Regenerative medicine-Direct and indirect cell reprogramming
My last three blog posts commented on direct cell reprogramming. You may be wondering if there is a technology called indirect cell reprogramming. The answer is yes. It is formally called induced pluripotent stem cell (iPSC, cells that have the ability to become any cell in the body)-mediated reprogramming, and it was developed earlier than direct cell reprogramming. In this blog post, I give a brief introduction of the two technologies and the challenges faced by both technologies.
Both reprogramming technologies involve the conversion of one cell type into another, target, cell type, intended for therapeutic use. Usually, the conversion starts by using the most abundant cells in the body, such as human fibroblast cells, a type of cell found in connective tissue.
Indirect cell reprogramming
Indirect cell reprogramming is a technology that converts somatic cells to another type of cell via first transitioning through an intermediate state, induced pluripotent stem cells (iPSCs).1 Therefore it is also called induced pluripotent stem cell (iPSC)-mediated reprogramming. The iPSCs have similar properties as embryonic stem cells in having unlimited differentiation capacity, while avoiding the ethical issues related to embryonic stem cells.2 Moreover, as iPSCs are generated from patients, this eliminates the problem of tissue rejection following transplantation in patients. Once iPSCs are generated, the cells can then be directed for development and differentiation, to form desired cells.
Four main transcription factors (Oct3/4, Sox2, Klf4, c-Myc), called Yamanaka factors, that induce somatic cells into pluripotent states, were first identified by Japanese scientists, and the finding was published in 2006.1 These transcription factors are highly expressed in embryonic stem cells, and are able to induce pluripotency in both mouse and human somatic cells.1
Since the generation of the first induced pluripotent cells, some progress has been made to improve the quality of the iPSCs generated and the efficiency of the technology. Methods include:
1) replacing of c-Myc, which is oncogenic and may cause tumour formation, in many protocols of pluripotent cell induction;3-6
2) using of viral delivery systems, such as an adenovirus, which does not integrate into the recipient’s DNA.7,8 This choice of virus is because a retrovirus, which is used for delivery of the four factors in the original protocol, would integrate into host’s DNA and cause insertional mutagenesis, which increases the risk of tumorigenicity;
3) replacing the original Yamanaka factors by small molecules and chemical compounds. This could also avoid the use of retrovirus;9
4) expressing Yamanaka factors for a shorter time for induction. This saves the time required to generate, expand and differentiate pluripotent cells, and avoids the creation of teratomas, a rare type of tumor that can contain fully-developed tissues and organs.10-12
Direct cell reprogramming
Direct cell reprogramming, also called transdifferentiation, is the conversion of a fully functional cell type into cell type of other lineage. The process bypasses a state of pluripotency. Transdifferentiation between some cell types can occur naturally in response to injury, and can be induced experimentally. I have made a brief introduction to transdifferentiation technology in a previous blog post.
A major limitation of direct cell reprogramming is the identification of reprogramming factors that promote conversion to a specific target cell type. The conventional strategy, which screens through a list of transcription factors that are important for the development of the target cells, is a time-consuming and laborious process. There are a few predictive software applications developed to facilitate the process. These include MOGRIFY13 and epiMOGRIFY14 which we mentioned in the last three blog posts, CellNet,15,16 and the one developed by Massachusetts Institute of Technology (MIT).17
Challenges faced by the two technologies
As you can see from the general description of the two technologies, direct cell reprogramming is faster, cost-effective and more accessible than iPSC-based protocols. Direct cell reprogramming protocols do not use Yamanaka factors, which are licensed, to induce pluripotency. The technology does not involve ex vivo cell expansion and transplantation, as in iPSC-mediated reprogramming.
