ZAR Proteins

Dopamine Neurons

Harlequin Ichthyosis & ABCA12

Multipotent Stromal Cell Homing

iPSCs & Recessive Dystrophic Epidermolysis Bullosa

Cellular Rejuvenation

Microscopy, HTS & Automation

Zygotic Arrest Proteins

Dr. Amanda Charlesworth’s lab is interested in elucidating the post-transcriptional regulatory effects of zygotic arrest proteins during fertilization and embryogenesis. As my first laboratory, I am eternally grateful to my first lab mates for teaching me the basics.

During early development, genes in the mother’s egg cells are translated into proteins necessary for embryo growth, but this translation is regulated. Zar1 and Zar2 are proteins that are important for this process. Zar2 binds to a specific region in maternal mRNAs called the Translational Control Sequence (TCS) and regulates translation. Zar1 has a similar function, but its molecular mechanism is poorly understood. Our team found that Zar1 and Zar2 bind to the TCS of specific mRNA molecules, using a zinc finger in the C-terminal domain. Furthermore, Zar1 had a higher affinity for RNA than Zar2. Our published work shows that Zar proteins have similar functions in regulating maternal mRNA translation but may have different roles in early development.

Dopamine Neurons

Dr. Freed’s lab specializes in treating Parkinson’s disease via two strategies. The first is using embryonic stem cells for cell replacement therapy, and the second is pharmaceutical activation of neuroprotective pathways which protect cells from oxidative stress.

In the 1990s Drs. Curt Freed and Robert Breeze performed the first-ever double-blind neurotransplantation surgery. As a proof-of-concept, they transplanted a series of four injections consisting of human fetal ventral mesencephalic cells, halting the progression of Parkinson’s Disease (PD). Tyrosine Hydroxylase (TH) neurons remained present in transplanted tissue for 20+ years providing sustainable production of dopamine for PD patients non-pharmaceutically.

Dopamine Neurons Engraft, Release Dopamine, and Persist for Decades

40 patients with severe PD, aged 34 to 75, were randomly assigned to receive transplanted tissue or a sham surgery. An important finding of this study was that responders to Levodopa had good outcome post procedure whereas participants with historically low response to L-dopa received less benefit on PD associated scoring.

The use of embryonic stem cells carries severe ethical considerations and with the advent of induced pluripotent stem cells (iPSCs), it became possible to harvest adult cells, reprogram them to a pluripotent state (meaning they can become virtually any cell of an organism), and obtain an endless supply of tissue to treat PD.

Limitations of Human iPSC Derived Dopamine Neurons

T. Kituchi et al., showed that without a purification protocol implanted human iPSCs form teratomas in monkey brains. Complete differentiation of iPSCs to DA Neurons or identification of a surface molecule for purification remains a persistent issue in the field.

In 2015, while a participant in NIH’s Blueprint ENDURE Program, I was lucky enough to work with Dr. Curt Feeed and Dr. Wenbo Zhou on a project to increase the feasibility of a cell replacement therapy for PD. Around this time, many reports on increasing reprogramming efficiency with microRNA were being published and we started to ask ourselves ‘What if it were possible to transdifferentiate fibroblasts directly into dopamine neurons with a microRNA cocktail?’

Poster Presented as Society for Neuroscience in 2014

MicroRNAs were isolated and sent for NGS sequencing to identify known and unknown microRNAs. CU Anschutz normalized pseudo reads with our in-house Biostatistics Department. A list of the top 16 significantly up-/down-regulated microRNAs was used to increase the conversion of hES and hiPSCs to dopamine neurons.

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Volcano Plot of Pitx3+ vs Pitx3- help us identify significantly up-/down-regulated micro RNAs. Similar Analysis was conducted across all experimental groups.
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Unilateral injection of 6-OHDA, a molecule similar to dopamine but with an additional hydroxyl group, selectively abolishes dopamine neurons in the midbrain (substantia nigra). Stem cells differentiated into dopamine neurons can then be transplanted to the Striatum, and the rats are evaluated for functional recovery. In A9, dopaminergic neurons have cell bodies (somas) in the substantia nigra and project axons to the striatum. As the cells die in the midbrain, their release of dopamine in the striatum is diminished. One day it may be possible to induct neurite growth  


Dr. Roops’s lab specializes in heritable blistering skin and connective tissue disorders. For this project, I completed previous post-doctoral students' work to complete manuscript publication.

