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Ling Campbell

Dr. Ely

Biology 303

31 October 2014

Regeneration at the Genetic Level

Tissue engineering and regenerative medicine are becoming increasingly popular.

The purpose of tissue engineering is to restore, maintain, or improve damaged tissues or

even whole organs through looking at the functional units of the body. Regenerative

medicine is a field that includes tissue engineering, but also looks to see how bodies can

naturally self-heal. Advances in this field could positively affect millions of lives. Tissue

engineering and regenerative medicine tend to focus on the basic unit of life, cells. Cells

are the building blocks of tissues, and understanding how cells work is the main

framework for any regenerative medicine advances. (“Tissue Engineering and

Regenerative Medicine”)

The central dogma of molecular biology states that DNA is transcribed to mRNA,

which is then translated to proteins. Thus, every functional unit of the cell begins with the

content of the DNA. Genes are the units of hereditary information that make up DNA. By

studying the genes associated with tissue regeneration, scientists will be able to see what

is causing the tissue degradation at a molecular level. Understanding how the problem

starts is the first step to solving it. Possible treatments can range from determining how

the body naturally solves a problem and capitalizing on it, to artificial regeneration of the

loss tissue. This paper will examine three articles that focus on genes specific to

regeneration of varying types of tissues.

According to “Limb Loss Statistics,” about 2 million people in the United States

are living with some form of amputation. The major causes of limb loss are vascular

diseases (such as diabetes), trauma, and cancer. Although natural, complete limb

regeneration is not possible for humans, other animals such as lizards and zebra fish

possess this amazing capability. In a paper by Elizabeth D. Hutchins et al. (2014),

scientists used a transcriptomic-wide analysis to study which genes are involved in tail

regeneration of the Lizard Anolis carolinensis. Lizards were chosen for this experiment

because of the models capable of regenerating limbs, lizards are the most closely related

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to humans evolutionarily. By studying the genetic regulation of regeneration, scientists

will have an advanced understanding of the process, which is relevant to developing

human therapies. In order to study the genes, sample cells were necessary. To obtain their

samples, autonomy (tail removal) of 5 reptile tails was performed by applying pressure to

the tails until it was separated. Sample sections of the regenerated tails were collected 25

days post autonomy (dpa).

RNA-Seq analysis was used to determine the levels of different genes’ expression

within the regenerating tails. A total of 326 genes were clustered in two distinctive

groups: genes whose expression increased towards the proximal base (Cluster I) and

genes whose expression increased towards the distal tip (Cluster 2) (Figure 1).

The RNA-Seq analysis also investigated Gene Ontology (GO) groups related to tissue

differentiation, specifically skeletal muscle growth, myogenesis, and biological adhesion,

in the base of the tail. The analysis found an increase in the expression of myogenic

regulatory factors related to muscle growth and repair. Some of these transcription

factors, such as pax, mowhawk (mkw), and lcf15, are typically found in myogenic stem or

progenitor cells (Anderson et al. 2009, Fan et al. 2012, Takahashi et al. 2007) while

others like the TGF family member myostain (msln) are involved in modifying muscles

mass (Manceau et al 2008). In particular, the increase of mkx, which demonstrated that

growth is possible with coordination between tendons and muscles in the tail cell, is

promising because this transcription factor is required for growth and repair in mammal

Figure 1. Heatmap of the 326 genes. The 25 dpa tissues were divided into 5 equal segments (S1-S5). Tissue was taken from each of the segments to perform RNA-Seq. There are significant differences in the density of certain genes along the tail. 129 genes had a higher level of expression distally towards the tip (Cluster II; S2 and S1). 197 genes had increased expression at the proximal base of the tail (Cluster I; S5, S4, S5).

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cells, indicating a similarity that could useful to human regeneration (Anderson et al.

2012) (Hutchins et al. 2014). The increased level of expression of these types of genes

and regulatory factors at the base of the tail is logical because the base of the tail is the

first area to grow and differentiate.

