Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins
Dohoon Kim, Chun-Hyung Kim, Jung-Il Moon, Young-Gie Chung, Mi-Yoon Chang,
Baek-Soo Han, Sanghyeok K, Eungi Yang, Kwang Yul Cha, Robert Lanza, and Kwang-Soo Kim
Introduction
Currently, all methods to derive induced pluripotent stem cells include risks associated with the use of viruses and mutation(s) during DNA transduction. However in this study, authors generated iPS cells from human fibroblasts by delivering the four reprogramming factors (Oct4, Sox2,Klf4, and c-Myc) with a cell penetrating peptide (CPP). The resulting cells maintained pluripotency for more than 35 passages and differentiated into all three germ layers both in vitro and teratomas.
The goal of the study was to generate pluripotency without viral and DNA vectors in order to make protein-induced human iPS (p-hiPS) cells suitable for clinical trials and patient-specific cells for regenerative medicine.
Procedure
To overcome the major obstacle for the delivery of proteins, which is their limited ability to cross the cellular membrane, authors hypothesized that the cell penetrating peptides can be used. Cell penetrating peptides can cross the membrane because they contain high amounts of basic amino acids such as lysine and arginine. To test the hypothesis, red fluorescent protein (RFP) was fused with a 9 arginine CPP and exposed to COS7 and human newborn fibroblasts (HNFs). Within a few hours,it was shown that the fluorescent protein was delivered into the cell (Fig. S1).
Figure S-1
Then, the four human reprogramming proteins were fused to a 9 arginine-CPP for HEK 293 cell lines, which resulted in high expression of the four proteins using Western blotting analyses (Fig. S2).
Figure S-2
16 hours treatment with the four proteins with 6 days incubation in ES Media resulted in iPS-like colonies with positive alkaline phosphatase (AP) activity after the 6th cycle. The overall process to generate iPS-like colonies took 8 weeks which is twice the amount of time required for virus transduction reprogramming process, and the efficiency of the process was 0.001% compared with 0.01% of the virus transduction.
Then to test for pluripotency, the expression of ES markers such as AP, Oct4, Nanog, tumor-rejection antigen (TRA)1-60, stagespecific embryonic antigen (SSEA) 3 and SSEA 4 was quantified using qRT-PCR. The expression pattern was identical to the embryonic stem cells. Furthermore, bisulfite sequencing analyses indicated that the promoter of the pluripotency
genes Nanog and Oct4 were significantly demethylated compared with the densely methylated parental HNF cells'.
At last, the p-hiPS cells formed embryoid bodies (EBs) by suspension culture and differentiated into cells of all three germ layers (Fig. 2d).
Figure 2d
Also, after transplantation of p-hiPS cells under the kidney capsule of nude mice for 6 to
8 weeks, teratoma formation was observed. The teratomas included neural tissues
(ectoderm), epidermal tissues (ectoderm), striated muscle (mesoderm), adipose tissue
(mesoderm), cartilage (mesoderm), respiratory epithelium (endoderm), and intestinal-like
epithelial tissues (endoderm) (Fig. 2e).
Figure 2e
(ectoderm), epidermal tissues (ectoderm), striated muscle (mesoderm), adipose tissue
(mesoderm), cartilage (mesoderm), respiratory epithelium (endoderm), and intestinal-like
epithelial tissues (endoderm) (Fig. 2e).
Figure 2e
6 comments:
The research that this paper presents is very interesting and seemingly very novel. Maybe it's make lack of familiarity with it, but I had a hard time following the figures. For example, for the first one, was there a time frame change from the first set to the second or in between the first and second row? Nonetheless, the concept is pretty refreshing and I had no problem following the potential of this field of research.
This idea is very novel comparing to the current method which involves using virus to change the genome of the original cell. Since the iPSC that been made are not carcinogenic, they might be used in medical application and it might be the potential treatment for many disease. However the low efficiency limited its application. I am curious of their differentiation. Will they differentiate after 35 passages under all kind of mechanical/chemical environment? Is there a way to keep them from differentiation? Also, I am curious about what's the reason for the low efficiency? Does the transmembrane process restrict the reprogramming or the transduction process restrict the reprogramming? Finally, does the author address if the CPP itself will affect the genome in any way?
This research novel in the sense that it uses nonviral vectors to reprogram the cells. However, I believe the efficiency is far too low for practical use. The method would need taylored proteins to be mass produced if it were to be for clinical use, which may be quite costly. The Harvard group that recently showed the synthetic mRNA method may be a better alternative to this one since the efficiency is much higher.
The concept of direct delivery reprogramming proteins is very interesting. I agree with the previous posters that this may lead to potential medical application for a wide variety of diseases diseases. Charlie brings up a great point about efficacy. What level of efficacy is needed to achieve any meaningful level of iPSC for clinical applications? It would be interesting how novel approaches in the future would increase both efficacy and avoid the use of viral vectors. Perhaps one can consider marking the proteins with specific factors to encourage targeted cellular uptake.
It seems that the insertion of protein rather than DNA for clinical use is a novel way to create iPS cells. The problem with this, which is not really mentioned in the study is how temporary this treatment would be. Although viral vector treatments certainly would have their drawbacks including cell mutation, they would not be temporary. In this sense, the paper talks about the clinical future of this research without considering all the limitations. The extremely low efficiency is mentioned and it is suggested that it be optimized, but they do not give any clear indication of a future work that would develop such a method. This is not to discount the many merits of this paper, which is the creation of iPS colonies without DNA viral vectors, but it seems to be farther from a clinical application than the paper seems to suggest.
Also, as Joanna mentioned the data is somewhat confusing, clumped closely together for several different types of analyses. It would be better in a future paper to have analysis that is more thorough and spread out with a specific statement of what each type of data suggests (microarray analysis, qt-pcr, etc).
Nonetheless, the novelty of this paper certainly makes it worthwhile as an interesting step forward in iPS cell research.
Post a Comment