine the plasticity of adult neural progenitors, rat hippocampal progenitor cells were xenotransplanted into embryonic zebrafish. These animals allow for easy detection of transplanted cells due to their external development and transparency at early stages. Adult neural progenitors were observed throughout the zebrafish for the duration of the experiment (at least five days post-transplantation). While the majority of transplanted cells were observed in the central nervous system, a large percentage of cells were located in superficial tissues. However, approximately one-third of these cells retained neural morphology and expression of the neuronal marker, Class III β-tubulin, indicating that the transplanted adult neural progenitors did not adapt alternate fates. A very small subset of cells demonstrated unique, non-neural flattened morphology, suggesting that adult neural progenitors may exhibit plasticity in this model, though at a very low rate. These findings demonstrate that the developing zebrafish may be an efficient model to explore plasticity of a variety of adult stem cell types and the role of external factors on cell fate

Representative image of transplanted AHPCs in the yolk periderm of a 1 dpf embryo exhibiting non-neural, flattened morphology.

<p>Representative image of transplanted AHPCs in the yolk periderm of a 1 dpf embryo exhibiting non-neural, flattened morphology.</p

Adult hippocampal progenitor cells transplanted at blastula stage are observed at least 5 days post-transplantation.

<p>A) Representative image of blastula containing transplanted cells. B) Zebrafish with transplanted cells at 1, 2, 3, 4 and 5 dpf. Green = GFP-expressing AHPCs. Scale bar = 250 μm.</p

A large percentage of transplanted cells retain neural progenitor phenotypes.

<p>Larvae at 3 dpf with transplanted AHPCs were immunolabeled for Nestin (red) at 3 dpf. Arrows indicate cells selected for higher magnification. A) Cells located at CNS and superficial regions were positive for Nestin. B) Cells in the zebrafish tail were Nestin positive. C) Quantification of average percent of Nestin⁺ cells/ location per fish at 3 dpf. N = 6. Error bars represent standard error of the mean.</p

Transplanted cells are retained in the CNS and superficial regions over time.

<p>Data represents the average percent of transplanted cells per fish at each location over time. At 5 dpf, a greater percentage of transplanted AHPCS were found in the CNS than in other non-superficial regions, and a greater proportion of cells were found in the CNS than at 1 dpf. **p≤ 0.01 Two-way ANOVA with Tukey’s multiple comparisons test. N = 6–13 animals per time point. Error bars represent standard error of the mean.</p

Expression of N-cadherin in the UROtsa parent cell line and the As⁺³ and Cd⁺² tumor transplants.

<p>(A and B). Real time-RT-PCR analysis of N-cadherin expression. The data is expressed as the number of transcripts of N-cadherin per 1000 transcripts of β-actin and is plotted as the mean ± SEM of triplicate determinations. (C and D). Western analysis of N-cadherin protein. * denotes significant difference from UROtsa parent cells (p<0.05).</p