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Can we reprogram senescent cells via antibody therapy?

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Antonio Carusillo
Antonio Carusillo Nov 11, 2020

Lately, I have re-called a relatively old paper (2017) where they were doing cell reprogramming. In particular, they tried to generate induced Pluripotent Stem Cells (iPSCs) from fibroblasts without the need of expressing the famous Yamanaka factors Oct4, SOX2, c-MYC and Klf4 .

Why did they want to avoid the Yamanaka factors?

There is one main reason: the Yamanaka factors can achieve the reprogramming cause they can alter the normal physiology of the cells. In particular, c-MYC is a well-known oncogene. This always poses a concern to the safety of the reprogrammed iPSCs since we may induce transformation in cancer-like cells and in fact, the process of re-programming and cancer transformation are very related

What do they do?

Interaction between the antibody and its cellular receptor may trigger a cellular response-able to change its physiology . In the study, they use a monoclonal antibody library (containing more than 10^11 different antibodies!) to screen for the ones able to induce cellular reprogramming from fibroblast to iPSCs. They found antibodies able to substitute for 3 out of 4 Yamanaka factors ( they could not identify one for Klf4). However, this proves the feasibility of such an approach.

Can we do the same with Senescent cells?

At this point, would it be possible to devise a similar approach to stimulate senescent cells? An experiment exactly similar could be devised in which an antibody library is screened for antibodies able to re-stimulate senescent cells. This stimulation may be assessed by doing transcriptomic analysis to look for a change in the expression of genes involved in cell division and metabolism, for example.
This approach would spare us from using more “invasive” approaches like genetic engineering which as we know – even though very promising – presents limitations in terms of accuracy and overall safety.

And can we also think about other possible strategies to achieve cell reprogramming?

[1]Blanchard, J., Xie, J., El-Mecharrafie, N. et al. Replacing reprogramming factors with antibodies selected from combinatorial antibody libraries. Nat Biotechnol 35, 960–968 (2017). https://doi.org/10.1038/nbt.3963

[2]Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors Takahashi, Kazutoshi et al. Cell, Volume 131, Issue 5, 861 - 872

[3]Wuputra, K., Ku, CC., Wu, DC. et al. Prevention of tumor risk associated with the reprogramming of human pluripotent stem cells. J Exp Clin Cancer Res 39, 100 (2020). https://doi.org/10.1186/s13046-020-01584-0

[4]Riggs JW, Barrilleaux BL, Varlakhanova N, Bush KM, Chan V, Knoepfler PS. Induced pluripotency and oncogenic transformation are related processes. Stem Cells Dev. 2013 Jan 1;22(1):37-50. doi: 10.1089/scd.2012.0375. Epub 2012 Oct 26. PMID: 22998387; PMCID: PMC3528096.

[5]Functional antibody selection in ES cells Anna N. Melidoni, Michael R. Dyson, Sam Wormald, John McCafferty Proceedings of the National Academy of Sciences Oct 2013, 110 (44) 17802-17807; DOI: 10.1073/pnas.1312062110

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Creative contributions

MicroRNA mediated cellular reprogramming - different contextual examples

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Subash Chapagain
Subash Chapagain Nov 13, 2020
Studies in different cellular contexts have revealed that microRNA are potent regulators of cell fate and they can direct differentiation/dedifferentiation in a contextual manner. miRNAs work not only by targeting a large repertoire of genes in different genetic networks but also they work as epigenetic regulators that are necessary for chromatin remodelling . This feature of miRNA functioning makes them a possible determinant of cell fate and lineage.

In a massively exciting study from 2011, it was seen that miR302/367 clusters could rapidly and efficiently reprogram mouse and human somatic cells to an iPSC state, without any necessity for exogenous transcription factors . What is even more exciting is that this miRNA based reprogramming approach is two-fold efficient than the standard Oct4/Sox2/Klf4/Myc (Yamanaka factors) mediated methods. These miRNA-induced iPSCs display equivalent characteristics to the classically induced iPSC- including pluripotency marker expression, teratoma formation and chimaera contribution (in the mouse cells). The study also reported that miR367 is essential for such reprogramming, and it acts by activating the Oct4 gene expression, as well as by suppression of Hdac2.

In another relevant research, working via their influence in chromatin remodelling, Yoo et al discovered that miR-9* and miR-124 direct the change of SWI/SNF-like BAF chromatin-remodelling complexes, a crucial process in the neuronal differentiation and function, a process facilitated by another factor NEUROD2. Furthermore, the addition of neurogenic transcription factors ASCL1 and MYT1L (two of the three above mentioned BAM factors) enhanced the rate of conversion and the maturation process of the converted neurons. It has to be noted that in the dearth of the miR-9*/miR-124, the factors alone could not effectively drive the induction into neuronal fate. This strongly suggests that microRNAs can have a strongly instructive role in neural fate determination . When lentiviral vector expressing precursors of miR9* and miR-124 along with turbo red fluorescent protein (tRFP) marker were used to infect human neonatal foreskin fibroblasts ( devoid of any neural progenitors, keratinocytes or melanocytes), these fibroblasts that expressed the microRNAs 9* and 124 showed a drastic reduction in proliferation and displayed neuronal morphologies post-infection, corroborated by the presence of MAP2, a marker protein of post-mitotic neurons. What was revealing was when the two miRNAs were separately expressed, MAP2 was not detected- suggesting that they have a synergistic effect on neural lineage generation. It was also noted from immunostaining that the induced neurons expressed SCN1a, a critical contributor to neuronal excitability, and also synapsin 1 and NMDA receptor 1. However, it shall be known that an estimate of 50% of total cells acquired neuronal fates ( 30 days post-infection), and finally, 5% of the total starting cells actually became physiologically functional neurons. This suggests a need for optimization and more rigorous research in using miRNAs as a fate changer of the cells.

