Deterministic versus stochastic model of reprogramming: new evidence from cellular barcoding technique.

Anastasia M Yunusova,Gennady V Vasiliev,Nariman R Battulin,Veniamin S Fishman

doi:10.1098/rsob.160311

Anastasia M Yunusova, Gennady V Vasiliev + Show 2 more

Open Access

https://doi.org/10.1098/rsob.160311

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Abstract

Factor-mediated reprogramming of somatic cells towards pluripotency is a low-efficiency process during which only small subsets of cells are successfully reprogrammed. Previous analyses of the determinants of the reprogramming potential are based on average measurements across a large population of cells or on monitoring a relatively small number of single cells with live imaging. Here, we applied lentiviral genetic barcoding, a powerful tool enabling the identification of familiar relationships in thousands of cells. High-throughput sequencing of barcodes from successfully reprogrammed cells revealed a significant number of barcodes from related cells. We developed a computer model, according to which a probability of synchronous reprogramming of sister cells equals 10–30%. We conclude that the reprogramming success is pre-established in some particular cells and, being a heritable trait, can be maintained through cell division. Thus, reprogramming progresses in a deterministic manner, at least at the level of cell lineages.

Highlights

Direct reprogramming of somatic cells to a pluripotent state can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc (OSKM) transcription factors [1,2]
Reprogramming is accompanied by resetting the epigenome of somatic cells, yielding induced pluripotent stem cells that functionally and molecularly resemble embryonic stem cells derived from early embryos [3,4,5,6]
Cells were transduced with lentivirus encoding M2 reverse tetracycline transactivator (M2rtTA) that drives reprogramming factors expression in the presence of DOX [18]

Summary

Introduction

Direct reprogramming of somatic cells to a pluripotent state can be achieved by overexpression of Oct, Sox, Klf and c-Myc (OSKM) transcription factors [1,2]. Monitoring the progression of cells through the reprogramming process revealed that almost all cells transit into the initiation phase with the downregulation of somatic cell genes followed by the activation of early pluripotency markers [7]. Only rare somatic cells pass through a second wave where the core pluripotency network is stably maintained [8]. The difference between these ‘lucky’ minority and cells that are refractory to reprogramming remains elusive

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