However, both technologies face the same problem of scalability, which could impede their therapeutic applications. For the direct cell conversion approach, most converted cells could either experience cell cycle arrest that limit the expansion of the cell populations, or they tend to revert back to what they were. This lowers the number of cells available for further use. Similarly, for the indirect cell reprogramming, forced differentiation of induced pluripotent stem cells to distinct lineages also leads to the problem of cell cycle arrest and inability to expand. Moreover, the efficiency of inducing mature cells into pluripotency is less than 4%, which is very low.18
Quality of induced pluripotent stem cells generated from the indirect cell reprogramming is also a concern. The iPSCs generated are highly tumorigenic.1 This maybe due to the fact that the iPSCs are mostly derived from somatic cells of aged individuals. The risk that comes with this source of cells is the incidence of spontaneously occurring tumours, which commonly increases exponentially with aging. Concerns related to tumorigenicity and teratoma formation by these cells hamper their direct applicability.
Conclusion
Both direct and indirect cell reprogramming have been developed for more than a decade, and the technologies provide a feasible method to generate cells for biological studies, tissue engineering or transplantation. Diseases such as diabetes, heart failure, arthritis, and many more, which affect a high proportion of the world population, are hopefully to be treatable from the cell reprogramming technologies. Although no cell-based therapy has yet been officially approved in any country in the world, once the challenges mentioned above are overcome, the clinical application of this technology may not be far away.
References
1. K. Takahashi, and S. Yamanaka. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126 (4): 663–76.
2. B. Lo, and L.Parham. Ethical issues in stem cell research. Endocrine Reviews, Volume 30, Issue 3, 1 May 2009, Pages 204–213.
3. J. Han, P. Yuan, H. Yang, et al. Tbx3 improves the germ-line competency of induced pluripotent stem cells. Nature, 2010;463:1096–100.
4. J. Chen, J. Liu, J. Yang, et al. BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone. Cell Res., 2011;21:205–12.
5. H.Y. Li, Y. Chien, Y.J. Chen, et al. Reprogramming induced pluripotent stem cells in the absence of c-Myc for differentiation into hepatocyte-like cells. Biomaterials. 2011;32:5994–6005.
6. M. Maekawa, K. Yamaguchi, T. Nakamura, et al. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. Nature, 2011;474:225–9.
7. N, Fusaki, H. Ban, A. Nishiyama, et al. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad. Ser.B., 2009;85:348–62.
8. W. Zhou, and C.R. Freed. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells, 2009;27(11): 2667–74.
9.X. Li, J. Xu, and H. Deng. Small molecule-induced cellular fate reprogramming: promising road leading to Rome. Curr. Opin. Genet. Dev., 2018, 52, 29–35.
10. L. Kurian, I. Sancho-Martinez, E. Nivet, et al. Conversion of human fibroblasts to angioblast-like progenitor cells. Nat. Methods, 2013; 10:77–83.
11. M. Thier, P. Wörsdörfer, Y.B. Lakes, et al. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell. 2012; 10:473–479.
12. A. Ocampo, P. Reddy, P. Martinez-Redondo, et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell, 2016 December 15; 167(7): 1719–1733.e12.
13.O.J.L. Rackham, J. Firas, and H. Fang, et al. A predictive computational framework for direct reprogramming between human cell types. Nature Genetics, March 2016, Vol. 48, No. 3.
14. U.S. Kamaraj, J. Chen, K. Katwadi, et al. EpiMogrify models H3K4me3 data to identify signaling molecules that improve cell fate control and maintenance. Cell Systems, 2020, 11, 509–522, November 18.
15. S.A. Morris, P. Cahan, H. Li, et al. Dissecting engineered cell types and enhancing cell fate conversion via CellNet. Cell, 2014, 158, 889–902.
16. P. Cahan, H.Li, S.A. Morris. et al. CellNet: network biology applied to stem cell engineering.Cell, 2014, 158, 903–915.
17. A.C. D’Alessio, Z.P. Fan, K.J. Wert, et al. A systematic approach to identify candidate transcription factors that control cell identity. Stem Cell Reports, 2015 Nov 10;5(5):763-775.
18. Rao MS, Malik N. Assessing iPSC reprogramming methods for their suitability in translational medicine. J Cell Biochem. 2012;113:3061–8.
