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HIP Neural Stem Cells FAQs

1. How long do you have to differentiate the HIP™ NSCs to have mostly MAP-2 positive and beta3-Tubulin positive cells?
The neural progenitor cells need to be differentiated 2 - 3 weeks to obtain a majority population of beta tubulin positive neurons by mitogen withdrawal.

2. Can I generate a single neuronal subtype from the HIP™ NSCs?
Yes. HIP™ NSCs can be directed to differentiate efficiently toward a single neuronal subtype, such as Tyrosine Hydroxylase (TH)-expressing Dopaminergic neurons. There are several published methods for generating these different cell types from similar iPSC-derived NSCs.

3. Is it possible to derive neurons, astrocytes and oligodendrocytes from your HIP NSCs?
Yes. While HIP™ NSCs are predisposed to spontaneously differentiate into neurons they are also capable of generating the other major cell types of the brain: astrocytes and oligodendrocytes. There are several published methods for generating these different cell types from similar iPSC-derived NSC.

4. Can I generate a masterbank from the neural progenitor cells?
Yes, HIP™ NSCs can be propagated and banked for later use. We have expanded our production lots over 5 passages and confirmed robust neuronal differentiation. Note that as the passage number increases there may be a reduced production of neurons relative to other cell fates. HIP™ Neurons cannot be propagated.

5. Do you have a protocol for differentiated the HIP™ NSC into neurons?
Upon withdrawal of the mitogen FGF2, the differentiating progeny of the HIP™ NSCs will be primarily neurons.

6. How do I differentiate HIP™ hNSC to Astrocytes?
HIP™ hNSC are biased toward neuronal differentiation. However, they will differentiate toward GFAP-positive astrocytes under certain conditions. Nearly pure astrocytes have been generated from human pluripotent stem cells (via NSC intermediates) in several laboratories:
1. Krencik R, Weick JP, Liu Y, Zhang ZJ, Zhang SC (2011). Specification of transplantable astroglial subtypes from human pluripotent stem cells. Nature Biotechnology 29(6):528-34.
2. Emdad L, D'Souza SL, Kothari HP, Qadeer ZA, Germano IM (2011). Efficient Differentiation of Human Embryonic and Induced Pluripotent Stem Cells into Functional Astrocytes. Stem Cells Dev. 2011 Jul 26. [Epub ahead of print].

7. How do I differentiate HIP™ hNSC to Oligodendrocytes?
As noted in the above question, hNSC will differentiate toward oligodendrocytes under certain conditions. Several protocols have been used to successfully generate oligodendrocytes from human pluripotent stem cells:
1. Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K, Steward O (2005). Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci. 2005 May 11;25(19):4694-705.
2. Hu BY, Du ZW, Zhang SC (2009). Differentiation of human oligodendrocytes from pluripotent stem cells. Nat Protoc. 2009;4(11):1614-22. Epub 2009 Oct 15.

8. How do I differentiate HIP™ hNSC to Neurons?
This will depend on the type of neurons you want to generate Upon withdrawal of bFGF from culture media HIP™ hNSCs spontaneously differentiate primarily into neurons, Various protocols can be used for directed differentiation to specific neural cell types:
1. Zeng H, Guo M, Martins-Taylor K, Wang X, Zhang Z, Park JW, Zhan S, Kronenberg MS, Lichtler A, Liu HX, Chen FP, Yue L, Li XJ, Xu RH (2010). Specification of region-specific neurons including forebrain glutamatergic neurons from human induced pluripotent stem cells. PLoS One. 2010 Jul 29;5(7):e11853.

9. How do I know if my cells are still undifferentiated?
Antibodies to Nestin and Sox1, or Sox2, are commonly used to assess the undifferentiated state of hNSC. These markers are downregulated upon differentiation. Early neuronal differentiation of the cells can be detected by antibodies for Beta-III Tubulin or Microtubule- Associated Protein 2 (MAP2).

10. How do I know if my cells are growing quickly enough?
When following the supplied protocol, you should expect to see a doubling time for HIP™ hNSC of 50 ± 6 hours. Many factors can lengthen the doubling time, especially higher passage number or poor quality FGF2. Poor recovery at passage can also affect the measurement of doubling time.

11. Should I add LIF?
If you have difficulty maintaining the hNSC in an undifferentiated state, adding 10 ng/mL of human leukemia inhibitory factor (LIF) to your media may help. Note: Only human LIF will work; mouse LIF will not bind the human receptor.

12. When should I passage the cells?
Cells should be allowed to become 100% confluent prior to subculturing. Gently dissociate the culture to single cells and replate at a density of 30,000-50,000 cells/cm2.

13. Can I passage HIP™ hNSC with Trypsin or TrypLE or scraping?
Yes, but it is not recommended. Accutase is commonly used and recommended for gentle dissociation of HIP™ hNSC. Be aware that the use of serum to inactivate trypsin may alter the performance of HIP™ hNSC.

14. How many times can I passage the cells?
When following our maintenance protocol, HIP™ hNSC are guaranteed to maintain their differentiation potential for at least 5 passages after thawing.