This document discusses novel factors involved in direct lineage reprogramming. It describes how epigenetic regulators, miRNAs, small molecules, and pluripotency factors can induce the conversion of one cell type into another without passing through a pluripotent state. Epigenetic regulators like chromatin modifiers and histone modifiers interact with lineage-specific transcription factors to activate master genes of the target cell type. miRNAs can also facilitate lineage conversion by downregulating non-specific gene expression. Small molecules provide advantages over genetic manipulation and can activate signaling pathways and transcription factors required for lineage specification. Indirect lineage reprogramming uses pluripotency factors to first generate an epigenetically unstable intermediate state that aids the conversion to various cell lineages

1. Direct Lineage Reprogramming: Novel Factors
Involved in Lineage Reprogramming
Assignment No. 01
Subject: Introduction to Nanobiotechnology
Submitted by: Ahmed Madni
Registration No.: SP14-BTY-011

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Direct Lineage Reprogramming: Novel Factors Involved in Lineage
Reprogramming
Introduction
Direct linage reprogramming has got a major focus in biomedical field. The production of
specific functional cell type from totally different cell lineage is called lineage reprogramming.
In other words, it is induction of functional cell type from another linage without passing through
intermediate stage of pluripotent. It is was first discovered when myoblast is produced from
fibroblast by expressing Myod, which is transcriptional factor of specific cell type, this specific-
cell type transcriptional factor idea has evolved as an ideal base to produce different cell linage
of interest just using specific cell type transcription factor. Transcription factor considered as cell
fate conversion.
iPSC (Induced pluripotent stem cells) are pluripotent cells produced from adult cell by
reprogramming process. In this, combinations of cell-type specific transcription factors are
involved in generating different cell linage. Direct linage reprogramming has carried out in mice
and human. Pancreatic insulin producing cells in mice which alleviated hyperglycemia is
reported. The neuron cells, cardiomycetes and hepatocytes have been induced by this
methodology. The strategy has got lot of attention in biomedical field. But improvement in the
maturation of converted cells is still in consideration of researcher.
Novel Factors Involved in Lineage Reprogramming
Expression of transcription factor induces the lineage conversion. Not only transcription factor
but other factors like epigenetic regulators, miRNA and small molecules are also used in lineage
reprogramming. Other factors are there but complete studied is still required.

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1. Epigenetic regulators
Epigenetic state is most important in lineage reprogramming. The exogenous factors interact
with epigenetic regulator result in activation of master genes of cell, so epigenetic regulator
involved in expression of desired cell type. Chromatin modifiers, it comprises to induce cell
conversion. It is experimented in mice that Gata4 and Tbx5, cardiac transcriptional factor which
has induced cardiomycetes from non-cardiac mesoderm in presence of BAF cardiac sub specific
unit. Another possibility, in which inhibition or removal of Chromatin modifier such as histone
deacetylases and polycomb repressor complex 2 (PRC2) facilitated the conversion of germ cells
into neuron cells in Caenorhabditis elegans. So interaction between epigenetic regulator and
extrinsic specific lineage factor produced specific cell type.
Lineage conversion can occur through manipulation of epigenetic regulator. In mice, DNA
methyltransferase Dnmt1 deficency in mice led the conversion of pancreatic cells to α cells. Arx
gene (master gene) which maintain α cell identity and methylation of this gene can be
responsible in cell conversion. Not only the disruption of DNA methylation involves but histone
modification could also be involved in lineage conversion.
The epigenetic regulator has been studied by analyzing of marker gene expression in target cell
and it is unclear to obtain mature cells. Most of conversion has be conducted in vivo but in vitro
is still required which will help in indication whether epigenetic regulators act as transcription
factor or not.
Two types of epigenetic regulators are involved in lineage reprogramming. First one is expressed
in specific cell lineage while other one is expressed in different cell types. Its mechanism is
unclear but one possibility is there, their function relies on signaling pathways and transcription

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factors. The interaction between these molecules can help in identification of epigenetic
regulators inducing lineage reprogramming.
2. miRNA
miRNA is a type of RNA which works as down regulator of gene expression by binding to
specific complementary sequence. In recent study, miRNAs have involved in neuron cells and
cardiac cells induction. Such as miR-9/9* and miR-124 involved in conversion of fibroblast to
neuron cell. Another aspect is that inhibition of miRNA regulator such as PTB. PTB inhibits the
miRNA-mediated activity of the REST complex.
miRNA in induction of cell lineage conversion is still inefficient as compared to transcription
factor. miRNA only induces neural marker gene in non-neural cell while transcription factors
involved in relatively complete expression of functional neural cell genes.
Two possibilities are there to study the mechanism of miRNA in lineage reprogramming by
activating master genes. First, the overexpression of lineage specific miRNA, in which
expression of specific gene decreases the level of many non-specific transcripts in non-specific
cell. Another is the expression of certain epigenetics regulators that promote the global
epigenetic changes in in vivo and in vitro context such as histone expression which will facilitate
the cell-type gene by heterochromatin or euchromatin formation. These possibilities still required
further study.
3. Small molecules
An organic compound of low molecular weight also involved in inducing cell lineage
reprogramming. The genetic manipulation in reprogramming is generating safety concern, so
using small molecules in induction of conversion remains good. Small molecules use has many
advantages over the conventional methods such as it is cell permeable, cost effective, easily

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synthesized and most important that its concentration can be controlled that will be in involved
in high degree of spatial and temporal expression control. Thus it reduces the requirement of
exogenous factors and directly induces cell fate conversion. Identification of small molecules is
big hurdle. It is identified in dedifferentiation or transdifferentiation process in vertebrate
species. The cellular states are the output of interaction of signal pathway, epigenetic regulator
and transcription factors. The signal pathway and epigenetic regulators are majorly involved in
natural conversion. For example, in Wnt signaling, small molecule is involved in upregulation of
histone deacetylase and pigmented epithelial cells converted to lens cell. Thus lens is regenerated
in adult newt. This phenomenon of induction is named as chemical iPSC induction.
Transcription factor mediated lineage reprogramming in the activation of core GRN of the target
cell type while small molecules mediate the activation of factors comprising the core GRN that’s
why the small molecules can replace the function of exogenous transcription factors. The core
GRN that determines one specific cell type which may be comprised of many several master
genes. Activation of cell linage master gene can trigger the feedback mechanism which leads to
form specific cell lineage. Small molecules are also involved in activation of transcription factor
such SaII4 and Sox2, which are two pluripotency genes for chemical reprogramming process. In
other report, small molecules have involved in activation of pancreatic regulator PDX1.
4. Pluripotency for indirect lineage reprogramming
Indirect lineage reprogramming is done by using pluripotency factor. This strategy has been
carried in formation of different cell lineage such as neural stem cell, cardiomyocytes, pancreatic
lineage, hepatocytes and epithelial cells in mouse and human. This reprograming is dependent on
the presence of an epigenetic unstable population at early and intermediate stages during
reprogramming. At initial stages of reprogramming, Oct4 a pluripotency factor that binds to

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regulatory regions of different genes. The pluripotency factor also interact with epigenetic
modulators like NuRD, BAF and the PRC complex result in reactivation of epigenetically
repressed specific gene of lineage. So, the pluripotency factor can act as lineage specifier in
reprogramming. In recent study it is discovered that the overexpression of pluripotency factor
can induce differentiation in embryonic stem cells (ESCs). The exact mechanism is still
inconclusive but it is suggested that the transient pluripotent state during the conversion process
is remained as possibility.