My last three blog posts commented on direct cell reprogramming. You may be wondering if there is a technology called indirect cell reprogramming. The answer is yes. It is formally called induced pluripotent stem cell (iPSC, cells that have the ability to become any cell in the body)-mediated reprogramming, and it was developed earlier than direct cell reprogramming. In this blog post, I give a brief introduction of the two technologies and the challenges faced by both technologies.
Both reprogramming technologies involve the conversion of one cell type into another, target, cell type, intended for therapeutic use. Usually, the conversion starts by using the most abundant cells in the body, such as human fibroblast cells, a type of cell found in connective tissue.
Indirect cell reprogramming
Indirect cell reprogramming is a technology that converts somatic cells to another type of cell via first transitioning through an intermediate state, induced pluripotent stem cells (iPSCs).1 Therefore it is also called induced pluripotent stem cell (iPSC)-mediated reprogramming. The iPSCs have similar properties as embryonic stem cells in having unlimited differentiation capacity, while avoiding the ethical issues related to embryonic stem cells.2 Moreover, as iPSCs are generated from patients, this eliminates the problem of tissue rejection following transplantation in patients. Once iPSCs are generated, the cells can then be directed for development and differentiation, to form desired cells.
Four main transcription factors (Oct3/4, Sox2, Klf4, c-Myc), called Yamanaka factors, that induce somatic cells into pluripotent states, were first identified by Japanese scientists, and the finding was published in 2006.1 These transcription factors are highly expressed in embryonic stem cells, and are able to induce pluripotency in both mouse and human somatic cells.1
Since the generation of the first induced pluripotent cells, some progress has been made to improve the quality of the iPSCs generated and the efficiency of the technology. Methods include:
1) replacing of c-Myc, which is oncogenic and may cause tumour formation, in many protocols of pluripotent cell induction;3-6
2) using of viral delivery systems, such as an adenovirus, which does not integrate into the recipient’s DNA.7,8 This choice of virus is because a retrovirus, which is used for delivery of the four factors in the original protocol, would integrate into host’s DNA and cause insertional mutagenesis, which increases the risk of tumorigenicity;
3) replacing the original Yamanaka factors by small molecules and chemical compounds. This could also avoid the use of retrovirus;9
4) expressing Yamanaka factors for a shorter time for induction. This saves the time required to generate, expand and differentiate pluripotent cells, and avoids the creation of teratomas, a rare type of tumor that can contain fully-developed tissues and organs.10-12
Direct cell reprogramming
Direct cell reprogramming, also called transdifferentiation, is the conversion of a fully functional cell type into cell type of other lineage. The process bypasses a state of pluripotency. Transdifferentiation between some cell types can occur naturally in response to injury, and can be induced experimentally. I have made a brief introduction to transdifferentiation technology in a previous blog post.
A major limitation of direct cell reprogramming is the identification of reprogramming factors that promote conversion to a specific target cell type. The conventional strategy, which screens through a list of transcription factors that are important for the development of the target cells, is a time-consuming and laborious process. There are a few predictive software applications developed to facilitate the process. These include MOGRIFY13 and epiMOGRIFY14 which we mentioned in the last three blog posts, CellNet,15,16 and the one developed by Massachusetts Institute of Technology (MIT).17
Challenges faced by the two technologies
As you can see from the general description of the two technologies, direct cell reprogramming is faster, cost-effective and more accessible than iPSC-based protocols. Direct cell reprogramming protocols do not use Yamanaka factors, which are licensed, to induce pluripotency. The technology does not involve ex vivo cell expansion and transplantation, as in iPSC-mediated reprogramming.
