I. microRNA for Stem Cells
- I have initial high transfection efficiency, but very few iPSC colonies. How can I increase the reprogramming efficiency?
- I've noticed a decrease in GFP expression overtime with my iPSC colonies. Does this decrease affect their pluripotency?
- What happens if the iPSCs differentiate? How can I prevent cells from differentiation? What are these weird looking cells on the fringe of iPSC colony?
- I have AMAXA device. What program would you recommend to use? For example, if I use foreskin fibroblasts, should I use the same program what I was using for reprogramming FSK/FBL with episomal vectors (7 factor) reprogramming?
- In your protocol there is recommendation to incubate 10' in ice before electroporation: how critical is this step? Should I follow this as well for AMAXA?
- When I was doing reprogramming with 7 factors, cells after nucleofection were cultured with fibroblast media and then at day 4 I switched to condition media. In your protocol Step3 p.2 you recommend to transfer colonies and switch media, "when iPSC colonies are larger". Could you be more specific and defined the size?
- Q1. How do you collect the mir-302-induced pluripotent stem (mirPS) cells?
A1. Due to a huge loss of cell adhesion molecules on the cell surface that occurs during somatic cell reprogramming, mirPS cells tend to attach to the gelatin-coated culture dish/flask very loosely. In around three days after electroporation, gently flush the loose cells with the medium inside and collect the loose cell suspension into a clean 15-ml tube. Spin down the loose cells and discard the old medium. Resuspend the loose cells in 2-ml of fresh knockout culture medium and continue to cultivate the cells in a 2-ml cell culture flask. At the mean time, add 2-ml of knockout culture medium to the old 35-mm diameter culture dish or into a.2-ml cell culture flask, and then place the cell culture dish/flask in a cell incubator. In time, many more reprogrammed cells will be generated. Please repeat the above procedure every three days for up to two weeks. Due to the fact that electroporation and/or reprogramming will kill many of the transfected cells, only a number of mirRPS cells will develop into egg-shaped cells with solid but loose nuclei surrounded by abundant cytoplasm. (Figure. 2)
Q2. How to isolate a good embryoid body?
A2. A two- to four-cell stage embryoid body is best for isolation to form a single mirPS cell line. The cell isolation is performed under an invert microscope using a micromanipulator in conjunction with a micro-injector/holder.
Q3. If the mirPS cells differentiate, what kind of cell will be see most often?
A3. Neuronal cells are one of the most often observed cell types in differentiated mirPS cell population (Figure.4).
- Q4. Why are the neuronal cells most observed from differentiated miRPS cells?
A4. Neuron cells may be the result of transfected cells having successfully undergone reprogramming and differentiation. To prevent this, avoid removing cells from incubator for extended periods of time. To further insure miRPS cells stay undifferentiated. Isolate and reculture embryo bodies quicker.
- Q5. How do you improve transfection efficiency?
A5. Low transfection efficiency may be the result of multiple factors including:
1) mycoplasma contamination
2) inadequate cell density
3) inadequate incubation time and electroporation condition
4) poor cell condition.
Determine if any of these factors were the case leading to low transfection efficiency.
- Q6. How do you reduce the extent of cell toxicity following electroporation?
A6. Presence of dead cells following electroporation is to be expected. Try reducing electroporation voltage and insure these cells are in good condition prior to electroporation.
Q7. How come the electroporation machine has no effect on my test cells?
A7. Electroporation machine may not be functioning properly in this case and re-calibration may be needed.
Q8. What cell lines can be reprogrammed using miR-302?
A8. The following human cell lines have been successfully reprogrammed by Mello Biotech and/or collaborating partners: HEK-293, Fibroblasts (NDFa/HFF), Keratinocytes, Melanocytes, Colo-829, LNCaP, MCF-7, HepG2 and Tera2. Murine embryonic fibroblasts have also been shown to work. Other cell types may work as well.
Q9. What type of tissue culture plates can be used?