Additionally, RNA-Seq analysis identified different levels of expressed genes in

the tip of the tail in GO groups associated with wound response, immune response,

hormonal regulation, and embryonic morphogenesis. The analysis found increased

expression of thyroid hormone generation genes, such as cga and dio2 that are central in

neuromuscular development, both in normal growth and after injury. Because there is a

need for a higher level of tissue differentiation and function at the tip of the tail, the the

higher level of expression of genes that code for these types of traits at this location is

fitting. The high levels of the wnt5a ligand and its receptor ror2 were especially

promising. Recent studies of regeneration have identified that Wnt signaling stimulates

differentiation of cells from skeletal muscles, osteogenic, and cardiogenic lineages

(Cruciat and Niehrs 2013).

At the tip and middle regions of the tail there was a high density of Wnt inhibitors such as

dkk2, igfbp4, wif1, and sgfrp2 (Figure 2). It is possible that the increased distribution of

these inhibitors at the end and center of the tail could create a gradient of Wnt signaling

throughout the tail that is required to constantly sustain the growth region of the

regenerating tail (Hutchins et al. 2014).

Figure 2. Heatmaps based on RNA-Sequence Analysis. Shows the 9 Wnt pathway genes. These genes and their inhibitors have a higher level of distribution at the tip and middle of regions of the regenerating tail, suggesting that the Wnt signaling can be transmitted throughout the entire tail.

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In order to determine the source of cells for tail regeneration, Hutchins and her

colleagues compared the cells from the regenerating tail to lizard satellite and embryo

cells. They found that the satellite and embryo cells have distinct patterns of gene

expression. Conversely, in the tail cells these genes are expressed at low levels without

any patterns along the length of the tail, suggesting that the tail stem cells are evenly

distributed throughout the tail (Figure 3A,B).

These findings are in accordance with the patterns of cell proliferation and specification

because it has been found by analyzing the distribution of minichromosome maintenance

complex component 3 (MCM3) and proliferating cell nuclear antigen (PCNA), two

markers of proliferation, that growth cells are evenly distributed throughout the

regenerated tail. The end result is that there is no single growth zone within the

regenerating tail (Hutchins et al. 2014). The information gained by this part of the study

is applicable to humans because satellite cells act as stem cells in mammals because some

have the ability to differentiate into skeletal muscle after injury (Askura et al. 2001).

Through the transcriptomic analysis of the lizard tail regeneration, Hutchins et al.

was able to identity genes, transcription factors, and signal pathways that play a pivotal

role in developmental roles such as myogenesis, chondrogenesis, and neurogenesis along

with processes reserved for adults such as responses to the immune system and wound

healing. The model of regeneration discovered in lizards shows a complex process of

formation of advanced developmental processes and specified wound repair that seem to

be unique to this amniote vertebrate. Some of the genes discovered have similar purposes

Figure 3. The expression of Satellite Cell Markers (A) and Embryonic Stem Cell markers (B). Expression of embryonic and satellite cell genes vary with position in the regenerating tail.

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in the human genome, which creates a bridge to applying the knowledge learned from

this study to the development of human therapies.

The study of genes for regenerative therapy does not have to involve regenerating

an entire entity. Instead, it is possible that the regeneration of one small area of tissue can

make a big difference in improving someone’s quality of life. For example, one of the

major causes of lower back pain is degenerative disc disease (DDD) (Luoma et al. 2000).

DDD is linked to intervetebral disc degeneration (IVDD), which decreases the function

and number of working cells in the disc and a reduction of extracellular matrix (ECM)

components such as aggrecan and type II collagen (Zhao et al. 2007). The current

treatments for the disease might temporarily alleviate pain, but in the end they do not fix

the actual problem. Studies have found that Nucleus pulposus (NP) cell transplantation

has been effective to slow down IVDD (Okuma et al. 2000). Unfortunately, it is not

possible to obtain NP cells from healthy normal disks because it might cause

degeneration in the original disc. But, NP cells have a chondrocyte-like phenotype similar

to bone marrow mesenchymal stem cells (BMSCs), which means BMSCs can also

differentiate to have the chondrocyte-like phenotype. Thus, BMSCs should be an

adequate cell source for IVD regeneration (Richardson et al. 2006, Steck et al. 2005,

Hiyama et al. 2008, Sakai et al. 2006, Sakai et al. 2003). Studies have also found that a

decrease in the transcription factor Sox9 found in NP cells, which is pivotal for

chondrogenesis and type II collagen synthesis, is linked to IVDD (Gruber et al. 2009).

