Blog Post 2: How does skipping weekend media changes affect cortical & subpallial patterning?
Question: As the interest in working with brain organoids grows, so do the questions of how to make organoid maintenance more user-friendly. One of the most frequent questions that we receive is
“Is it really necessary to feed on weekends and
follow strict every-day or every-other-day schedules?”
The answer to this question not only impacts work-load requirements, but could also offer significant financial savings. In this blog post, we aim to address this question for cortical and subpallial organoids.
Figure 1
Design: We used two hiPSC lines from different sexes and followed either the Cortical (hCS) or Subpallial (hSS) protocols for differentiation (Figure 1A). To compare the effects of skipping media changes over the weekend, we compared our typical hCS and hSS feeding schedule (Figure 1B) with a protocol that skips media changes over the weekend after the primary neural fate induction stage (i.e. after Day 5, Figure 1B) for both regions.
Organoids were collected in triplicate on Day 30 and Day 60 for bulk RNA sequencing to compare potential gene expression differences between protocols. We also repeated this experimental protocol with an entirely separate differentiation at day 30. In addition to transcriptomics, we also collected conditioned media from the hSS plates to directly measure SAG levels, an ventral forebrain patterning small molecule.
Results
Figure 2
Figure 3
Nov 6th, 2023
We optimized a mass spectrometry (MS) protocol to sensitively quantify SAG levels in media. When comparing SAG levels from days 12-21 of the Subpallial (hSS) protocol, we could not detect any significant differences between the conditioned media of organoids that were fed over the weekend (days 15-16) versus those that received scheduled media changes on both days (Figure 2B). This finding suggests that, in the case of SAG, skipping a weekend feed doesn’t seem to change the amount of SAG available to the organoids during this crucial patterning stage of the protocol. We have subsequently begun similar assessements of other small patterning molecules like IWP-2 and RA.
Perhaps the most critical developmental stages for cortical and subpallial spheroids are the early patterning steps between hiPSC aggregation (Day -1) and neural fate commitment (by around Day 30). During this time, cells respond to a variety of forebrain patterning molecules and begin adopting a neural ectoderm fate marked by the presence of PAX6+ VZ progenitors (in the case of hCS) or NKX2-1 ventral VZ progenitors (in the case of hSS).
Therefore, we chose to perform bulk RNA-sequencing at day 30 for all of our conditions as a readout of potential differentiation consequences with each feeding schedule. When we compare our samples (including spinal organoids as an additional contrast) via PCA (generated using a select number of cortical, subpallial and spinal cord marker genes), we observe almost no differential effects of the skipping weekend feeding condition. Of note, we observed regional and line-dependent variability to be far greater than the effects of different feeding schedules (Figure 3).
When we investigated differential gene expression between feeding schedules at Day 30 in hCS & hSS, we noticed that there were virtually no significant changes across conditions (Volcono Plots, Figure 4).
Zooming in on early developmental markers also showed no substantive changes, including PAX6 or SOX2 in the hCS, and ventral markers NKX2-1 and DLX1 in the hSS (Heatmaps, Figure 4).
This led us to conclude that there are no early patterning issues that arise in the early stages of differentiation using weekend-skipped patterning recipes.
To ensure that our hCS and hSS were continuing to develop and mature within their respective dorsal and ventral forebrain lineages, we next performed a second round of bulk RNA sequencing at a later timepoint (Day 60). Similar to the results at Day 30, we observed no significant differences in gene expression at Day 60—whether looking at targeted regional markers (eg. TBR1, CUX1, GAD-1; heatmaps, Figure 5), or whole transcriptomic data (Volcano plots, Figure 5).
In summary, we found that simplified feeding schedules after Day 5 in hCS and hSS have no significant effects on the patterning and differentiation of hCS and hSS.
Figure 4
Figure 5
Verdict:
A weekend-skipping media change schedule after Day 5
has no significant effect on the developmental trajectory of
cortical and subpallial organoids.
Based on these results, the Hub has now adopted an alternative feeding protocol for hCS and hSS where media changes do not occur over the weekend after Day 5 (see Recipes & Protocols for recipe templates). Following the initial patterning phase, organoids receive media on Monday, Wednesday, and Friday once the feeding protocols move to an every-other-day feeding schedule (day 16 for hCS and day 18 for hSS).
Thank you for reading!
Questions, comments, suggestions?
Please email our manager Alexia King at alexia.tyler.king@emory.edu
See you at the next post!