A9. Several types of plates work well. For feeder culture conditions, use a plate optimized for the feeders. For feeder-free conditions, consider the following types.
1) BD cell culture plates
2) Gelatin coated plates
3) Collagen-IV coated plates during transfection. (Avoid collagen-IV coated plate one week after transfection as they may lead to differentiation of iPSC colonies).
Q10. Do we have to purchase media from Mello Biotech?
A10. Under feeder-free conditions, we recommend using our feeder-free media 1 to 2 weeks after transfection or after isolation of iPSC colonies. Use media optimized for the target cell line before switching to feeder-free media. Culturing under feeder conditions may significantly improve the survival rate of iPSC colonies. Mello Biotech media can also we used for feeder conditions.
- Q11. Is the transfection reagent cytotoxic?
A11. We have not observed any cytotoxicity under recommended conditions. If you experience cytotoxicity, try decreasing the amount of transfection reagent.
Q12. How many cells do you need per reaction?
A12. The number of cells required varies and depends on the cell type. We suggest using 40-60% confluency as standard for cell preparation.
Q13. Does the miR-302/367 plasmid integrate into the genome?
A13. Integration depends on the delivery methods. Liposomal transfection is transient in nature, and it should not integrate into the genome. Transfection by electroporation should not cause integration. However, under conditions of high voltage or long duration, some integration (
- Q14. How can I improve the transfection efficiency?
A14. GFP should be observed within 1~2 days after transfection. Some cells are difficult to transfect. Try different ratios of transfection reagent to plasmid.
1) Try increasing the plasmid concentration
2) Multiple transfections (every 3 days) may be necessary for difficult cell lines.
3) If you are experiencing problems:
i. Confluency may be too high. Optimal confluency is between 40%~60%.
ii. Remember serum-free medium must be used for transfection!
iii. During transfection, apply liposomal complex evenly drop-wise over target cells
4) Avoid the following conditions:
i. Inadequate cell density
ii. Inadequate incubation time to form the liposomal complex
iii. Poor cell condition such as contamination
Q15. What is the morphology of a miR-302-induced pluripotent stem cell?
A15. The iPSCs look like spherical cells with loose nuclei in the center and are surrounded by abundant cytoplasm.
Q16. I have initial high transfection efficiency, but very few iPSC colonies. How can I increase the reprogramming efficiency?
A16. There are several ways you can try:
1) Reprogramming is largely dependent on cell type and concentration of miR-302 (Lin et al., 2010).
2) Make sure puromycin selection is performed. Wait 3~4 days after Puromycin selection before transferring colonies to feeder-free medium. If you switch cells to feeder-free medium too soon, cells may detach and form aggregates which may reduce iPSC survival.
3) We recommend culturing iPSC colonies under feeder-containing conditions to improve reprogramming efficiency.
Q17. I've noticed a decrease in GFP expression overtime with my iPSC colonies. Does this decrease affect their pluripotency?
A17. In some cases, we observe the decrease of GFP over time (~3-4 weeks post-transfection). However, this may be due to the silencing of the CMV promoter as a natural part of the reprogramming process. However, the internal miR-302 of reprogrammed cell will continue the reprogramming process. You can evaluate the pluripotency of the iPSC colonies by methods suggested in this FAQ.
Q18. How can I isolate an iPSC colony to produce a cell line?
A18. Please try the following methods:
1) A two- to eight-cell stage iPSC colony is the best choice to isolate for the formation of a single iPSC line. Cell isolation can be performed under an inverted microscope using a micromanipulator in conjunction with a micro-injector/holder.
2) Alternatively, you can select colonies with pipette and very sharp eyes.
3) Fluroesence activated cell sorting (FACS) can also be used to isolate iPSC colonies.
Q19. Why does the embryoid body generated looks different from OSKM or hESC?
A19. As long as mir-302 is expressed, the EB will have homogeneous Oct4/Sox2/Nanog expression. Thus, these miR-302 EBs have a more spherical shape.
1) Most often, iPSC will differentiate into neuronal or fibroblast-like cells along the edges of the colonies.
