This study aimed to generate induced pluripotent stem cells (iPSCs) from urine cells of patients with fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder. Urine samples were collected from three patients, cultured, and characterized. The cells were then to be reprogrammed into iPSCs via electroporation of plasmids containing reprogramming factors. While initial culturing of control urine cells was unsuccessful, culturing of cells from a second FOP patient showed promise. Future work includes characterizing the urine cells and reprogramming them to produce patient-specific iPSCs for studying FOP.

1. Generating Induced Pluripotent Stem Cells from Urine Cells in Patients with
Fibrodysplasia Ossificans Progressiva (FOP)
Charles Malcolm Roberson1,3, Corey Joseph Cain, Ph.D2, Marcela Morales2, Edward Hsiao, M.D., Ph.D2.
1Morehouse College, Atlanta, Georgia, 30314
2Department of Medicine, Division of Endocrinology and Metabolism; Institute for Human Genetics & Program in Craniofacial
Biology; University of California, San Francisco, San Francisco, CA 94143
32015 UCSF Summer Research Training Program
Experimental Design
• Cells from 250mL Urine Sample Cultured & Expanded
• Plasmid DNA Amplified
• Restriction Digest
• Characterization of Urine Cells via qPCR
• Reprogramming into iPS Cells via electroporation
Abstract
We are investigating the potential usage of urine cells to produce Induced Pluripotent Stem (iPS)
Cells in patients dealing with Fibrous Dysplasia Ossificans Progressiva (FOP), a rare genetic
disorder caused by a mutation in activing A Type I BMP receptor. In addition to the signature
characteristic of progressive heterotopic ossification, patients display a very limited range of
movement, and experience large amounts of pain during flare-ups that result in abnormal bone
formation. Physical injury can also stimulate flare-ups in these patients, making the collection of
dermal fibroblasts as a source of iPS Cells risky due to the damage that can be caused. We
hypothesized that electroporation of episomal plasmids containing the desired transcription
factors would allow the plasmid DNA to enter the somatic cell and drive reprogramming of renal
tubular epithelial cells found in urine into iPS cells. We collected urine samples from three
patients, one control and two with FOP, expanding the cells in culture until confluent enough to
characterize. In the future, we plan on reprogramming the cells via electroporation of bacterial
plasmids to introduce four transcription factors (KLF4, SOX2, OCT4, and LIN28) known to drive
iPSC formation into the cell. Following reprogramming, we will characterize the resulting iPSCs
via qPCR and compared them to the initial urine cells, as well as asses their potential usage in
treatments.
Preparation of Plasmid DNA
Plasmid # of Cut Sites Total BP Cut Locations (BP) Band Sizes (BP)
KLF4 2 11,981 1,727/3,528 1,801/10,180
LIN28 2 13,478 1,727/3,598 1,871/11,607
SOX2 2 12,475 1,727/4,021 2,295/10,180
OCT4 4 14,288 1,727/2,835/4,926/12,286 1,108/2,091/3,729/7,360
Following plasmid amplification, we performed a
restriction digest to verify the genetic structure of the
plasmid DNA following amplification. Following exposure
to the ECOR1 enzyme, we ran gel electrophoresis, and it
was expected that bands would show up corresponding
to each of the cuts made on the bacterial plasmids. The
size of the uncut plasmid, as well as the expected sizes
of resulting bands following the restriction digest, are
shown above.
Culturing of Urine Cells from Sample
Sample 1 (Control) Sample 2 (FOP) Sample 3 (FOP)
In our initial sample from a control patient, we collected a
much lower number of cells than we later would . We saw an
increase in cell density over the first few days , and even a few
colonies that formed, but around the 10th day the cells started
to lift off from the plates and die, to the point where by day 16
there were no cells left alive .
One of the reasons that we think this happened was the size
of plate the cells were initially placed in, which was much
larger than we would use for later collections which had a
higher number of cells. In addition to this, we added more
Pen/Strep on the 7th day as a precaution, seeing that cells
were starting to look unhealthy. This may have also resulted in
the decrease of proliferation in this sample.
