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Dr. Andrew Gaffney: SCTi003-A is a parent cell line that we’ve used to make other products. ���ʳ���徱���𳦳�™ is manufactured from the SCTi003-A cell line; however, it is a high-density and single-use version of the SCTi003-A cell line. If you procure the SCTi003-A cell line, it is frozen in clumps/aggregates with approximately one million viable cells per vial. In contrast, ���ʳ���徱���𳦳�™ is provided as a single-cell suspension with approximately ten million viable cells per vial, so you can count exactly how many cells you’re placing into each vessel. The choice of which product to use depends on your application: if you want to keep your cells in long-term culture, use the SCTi003-A cell line, and if you’re looking to immediately initiate downstream experiments, such as differentiation or transfection 24 hours after thawing your cells, we’d recommend using ���ʳ���徱���𳦳�™.

Dr. Andrew Gaffney: The main difference between the two products, SCTi003-A and ���ʳ���徱���𳦳�™, is the format in which we’ve generated the product. SCTi003-A is intended for long-term culture—you can keep the cells going for many passages and use the cells whenever they are needed. What we mean by single-use for ���ʳ���徱���𳦳�™ is that you simply take a vial out of the freezer whenever you want to use it and immediately perform your downstream experiments. There are also differences in the permitted use and licensing for the two products; long-term culture for both products is not permitted without a license. There is a license fee associated with the SCTi003-A cell line, whereas there isn’t for ���ʳ���徱���𳦳�™, because it’s not intended for long-term culture.

Dr. Andrew Gaffney: One vial of cryopreserved cells is included in each order.

Dr. Andrew Gaffney: There is a fee with the SCTi003-A cell line, which is $5000 USD per year for commercial entities. Non-profit organizations interested in using SCTi003-A may apply for a license fee waiver. There are no fees associated with ���ʳ���徱���𳦳�™.

Dr. Andrew Gaffney: The SCTi003-A cell line is frozen at passage 32 and ���ʳ���徱���𳦳�™ is frozen at passage 35.

Dr. Andrew Gaffney: We freeze the parent line as clumps or small aggregates at approximately one million viable cells per vial. When you thaw this vial for the first time in your lab, we recommend seeding at a range of densities across the 6-well plate, allowing them to recover for a week, and picking the well with the optimal clump morphology to continue your long-term passages with.

Dr. Andrew Gaffney: Yes, we do and we hope to make many more lines in the future that are similar in quality standards to the SCTi003-A cell line.

Dr. Andrew Gaffney: A classic feature of iPSCs is that they can be expanded in the undifferentiated state indefinitely. As long as the iPSCs are still expanding in this undifferentiated state, they remain viable. Differentiation experiments could be initiated at any passage number. One risk is that with human pluripotent stem cells, there is an association between higher passage numbers and links to commonly acquired karyotypic abnormalities, which is why it is important to check the cells fairly frequently. It’s also been noted that some of these abnormalities have been linked to skewed or limited differentiation capacities. We provide the cells at passage 32 and we perform a lot of quality control on these cells; so, after initiating these cultures, these cells would be fine to differentiate straight away. We’ve previously kept them going for around 20 passages so far in-house and they’ve remained karyotypically stable. As long as our protocols are followed, differentiation should be fine at passage 47, but you should still be checking the quality of your cells.

Dr. Andrew Gaffney: We developed the SCTi003-A cell line in mTeSR™ Plus medium, which is also the medium that the cells were expanded in before banking. We strongly recommend that you recover the iPSCs in the same medium in which they were maintained before freezing, i.e. mTeSR™ Plus. If you do need to switch media downstream, I don’t see why it wouldn’t work; but we haven’t tried or validated other downstream media ourselves. The only thing that I would stress is that when the cells are thawed for the first time, they should go into mTeSR™ Plus.

Dr. Andrew Gaffney: The iPSCs are maintained in mTeSR™ Plus on hESC-qualified Matrigel® during manufacturing. This can be switched downstream, but we recommend thawing into this same system used previously in manufacturing. Thawing onto Vitronectin XF™ matrix has also worked well—please get in touch if you’re looking for the protocol.

Dr. Erin Knock: There are no fees associated with the Human iPSC-Derived Neural Progenitor Cells (NPCs).

Dr. Erin Knock: Our STEMdiff™ SMADi Neural Induction Media protocol is done on hESC-qualified Matrigel®, but for the NPCs, you can choose to thaw and expand on hESC-qualified Matrigel® or PLO-Laminin. For neuronal differentiation, we generally recommend PLO-Laminin- (Sigma P4957) or PDL-Laminin-coated plates (Sigma L2020).

Dr. Erin Knock: The advantage of starting from NPCs is that you have some expansion time before you differentiate into your neurons. For generating a large number of neurons, start by thawing the NPCs, expanding them to generate a large number of NPCs, and then differentiating into a large number of neurons. Alternately, if you have uncertain timelines and wish to expand your NPC line, you can freeze them down, thaw them again, and start later. So NPCs give you flexibility. On the other hand, neuron precursors are already on that path to becoming a neuron; they’re not proliferative anymore and you won’t be able to expand them. You can also freeze them down, but they’re not going to be as robust as the NPCs when you freeze and thaw them. This might lead to a small reduction in yield, but in terms of purity and efficiency of differentiation, they’re pretty equivalent.

