direct reprogramming of hematopoietic progenitor cells into neural stem cells

Direct Reprogramming of Hematopoietic Progenitor Cells into Neural Stem Cells

When the cells are 30 to 50% confluent, aspirate the supernatant and reserve, and then add 1 milliliter of neural progenitor medium to each well. To make a backup plate using the spheres in the supernatant, centrifuge the reserve supernatant at 170 times g for 10 minutes.
Then, aspirate the supernatant and resuspend the pellet in neural progenitor medium. Plate in a matrigel-coated 24-well plate; change the medium in each plate of cells every other day until the cells reach 60% to 80% confluence, usually after one week.
Once the cells reach the required confluence, aspirate the supernatant, and add 1 milliliter of neural stem cell medium to each well. Then, dissociate the cells by using a cell scraper followed by very gentle pipetting.
Next, transfer the cells from one well of the 24-well plate into one well of a 6-well plate. Add another milliliter of medium to the well, and after repeating the procedure for all wells, place the 6-well plate into the 37 degrees Celsius 5% carbon dioxide incubator.
Change the full volume of medium every other day until the cells reach 60% confluence. After each time, dissociate and replate the cells at a 1-to-3 ratio as just shown.

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1. Neural stem cell induction
NOTE: Depending on cell conditions, about 5 or 7 days after transfection, monolayer adherent cells will reach 30 - 50% confluence (Figure 1C).
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	<li>Remove the supernatants containing floating cells and spheres and then add 1 ml of neural progenitor medium (Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12), containing 1x N2 supplement, 0.1% (w/v) bovine serum albumin (BSA), 1% (v/v) anti...

Source: Wang, T., et al. <a href="https://app.jove.com/v/52298">Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells.</a> J. Vis. Exp, (2015)
This video demonstrates the direct reprogramming of genetically modified hematopoietic progenitor cells into neuronal stem cells. Transcription factors expressed by hematopoietic progenitor cells trigger a cascade of molecular events facilitating their transformation. Further specific media is used to select and grow induced neuronal stem cells.

<img alt="Figure 1" src="/files/ftp_upload/22403/22403_Figure1.jpg" /> 
Figure 1. Morphological changes of CD34 cells after Sendai virus transfection. (A) Significant number of CD34 cells can be observed at the time of transfection. (B) Sphere-like cell aggregates can be observed 24 hr after Sendai virus transfection, which expands during time. (C) Mostly, bipolar adherent cells appeared after 5 days of transfection....

1. Neural stem cell induction
NOTE: Depending on cell conditions, about 5 or 7 days after transfection, monolayer adherent cells will reach 30 - 50% ...

Begin with a cell suspension of genetically altered hematopoietic progenitor cells.
These cells express transcription factors required for their reprogramming into neural stem cells. This process is called direct reprogramming.
Transfer the cells to an extracellular matrix-coated multiwell plate with a neural progenitor medium, and incubate.
The extracellular matrix provides a three-dimensional scaffold for cell attachment and growth.
During incubation, transcription factors expressed by the hematopoietic progenitor cell initiate a sequence of molecular events that allow its transformation into a neural stem cell.
The neural progenitor medium contains factors that favor the survival of induced neural stem cells.
After incubation, replace this medium with a fresh neural progenitor medium to promote cell multiplication.
Detach the cells using a cell scraper.
Collect this suspension and transfer the cells to a well containing neural stem cell medium.
Incubate for maintenance of induced neural stem cells.

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