Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Summary

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Abstract

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy¹. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2–3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

Introduction

Cardiomyopathy is the second leading cause of death in patients with Duchenne and Becker muscular dystrophy (MD). Although mutations in the X-linked dystrophin gene occur in 1:3,500 male births, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Human induced pluripotent stem cells derived from muscular dystrophy patients have emerged as a novel tool to study the underlying disease mechanisms and to use for drug screening^1,2.

The anticipated discomfort of skin biopsies or blood samples may dissuade young patients and/or their guardians to give consent for study participation. Urine samples are a non-invasive source of somatic cells that are amendable to reprogramming methods. We have recently shown that urine cells collected from muscular dystrophy patients may be cultured and efficiently reprogrammed into iPSCs using retroviral transduction with the Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc; OSKM)¹. The disadvantage of retroviral gene delivery is the random integration of the reprogramming genes into the host chromosomes. To overcome this limitation, we have used the non-integrating Sendai virus for urine cell reprogramming.

This protocol details the Sendai virus reprogramming of isolated urine cells from muscular dystrophy patients which can then be differentiated into cardiomyocytes or other cell types for further study. This protocol can also be adapted for other patient specific diseases.

Protocol

NOTE: Patients and/or their guardians should give informed consent to participate in an Institutional Review Board approved study.

1. Buffers and Media Preparations

1. Washing buffer: To prepare 100 ml of washing buffer, add 1 ml of 100x pen/strep Solution (100 U/ml penicillin + 100 µg/ml of Streptomycin) to 99 ml of phosphate buffer saline (PBS).
2. Urinary Progenitor Cell (UPC) Medium: To prepare UPC medium, mix equal volumes of Keratinocyte Serum Free (KSF) Medium + Progenitor Cell Medium.
   1. KSF Medium: To prepare keratinocyte medium, add 5 ng/ml of epidermal growth factor (EGF), 50 ng/ml of bovine pituitary extract (BPE), 30 ng/ml of cholera toxin, and pen/strep solution (100 U/ml penicillin, 100 µg/ml streptomycin) to 500 ml KSF medium.
   2. Progenitor Cell Medium: Add three quarters of DMEM with one quarter of Ham-F12 media and supplement with 10% FBS, 0.4 µg/ml hydrocortisone, 0.1 nM cholera toxin, 5 ng/ml insulin, 1.8 x 10^-4 M adenine, 5 µg/ml transferrin, 2 x 10^-9 M triiodo thyronine, 10 ng/ml EGF, and pen/strep solution.
3. hES Medium: Add 20% KO-serum replacement (K-SR), 1x MEM non-essential amino acids, 2 mM l-glutamine, 100 µM β-mercaptoethanol, 20 ng/ml bFGF and pen/strep to 400 ml DMEM/F12 medium.

2. Urine Sample Collection

1. Instruct the patients to drink fluids 30 min prior to urine collection to ensure that an adequate amount (~30–40 ml) of urine can be collected.
2. Give the patients and/or their guardians a urine collection kit containing the following items: (1) written instructions on how to obtain sterile or clean catch urine sample, (2) moist anti-bacterial toilettes, and (3) a 100 ml sterile specimen collection cup. Instruct patients to collect sample.
3. Immediately place the urine samples on ice and transfer to the laboratory in a cooler. Process the urine for cell isolation immediately. If a delay is unavoidable, store the specimen on ice for up to 4 hr with minimal loss of cell viability.

3. Isolation and Expansion of Urine Cells

NOTE: Perform the following steps under sterile conditions in a BSL2 Biological Safety Cabinet.

1. Using sterile pipettes, transfer urine samples to sterile 50-ml centrifuge tubes. Centrifuge at 400 × g for 10 min at RT.
2. Aspirate the supernatant leaving 1 ml in the tube; be careful not to disturb the cell pellet.
3. Resuspend and combine the pellets from multiple tubes into 7 ml of washing buffer and repeat centrifugation at 400 × g for 10 min at RT.
4. Carefully aspirate the supernatant, leaving the cell pellet and ~0.2–0.5 ml of supernatant. Resuspend in 2–3 ml of Urinary Progenitor Cell (UPC) medium and transfer the suspension into 4–6 wells of an uncoated 24-well plate.
5. After 72 hr, supplement the culture with 0.5 ml of fresh UPC medium per well and change medium every 2–3 days after. The erythrocytes and squamous cells that do not attach to the plate will be removed with the medium changes.
6. Monitor culture after 4–6 days.
   NOTE: Small 2–4 cell colonies will start to appear. Cells will be rounded or elongated which represent type I or type II of renal epithelial (RE) cells respectively³.
7. Change the medium every 2–3 days since once the cells appear, they will undergo a rapid expansion phase.
8. Once the cells reach 80–90% confluence, around 9–15 days after plating, dissociate and passage the cells (15,000–18,000 cells/cm²) onto 2–4 wells of 24 well plate for further expansion. Mark this as cell passage 1 (P1).
9. Characterize urine cells for lineage specifications through flow cytometric analysis, immunohistochemical staining or through reverse transcriptase-polymerase chain reaction (RT-PCR).
10. Reprogram isolated urine cells (section 4) or freeze them down in Freezing Medium (DMEM supplemented with 10% serum and 10% DMSO) for long-term cryostorage.