• 4.0 x 104 Cells initially collected
In our first FOP sample, we saw a much greater number
of live cells, as well as a higher rate of overall
proliferation, enough to the point where we decided to
split the cells after only 6 days , much earlier than the
previous plan. This early split, combined with the 1:2 split
into a larger plate, resulted in an density that was very
low, and we think this negatively affected cell growth
rates. Following the split, many cells started to die, and at
the moment, there are very few cells left from this
sample.
• 1.0 x 105 Cells initially collected
When we got a sample from our 2nd FOP patient and 3rd
overall, we felt that we had a general idea of what not to
do, and planned on strictly following this protocol. We
collected more than double the number of live cells
compared to the previous FOP sample , but we
discovered some contamination in the plates, which
forced us to add gentamycin to the plates after the
second day. After this, we saw an increase in proliferation
and a decrease in the apparent contamination of the
culture. As of the 8th day, we saw 5-6 colonies of live cells,
which it looks like we will be able to use in further
continuing this project.
Cells were initially placed in the primary media, and
the media was slowly changed to the Renal Growth
media as the culture began to become more
confluent.
Primary Media
DMEM/Ham´s F12 1:1
10% Fetal Bovine Serum
SingleQuot Kit CC-4127 REGM
1X Amphotericin B
5X Penicillin/Streptomycin
Renal Growth Media
SingleQuot Kit CC-4127 REGM
Renal Epithelial Basal Medium
SingleQuot Kit
rhEGF
Insulin
Hydrocortisone
GA-1000
FBS
Epinephrine
T3
Transferrin
Conclusions/Future Directions
Unfortunately, we experienced great difficulty in culturing the urine cells, and very few colonies were
formed, slowing down the timetable of the project. However, given the current success in the 2nd FOP
sample, this project should be able to advance smoothly and efficiently.
qPCR will conducted to characterize the Urine Cells, testing for markers found primarily in epithelial
cells, fibroblasts, and renal tubular cells. It is hypothesized that the isolated cells will most resemble
renal tubular epithelial cells, based on the cells seen during the cell expansion, as well as previous
studies involving cells collected from urine samples.
Following Characterization, the cells will be reprogrammed via electroporation, as described above.
Fibrodysplasia Ossificans Progresiva (FOP)
FOP is a genetic disease affecting 1 in 2 million people throughout the world. The most noticeable effect
resulting from FOP is congenital heterotopic ossification, which is the formation of bone outside of the
skeleton. This is caused by painful flare-ups, in which swelling in the effected areas is followed by bone
formation, and these can either occur randomly, or be induced by trauma, such as an injury. At birth,
there are no noticeable traits caused by FOP other than malformation of the big toes and sometimes of
the thumbs. FOP is caused by a single nucleotide substitution in the gene coding for Activin Receptor 1,
which is a type 1 BMP Receptor. This mutation causes constitutive activation of the BMP signaling
pathway, ultimately resulting in an increase in chondrogenesis and osteogenesis. Currently, there is no
effective treatment, although anti-inflammatory drugs and pain medications are used to mediate the
symptoms. Using stem cells that are derived from FOP patients allows us a method of studying this
disease in-vitro over a long period of time.
Reprogramming of Urine Cells
*btxonline.com
Of the available methods for reprogramming to make iPS Cells, we have chosen to use
electroporation to introduce certain transcription factors into a cell. OCT4, SOX2, KLF4, and LIN28
are four transcription factors that, together, have been shown to drive iPS Cell formation. During
electroporation, cells are given an electric shock which disrupts their plasma membranes, allowing
the influx of extracellular contents into the cell. For this process to be successful, plasmid DNA
containing each of the four transcription factors needs to successfully enter the cell. The likelihood of
this happening is fairly low due to the fact that this process occurs almost completely randomly.
One of the advantages about electroporation as compared to another common method of viral
infection is that this is an integration-free method of reprogramming, which greatly reduces the risk
of tumor formation due to persistent transgene reactivation.
Overall Objective
To investigate If Urine Cells from patients with FOP can be reprogrammed into
iPS Cells through electroporation of plasmid DNA.