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

AbstractWe 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 geneticdisorder caused by a mutation in activing A Type I BMP receptor. In addition to the signaturecharacteristic of progressive heterotopic ossification, patients display a very limited range ofmovement, and experience large amounts of pain during flare-ups that result in abnormal boneformation. Physical injury can also stimulate flare-ups in these patients, making the collection ofdermal fibroblasts as a source of iPS Cells risky due to the damage that can be caused. Wehypothesized that electroporation of episomal plasmids containing the desired transcriptionfactors would allow the plasmid DNA to enter the somatic cell and drive reprogramming of renaltubular epithelial cells found in urine into iPS cells. We collected urine samples from threepatients, one control and two with FOP, expanding the cells in culture until confluent enough tocharacterize. In the future, we plan on reprogramming the cells via electroporation of bacterialplasmids to introduce four transcription factors (KLF4, SOX2, OCT4, and LIN28) known to driveiPSC formation into the cell. Following reprogramming, we will characterize the resulting iPSCsvia qPCR and compared them to the initial urine cells, as well as asses their potential usage intreatments.

Preparation of Plasmid DNAPlasmid # of Cut Sites Total BP Cut Locations (BP) Band Sizes (BP)

KLF4 2 11,981 1,727/3,528 1,801/10,180LIN28 2 13,478 1,727/3,598 1,871/11,607SOX2 2 12,475 1,727/4,021 2,295/10,180OCT4 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 collectedIn 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 collectedWhen 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 MediaDMEM/Ham´s F12 1:110% Fetal Bovine SerumSingleQuot Kit CC-4127 REGM1X Amphotericin B 5X Penicillin/Streptomycin

Renal Growth MediaSingleQuot Kit CC-4127 REGMRenal Epithelial Basal Medium

SingleQuot Kit

rhEGFInsulinHydrocortisoneGA-1000

FBSEpinephrineT3Transferrin

Conclusions/Future DirectionsUnfortunately, 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 ObjectiveTo investigate If Urine Cells from patients with FOP can be reprogrammed into iPS Cells through electroporation of plasmid DNA.