Similarly, in what was the first reporting of direct cardiac reprogramming in vivo, researchers have found that miRNAs 1, 133, 208 and 499 are capable of inducing direct cellular reprogramming of fibroblasts in cardiomyocyte-like cells in vitro. The induced cells were reported to show the expression of mature cardiomyocyte markers, sarcomeric organisation and spontaneous calcium flux characteristic of cardiomyocyte-like phenotype .

All of these examples strongly suggest that miRNAs might be the next big thing in cellular reprogramming research. Hopefully, as more research data is generated, we can exploit this approach in the future and effectively apply for clinical usages as well.

[1] Yoo, A. S., Staahl, B. T., Chen, L., and Crabtree, G. R. (2009). MicroRNA-mediated switching of chromatin-remodelling complexes in neural development. Nature 460, 642–646. doi: 10.1038/nature08139

[2]Anokye-Danso F, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y, Zhang Y, Yang W, Gruber PJ, Epstein JA, Morrisey EE. Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell. 2011 Apr 8;8(4):376-88. doi: 10.1016/j.stem.2011.03.001. Erratum in: Cell Stem Cell. 2012 Dec 7;11(6):853. PMID: 21474102; PMCID: PMC3090650.

[3]Yoo AS, Sun AX, Li L, et al. MicroRNA-mediated conversion of human fibroblasts to neurons. Nature. 2011;476(7359):228-231. Published 2011 Jul 13. doi:10.1038/nature10323

[4]Jayawardena TM, Egemnazarov B, Finch EA, Zhang L, Payne JA, Pandya K, Zhang Z, Rosenberg P, Mirotsou M, Dzau VJ. MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes. Circ Res. 2012 May 25;110(11):1465-73. doi: 10.1161/CIRCRESAHA.112.269035.

BAM factors and direct neuronal reprogramming

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Subash Chapagain
Subash Chapagain Nov 13, 2020
Though the Yamanaka factors have been established as the go-to agents of the cellular dedifferentiation to induce PSCs, there can be alternatives to them, depending upon what final cell fate we aim to achieve by such a reprogramming. The technique referred to as direct reprogramming/transdifferentiation, for instance in the case of neuronal cells, has proven to be a robust and reproducible method to generate mature neurons of many subtypes from multiple cell sources .

One research used an approach equivalent to the Yamanaka research, and the group discovered that when three transcription factors Ascl1, Brn2 and Myt1l (collectively called the BAM factors) were expressed ectopically, they could produce induced neurons (iN) from the mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) .

The team was also able to induce the neuronal-like cell fate using several other combinations of the related factors. These induced Neurons expressed neuron-specific (marker) and even showed physiological properties of neurons ( they were able to have action potential firing and they could form functional synapses) in vitro. When applied to human cells, this method was found to be applicable, with the addition of NeuroD1- producing mature neurons with electrophysiological properties . Similarly, it was found that the BAM factors could also reprogram mouse hepatocytes into iNs, an indication that both the mesodermal and endodermal lineages are capable of transdifferentiation into the neural, ectodermal lineage .

It was revealed from single-cell and genome-wide expression studies that direct reprogramming of each lineage involves a coordinated activation of neuronal transcriptional pathways, and simultaneously silencing of the transcriptional program of the source cell. Interestingly, these protocols did not yield any dividing precursor or any natural stem cell intermediate. It is exciting to see that though this transient expression of only a few transcription factors in a non-neural cell can irreversibly reprogram a cell fate and produce functional neurons whereas such a phenomenon is not observed during normal development of an organism. One plausible hypothesis attached to this is that the direct reprogramming makes use of the conserved feed-forward transcriptional circuits that may be used to maintain the neuronal identity in mature neurons all the while heavily silencing/overriding the ongoing transcriptional networks associated with the cell type’s own identity. In this manner, if we know what we want as the final fate of our cells to be, can we use similar equivalent approach to generate other cell types thereby omitting the dependency on Yamanaka factors and hence the associated risks of tumorigenicity?

[1]Tsunemoto RK, Eade KT, Blanchard JW, Baldwin KK. Forward engineering neuronal diversity using direct reprogramming. The EMBO Journal. 2015 Jun;34(11):1445-1455. DOI: 10.15252/embj.201591402.

[2]Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors. Nature. 2010;463:1035–1041.

[3]Pang ZP, Yang N, Vierbuchen T, Ostermeier A, Fuentes DR, Yang TQ, Citri A, Sebastiano V, Marro S, Sudhof TC, Wernig M. Induction of human neuronal cells by defined transcription factors. Nature. 2011;476:220–223.

[4]Marro S, Pang ZP, Yang N, Tsai MC, Qu K, Chang HY, Sudhof TC, Wernig M. Direct lineage conversion of terminally differentiated hepatocytes to functional neurons. Cell Stem Cell. 2011;9:374–382

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General comments

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J
Juran9 months ago
It sounds like a concrete experiment. Concerning the part of proving the activation/stimulus of senescent cells reprogramming, we could use cell cycle analysis using flow cytometry to see if cells entered the cell cycle. This, followed by transcriptomic data could be a more convincing proof of activation.