Other research has determined that Sox9 gene transfer could stimulate cartilage growth of

the BMSCs (Paul et al. 2003, Tsuchiya et al. 2003). The paper by Sun et al. (2014)

hypothesizes that Sox9-induced BMSCs will have positive effect on IVD repair and

regeneration.

To study the effects of the Sox9 transcription factor, BMSCs were collected from

white rabbits and then cultured. These cells were then either transduced with just green

fluorescent protein (GFP) or both GFP and Sox9 genes. The results of the Sox-9 goups

showed the expression of Sox9, Col II, and Aggrecan increased substantially without

causing hypertrophy, cell enlargement. The BMSCs (both with the Sox9 and without)

were then embedded into chitosan-glycerophosphate (C/Gp) gels. The scientists found a

significant increase in the expression levels of Col II, Aggrecan and Sox9 in Sox9-

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transduced BMSCs in C/Gp gels compared to the increase on GFP-transduced BMSCs at

21 days after transduction. These findings show that Sox-9 transduction can induce

chondrogenic differentiation of BMSCs using the C/Gp gels as a growth medium.

The 50 rabbits in this study were induced with disk degeneration two weeks

before the beginning of treatment using the puncture method. The animals were then

divided into 5 different groups according to their treatments: rabbits with no surgery

(normal), rabbits who had the puncture surgery (degeneration), rabbits with the puncture

surgery and a chitosan-glycerophosphate (C/Gp) injection (C/Gp), rabbits with the

puncture surgery and then GFP-transduced BMSCs on C/Gp gels, and rabbits with Sox-9

transduced BMSCs using the C/Gp transplantation (Sox9). Sun et al. (2014) used

Magnetic resonance imaging (MRI) to visualize the effects of different induced BMSCs

on disk degeneration, using the amount of NP cells as an indicator (Figure 4).

In Figure 4A, the T2-weighted MRI images show that there was a significant decrease in

the size of the NP area for untreated degenerative cells and for the C/Gp groups that had

no additional BMSCs. Conversely, the Sox9 and GFP group showed decreased amounts

of NP degradation. In Figure 4B, the average T2 of the NP cells was higher for the Sox9

Figure 4. Imaging Studies. C/Gp group (A) T2- weighted MRI images at 6 and 12 weeks. (B) The mean of NP at 6 and 12 weeks.

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group than the other three groups. These findings suggest that the addition of Sox9 to

BMSCs can significantly slow down the amount of IVDD.

Through histological evaluations Sun et al. (2014) evaluated the amount of

chondrocyte-like cells and the NP structures. After 6 weeks of treatment, in the

degeneration group and C/Gp group the distinction between the disks was hard to see, but

still present and the number of BMSCs had decreased (Figure 5A-J). In contrast, the

distinction between disks was easily identifiable in the GFP and Sox9 group, and the NP

structure was comparatively well intact. 12 weeks after treatment, the degeneration and

C/Gp groups had no distinct border between disks and there was an inflow of connective

tissue in the gap. The GFP and Sox9 groups managed to maintain divided disks, with the

Sox9 group being slightly more successful. The results from this analysis augment the

idea that Sox9 transduced BMSCs are more capable of preventing disk degeneration.

The results of this experiment suggest that BMSCs exposed to higher levels of

Sox9 can decrease IVDD, meaning that successful stem therapies for this disease could

be possible with the right implantation procedure. Still, there are some limitations to this

study. For example, this injury model might not be similar enough to IVDD in humans.