However, both technologies face the same problem of scalability, which could impede their therapeutic applications. For the direct cell conversion approach, most converted cells could either experience cell cycle arrest that limit the expansion of the cell populations, or they tend to revert back to what they were. This lowers the number of cells available for further use. Similarly, for the indirect cell reprogramming, forced differentiation of induced pluripotent stem cells to distinct lineages also leads to the problem of cell cycle arrest and inability to expand. Moreover, the efficiency of inducing mature cells into pluripotency is less than 4%, which is very low.18
Quality of induced pluripotent stem cells generated from the indirect cell reprogramming is also a concern. The iPSCs generated are highly tumorigenic.1 This maybe due to the fact that the iPSCs are mostly derived from somatic cells of aged individuals. The risk that comes with this source of cells is the incidence of spontaneously occurring tumours, which commonly increases exponentially with aging. Concerns related to tumorigenicity and teratoma formation by these cells hamper their direct applicability.
Conclusion
Both direct and indirect cell reprogramming have been developed for more than a decade, and the technologies provide a feasible method to generate cells for biological studies, tissue engineering or transplantation. Diseases such as diabetes, heart failure, arthritis, and many more, which affect a high proportion of the world population, are hopefully to be treatable from the cell reprogramming technologies. Although no cell-based therapy has yet been officially approved in any country in the world, once the challenges mentioned above are overcome, the clinical application of this technology may not be far away.
References
1. K. Takahashi, and S. Yamanaka. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126 (4): 663–76.
2. B. Lo, and L.Parham. Ethical issues in stem cell research. Endocrine Reviews, Volume 30, Issue 3, 1 May 2009, Pages 204–213.
3. J. Han, P. Yuan, H. Yang, et al. Tbx3 improves the germ-line competency of induced pluripotent stem cells. Nature, 2010;463:1096–100.
4. J. Chen, J. Liu, J. Yang, et al. BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone. Cell Res., 2011;21:205–12.
5. H.Y. Li, Y. Chien, Y.J. Chen, et al. Reprogramming induced pluripotent stem cells in the absence of c-Myc for differentiation into hepatocyte-like cells. Biomaterials. 2011;32:5994–6005.
6. M. Maekawa, K. Yamaguchi, T. Nakamura, et al. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. Nature, 2011;474:225–9.
7. N, Fusaki, H. Ban, A. Nishiyama, et al. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad. Ser.B., 2009;85:348–62.
8. W. Zhou, and C.R. Freed. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells, 2009;27(11): 2667–74.
9.X. Li, J. Xu, and H. Deng. Small molecule-induced cellular fate reprogramming: promising road leading to Rome. Curr. Opin. Genet. Dev., 2018, 52, 29–35.
10. L. Kurian, I. Sancho-Martinez, E. Nivet, et al. Conversion of human fibroblasts to angioblast-like progenitor cells. Nat. Methods, 2013; 10:77–83.
11. M. Thier, P. Wörsdörfer, Y.B. Lakes, et al. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell. 2012; 10:473–479.
12. A. Ocampo, P. Reddy, P. Martinez-Redondo, et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell, 2016 December 15; 167(7): 1719–1733.e12.
13.O.J.L. Rackham, J. Firas, and H. Fang, et al. A predictive computational framework for direct reprogramming between human cell types. Nature Genetics, March 2016, Vol. 48, No. 3.
14. U.S. Kamaraj, J. Chen, K. Katwadi, et al. EpiMogrify models H3K4me3 data to identify signaling molecules that improve cell fate control and maintenance. Cell Systems, 2020, 11, 509–522, November 18.
15. S.A. Morris, P. Cahan, H. Li, et al. Dissecting engineered cell types and enhancing cell fate conversion via CellNet. Cell, 2014, 158, 889–902.
16. P. Cahan, H.Li, S.A. Morris. et al. CellNet: network biology applied to stem cell engineering.Cell, 2014, 158, 903–915.
17. A.C. D’Alessio, Z.P. Fan, K.J. Wert, et al. A systematic approach to identify candidate transcription factors that control cell identity. Stem Cell Reports, 2015 Nov 10;5(5):763-775.
18. Rao MS, Malik N. Assessing iPSC reprogramming methods for their suitability in translational medicine. J Cell Biochem. 2012;113:3061–8.
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