2) To prevent differentiation, keep iPSC colonies small by mechanical passage. Do not treat with trypsin.
3) Use of feeder cells may also reduce differentiation.
4) Mello Biotech also offers a proprietary media optimized for iPSCs to reduce differentiation.
1) A maximum of 26 passages is observed so far under feeder-free conditions.
2) Additional passages may be possible under feeder-containing conditions.
Q22. How long is the cell-cycle of mirPS?
A22. Cell-cycle rate depends on the concentration of mir-302. Typically range from 16 hours to 24 hours. The greater the miR-302 concentration, the slower the cell cycle.
1) Store the kit at -80 degrees Celsius freezer to maintain quality. The vector is stable for one year under this storage condition. Avoid repeated freeze-thaw of the vector.
2) Due to the structural complexity of mir-302 cluster, amplification of the vector may cause mutations.
3) If you would like to sequence the vector to insure its quality, please contact Mello Biotech for primer information.
1) Antibody staining
2) Oct4/Sox2/Nanog expression
3) Check for global demethylation
4) Teratoma formation
5) Germline transmission (for non-human cell-lines)
Q25. Why does the Klf4 expression not shown in the western blot?
A25. Mir-302 does not affect Klf4 expression 10 days after transfection. You will see the same amount of Klf4 in target cells pre and post transfection.
Q26. Have you done any studies on the chromosomal stability?
A26. We have not yet studied the chromosomal stability, but there are no indications that it would have affect. Chromosomal stability would depend on cell source.
Q27. We have the AMAXA device. What program would you recommend to use? For example, if I use foreskin fibroblasts, should I use the same program what I was using for reprogramming FSK/FBL with episomal vectors (7 factor) reprogramming?
A27. We do not currently have a written protocol for the AMAXA. However, I recommend testing conditions for HFF cells using the control plasmid. Once you have determined transfection conditions you can then move forward using the miR302/367 plasmid. Please alert us out your findings so we can develop a protocol for AMAXA.
Q28. Should I use the hypoosmolar buffer for AMAXA?
A28. We recommend to use the type of buffer that you normally use with the AMAXA. Our hypoosmolar is optimized for the Mulitporator.
Q29. In your protocol there is recommendation to incubate 10' in ice before electroporation: how critical is this step? Should I follow this as well for AMAXA?
A29. This step is non-critical and optional. We find that it sometimes decreases cell death associated with electroporation shock in some cell types.
Q30. When I was doing reprogramming with 7 factors, cells after nucleofection were cultured with fibroblast media and then at day 4 I switched to condition media. In your protocol Step3 p.2 you recommend to transfer colonies and switch media, "when iPSC colonies are larger". Could you be more specific and defined the size?
A30. Colonies can be transferred at any size, but since we don't have a micro-manipulator in our lab, we wait until they are large enough to be selected by hand with a pipette tip. The important thing is to make sure there are not a lot of untransfected cells transferred over to Feeder-Free Medium as they can form balls of cells around the transfected cells and prevent the colony from growing. You can also perform Fluorescence Activated Cell Sorting (FACS) or limiting dilutions to insure a pure cell population before using Feeder-Free Medium. Keep in mind, once the cells are transferred into the Feeder-Free Medium, they form embroid bodies that are free-floating. These should not be confused with differentiating cells--these are your iPSCs.
II. Natural microRNA Precursors
- Q1. What is a CCE Precursor?
A1. It is a RNA Cell Extract.
- Q2. What is mir-302 Precursor?
A2. It is a purified microRNA.
- Q3. What makes is natural ?
A3. Our Natural microRNA Precursors are un-synthesized. They are solated from dicer negative cells.
- Q4. How does it compare to other products?
A4. There are no other natural products in the current market.x
III. mirTyro & Skin Lightening
- Q1. How should mirTyro be stored and maintained?
A1. mirTyro is most stable when dissolved in water-glycerin solution at neutral pH and temperature between 4 degrees and 25 degree Celsius.