But although the results from this study are not the absolute cure, Sun et al. were able to

demonstrate that Sox9 gene transfer could enhance the repair effects of BMSCs through

an increase in ECM production. Further studies will be necessary to determine NP-

specific markers in order to test which additional phenotypes should be targeted.

Furthermore, additional studies will be necessary to test if this or any therapy is plausible

long-term.

Another way gene expression can help scientists study regeneration is through the

examination of the genes used to monitor a certain pathway within the cell. The paper by

Zhang et al. (2014) looks at how the Il-18 pathway positively stimulates liver

regeneration. Hepatocyte cells make up the majority of the liver (Sell 2013). When the

liver has undergone a partial hepatectomy (PH) and therefore has lost liver cell mass, the

hapatocytes are activated and replenish this cell mass through liver regeneration (LR)

(Taub 2004). The LR process is divided into three phases: the priming phase that is

typically a half hour to six hours after liver cell removal, the progressing phase that is 6

hours to 72 hours after PH, and the terminal phase that is 72 hours to 168 hours after PH

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(Ma et al. 2009). Interleukin-18 (Il-18), typically associated with defense against

infection, has been linked to liver regeneration. Shiraki et al. (2007) found that the

concentration of Il-18 increased significantly after PH. Other studies have shown that Il-

18 can increase the amount of NF-B and X-linked inhibitors of apoptosis (Yamamoto et

al. 2008).

In order to determine what pathways were used to stimulate Il-18, J. Zhang et al.

(2014) performed PCR. They determined that there were three branches consisting of 13

genes involved with Il-18 signaling. The genes associated with these pathways can be

seen in Table 1. By looking at these results, it is possible to see that the genes associated

with regeneration were upregulated.

Table 1.Change in genes related to the Il-18 pathway during liver regeneration.

1, 2, and 3 represent respectively the NF-B signaling branch, p38 branch, and JNK branch. The values of the red and green indicate values of up regulation or deregulation that have significantly changed. The arrows denote genes that have been linked to liver regeneration.

Zhang et al. (2014) performed a gene synergy analysis using the expression

profile function Et, which is a mathematical model used to measure gene synergy

between related genes within a certain time (Tong 1978). The Et analysis looked at the

interaction between genes related to the Il-18 signal pathway and genes involved with

hepatocytic cell proliferation. In accordance to Table 1, the NF-B signaling branch, p38

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branch, and JNK branch were increased in liver cells with a PH compared to liver cells in

a completely intact liver, meaning that Il-18 could promote regeneration of hepatocytes

through these branches (Zhang et al. 2014).

Additionally, Zhang et al. treated his rats with injections of rrIl-18, an activated

form of recombinant Il-18. There were no significant differences in the mitotic indexes of

rats treated with low dosages of 0.5l or 1l rrIl-18. But, once the amount of rrIl-18 was

increased to 4l, there was a significant increase in mitotic activity (Figure A, B),

suggesting that high concentrations of Il-18 can increase the rate of hepatocyte

regeneration after PH.

In this study, Zhang and his colleagues determined that the Il-18 pathways

associated with increased LR can be viewed at a genetic level through the upregulation

and downregulation of associated genes. By understanding the genetic component driving

the Il-18 pathway in rabbits, scientists can seek similarities in the human genome.

Regulation of Il-18 production and signaling pathways has potential for liver failure and

regeneration treatments.

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In conclusion, the papers by Hutchins et al. (2014), Sun et

al. (2014), and Zhang et al. (2014) has offered new insight into

the genetic components of different types of tissue regeneration. The paper by Hutchins

et al. identified genes, regulatory factors, and signal pathways that were linked to lizard

tail regeneration. Sun et al. investigated the effect of increased quantities of Sox-9 on

decreasing the progress of IVDD. Finally, Zhang et al. determined that controlling the Il-

18 pathway and production has a direct correlation to liver regeneration. Unfortunately,

all these studies have limited application because the scientists are using animal models,

not human subjects. But, identifying the genes, transcription factors, and signal pathways

(in addition to any other genetics regulators) responsible for specific cell proliferation can

increase understanding of how degeneration occurs and provide insight into future

therapies.

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