Harlequin Ichthyosis is a severe skin disease caused by mutations in the human gene ABCA12. Researchers have identified a novel mutation in the mouse Abca12 gene that leads to a similar disease in mice, which can be used to study potential treatments for this debilitating neonatal disease. The mutation affects the delivery of glucosylceramides and CORNEODESMOSIN, leading to abnormal skin development and the absence of lipid lamellae in the epidermis. The mutant mice also have reduced levels of KALLIKREIN 5 and -7, which are proteolytic enzymes required for normal skin shedding. This research highlights the essential role of ABCA12 in transferring lipids and proteolytic enzymes required for normal skin development and provides a model for exploring potential treatments for this potentially lethal disease.

MSC Homing

While at The Gates Center for Regenerative Medicine, we wondered how to treat internal epithelium of skin and connective tissue disorders like RDEB and EDS.

Multipotent Stromal Cells (MSCs) can be isolated from bone marrow, adipose (fat from liposuction), Wharton’s Jelly (umbilical cord tissue), and easily derived from induced pluripotent stem cells. MSCs have received significant attention due to their immune-privileged nature (lack of Major Histone Complex II (MHCII), making them suitable for off-the-shelf allogenic therapies) and their ability to regulate & improve wound healing. MSCs reduce inflammation and attenuate scar tissue through their release of exosomes, extracellular vesicles containing cargo of peptides and RNAs.

Multipoint Stromal Cell Replacement for RDEB

A significant hurdle for MSC therapy is their inability to efficiently engraft into the host tissue and their failure to be delivered intravenously and home to the tissue necessary. Separately, many groups have attempted to perform Glycosyltransferase-Programmed Stereosubstitution (GPS) of CD44 to increase hematopoietic stem cell engraftment following bone marrow transplantation to Hematopoietic Cell E- and L-Selectn Ligand (HCELL). Fucosyltransferases (FUT6 and FUT7) perform this glycosylation with excellent efficiency. Could MSCs be engineered to increase homing and therapeutic potential?

HCELL acts as a grappling hook for circulating cells. When inflammation occurs, E- and L-selectin are expressed in endothelial beds (blood vessels). HCELLs affinity for these selections slowdown cells and allow for activation, arrest, and ultimately complete extravasation (aka leaving the bloodstream to perform its task) 

Since our lab focuses on RNA, holding patents on both RNA reprogramming and a consolidated non-integrating gene correction and reprogramming protocol, we wondered if transfecting MSCs with FUT6 RNA would allow cells to glycosylate themselves. If so, how long would it take for HCELL expression, and how long would it last?

We were successfully able to induce HCELL expression on both human and mouse MSCs; furthermore, The expression peaked at 48 hours and remained in high expression for an additional 48 hours.

HCELL on MSCs is cool but will this enhance homing and engraftment?

To evaluate homing efficiency, I performed a subcutaneous injection of TPA (an irritant) into a mouse modified to express a red fluorescent protein (tdTomato). I then delivered MSCs that had been modified to express HCELL and a green fluorescent protein (GFP) through a tail vein injection. Using a point-scanning confocal microscope and a homemade intravital imaging chamber, I was able to look for green cells in the ear following injection.

Only hours after the infusion, we were able to see cells that had migrated from the tail to the ear systemically. One cell even left the bloodstream while imaging.

A significant benefit of intravital imaging is the ability to monitor the same mouse over extended periods.

Will multiple infusions lead to more engraftment?


Drs. Biluosova and Kogut have patented a highly effective RNA reprogramming technique and a single-step iPSC gene correction editing protocol that utilizes a reverse transcriptase Cas system to increase cutting efficiency with low indel (prime editing).

Coming Soon

Cellular Rejuvenation

Drs. Bilousova and Kogut have developed an RND rejuvenation cocktail that utilizes CRISPR activation to upregulate factors that increase telomere length and stabilize metabolic functions.

Coming Soon – A Little teaser just for you

Young on the left / Old on the right

Microscopes, HTS & Automation

At the Gates Center, I developed a Transendothelial Migration Assay (TEM) for high throughput screening (HTS), two sheer flow assays to evaluate homing efficiency in vitro, and tools to assess in vivo homing.

Using an ibidi pump system and μ sticky slides, I was able to confirm that modified MSCs had an affinity to E- and L-selectin. By inserting a western blot membrane with recombinant (manufactured) protein between the slide and flowing modified MSCs over the protein, I was able to see whether the cells were impacted.

Some AMAZING donors allowed me invest in some new Equipment

Now that we can slow then down, let's figure out how to increase engraftment

To evaluate which modifications increased extravasation, I modified a migration assay for high-content analysis.

Now I can evaluate many different conditions quickly