Dr. Erin Knock: The best way to get NPCs from iPSCs is by using STEMdiff™ SMADi Neural Induction Media. Two different protocols can be used for this: the embryoid body protocol and the monolayer protocol. The embryoid body protocol is best to start with if you don’t know the neurogenic capacity of your iPSC line. For example, if you are not using the SCTi003-A cell line and you don’t know if it’s going to make high-quality NPCs or not, the embryoid body protocol is robust. It will get you some neuroectoderm, and you can judge the efficiency based on your starting cell line. For a line that you already know has good neurogenic capacity (i.e. it already differentiates well into NPCs and neurons, similar to the SCTi003-A cell line), the monolayer protocol is technically simpler. It is more efficient, resulting in less technical variability. So for certainty, lean toward the embryoid body protocol, and for reproducibility, lean toward the monolayer protocol. You can also purchase iPSC-derived NPCs directly instead of differentiating your own.

Dr. Erin Knock: Use either an electroporation or lipid-based transfection system to transfect iPSC cells. We recommend that if you’re plating cells down close to transfection, do so in ����DzԱ��™2, as that will greatly increase the survival of your cells post-transfection.

Dr. Erin Knock: We can’t divulge the formulation of the STEMdiff™ SMADi Neural Induction Media, but the main driving force behind the media kit is the dual SMAD inhibition. As guided by the literature looking at PSC-to-neuroectoderm differentiation, we’re targeting these pathways with dual SMAD inhibitors in the kit. Additionally, the basal media is formulated to increase the optimal health and survival of the cells during that PSC-to-NPC transition.

Dr. Erin Knock: You’ll see a few of them moving around but they are not migratory by any means. The NPCs will generally stay within the confined area; they might move around a little bit, but as they start differentiating, they will elongate and then remain fixed in place.

Dr. Erin Knock: After passage 5, the NPCs will start to transition from a neurogenic fate to a gliagenic fate. That transition to gliogenesis is often associated with a change in morphology, where you’ll see cells that were very small, phase-dark, and defined, becoming larger and more spread out. There are a wide range of astrocyte morphologies, from something that is very protoplasmic-looking, to something that is very spiky, to radial glia that are neural in morphology. The abnormal morphology observed after passage 5 is that range in glial morphology that is starting to show as that transition from primarily neurogenesis to primarily gliogenesis is occurring. You can continue to passage the NPCs beyond passage 5, but note that you’re going to see a shift as the percentage of neurons being generated decreases and the percentage of glia being generated increases.

Dr. Erin Knock: Mature astrocytes can be obtained from NPCs up to passage 10. However, once you've derived astrocytes, they can be passaged for an additional 10 passages after the STEMdiff™ astrocyte maturation phase, depending on the cell line used. Deriving astrocytes from later-passage SCTi003-A-derived NPCs (due to the switch from neurogenesis to gliogenesis) will result in a higher rate of gliogenesis, and the resulting astrocytes can be passaged 10 – 20 times.

Dr. Erin Knock: There are protocols out there for both. Starting from NPCs is faster because they’ve already gone through the neuroectoderm phase. So, you can generally get neurons from NPCs in 3 – 5 days. Starting from iPSCs, such as SCTi003-A, might take a little longer and the usual protocols take 7 – 10 days. If you start from iPSCs, you’re going to have almost indefinite expansion beforehand whereas with NPCs, after passage 5 you will start seeing a lot more spontaneous differentiation toward the glial lineage, making neuron differentiation less efficient.

Dr. Erin Knock: The success of differentiation downstream of CRISPR editing depends on the gene you are editing and the potential off-target edits. If you knock out a gene that is critical for neural differentiation, you will see poor differentiation. Similarly, if you have an off-target edit in a gene that regulates differentiation, you may see poor differentiation. You must assess the pluripotency via trilineage differentiation and perform whole genome sequencing as part of your quality control when creating an edited PSC line.

Dr. Erin Knock: In 2D culture, we typically see apical progenitors, because 2D cultures often lack the organization required for basal progenitors to form. Occasionally, if you have a high amount of rosette-like structures forming in your monolayer NPC culture, you can see the presence of basal progenitors. If you want more physiological ratios of apical and basal progenitors, an organoid system will be more efficient.

Dr. Erin Knock: We do tend to find that NPCs survive better at higher densities and we recommend keeping them to “> 100%” confluence before passaging, meaning that they are starting to multilayer over themselves. If you seed at a lower-than-recommended density, you may see poorer survival. However, if you thaw on a Friday at the recommended density and do a double feed with STEMdiff™ Neural Progenitor Medium, you can probably change the medium again on Monday with minimal effects.

Dr. Erin Knock: There isn’t a good way to stain for DCX in live cultures. We do have NeuroFluor™ NeuO, which will start being expressed after 7 days of differentiation, so it may be a bit late for “newborn” neurons.

Dr. Erin Knock: Holes, in themselves, are not a deal breaker, as long as your remaining cells consist of a high percentage of PAX6- and SOX1-positive cells. To keep the density high and retain good expansion and survival, my recommendation is to keep the cells a day or two past the recommended 6 – 7 day passage time, then passage and re-seed at a slightly higher density than your previous passage.