4. Urine Cell Reprogramming Using Sendai Virus

NOTE: The proper handling and the use of PPE (personal protective equipment) is recommended while manipulating the transfecting agents. Perform the following steps under sterile conditions in a BSL2 Biological Safety Cabinet. The proper disposal of transfecting agent and/or transfected cells is recommended to avoid risk of environmental and health hazards. For urine cell reprogramming, use a Sendai Reprogramming Kit with modifications to the manufacturer’s feeder-dependent protocol as detailed below.

1. To ensure high efficiency reprogramming using the Sendai virus, use passages 1–5 of urine cells that are rapidly dividing. If the cells do not replicate well or become senescent, discard them as they will not reprogram efficiently.
2. Seed 60,000 cells per well in two wells of a 6 well plate. Mark this as Day -2. Adjust cell seeding density (5 x 10⁴ – 9 x 10⁴ cells per well) as needed to reach 80–90% confluence by Day 0.
3. On Day 0 (48 hr after plating the cells), prepare the Sendai reprogramming vectors (SeV) containing each of the four OSKM factors. Add each of the vectors to 1 ml of pre-warmed UPC medium. Use an MOI of 1–1.5 (5–7.5 x 10⁵ CIU), which is sufficient for reprogramming urine cells. Aspirate the medium and slowly add the 1 ml UPC + SeV to one well of the urine cells. Use the second well as the transduction negative control.
4. On Day 1, replace the UPC + SeV medium with fresh UPC medium. Some cell death is seen after 24 h due to viral cytotoxicity.
5. Depending on efficiency of reprogrammed clone formations and the compact morphology, keep changing the medium daily until day 6 or 7.
6. One day prior to dissociation of transduced cells (day 5 or 6), prepare MEF feeder plates by plating Mitomycin-C (10 µg/ml for 3 hr) treated-MEFs at density of 5 x 10⁴ cells/cm², on 0.1% gelatin coated 100 mm² culture dishes.
7. Next day (day 6 or 7), dissociate the cells with 0.25% trypsin, resuspend the cells in UPC medium and plate 5 x 10⁴ – 2 x 10⁵ cells/100 mm² MEF feeder plate.
8. The following day, switch to hES medium and change the medium every day. Observe the cells under a microscope to monitor transformed cells.
   NOTE: The cells form clonal aggregates with characteristic cobblestone morphology and having higher nucleus to cytoplasmic ratio (12–18 days post transduction).
9. Within two to three weeks after transduction, pick colonies and transfer to new plates for clonal expansion.
10. Perform live cell staining for selecting iPSC clones by incubating cells with TRA-1-81 antibody for 1 hr at 37 °C in CO₂ incubator followed by counter staining with specific florescent dye conjugated secondary antibody for 1 hr at 37 °C in CO₂ incubator.
11. Use a 26 G 1½ inch needle to cross-hatch the larger TRA-1-81 positive iPSC clones into small equal sized pieces (4–6 pieces per clone).
12. Use a sterile pipette-tip to pick and transfer 10–20 cross-hatched pieces from multiple clones into each well of Matrigel (10 µg/cm²)-coated 24 well plate with hES medium.
    NOTE: Pieces from a single clone plated individually into a single well do not expand or maintain pluripotency.
13. Change from hES to iPSC medium 24–48 hr after the reprogrammed clones are attached to the Matrigel surface.
14. During maintenance on Matrigel-coated plates, manually scrape-off any differentiated cells or contaminating MEF feeder cells using a pipette-tip to enrich for fully reprogrammed and pluripotent dystrophic iPSC (MD-iPSC) clones.
15. After individual clones have reached ~100 μm size, expand the clones by dissociating with 0.48 mM EDTA (ethylenediaminetetraacetic acid) solution for 3 min and plating small cell aggregates of MD-iPSCs suspended in iPSC medium (supplemented with 5 μM Y27632, RhO Kinase inhibitor) on fresh matrigel (10 µg/cm²) coated 12 well plate. Change iPSC medium daily.
16. Full reprogramming of each clone can be determined by both gene expression analysis of OSKM genes and immunohistochemical staining of pluripotency markers (Oct3/4 and TRA-1-81). A rigorous evaluation of pluripotency potential should be employed to confirm fully reprogrammed iPSC lines can differentiate into three germ layers. This can be accomplished either by embryoid body (EB) formation and differentiation assay or a teratoma formation assay.

Results

Most progenitor cells isolated from human urine are positive for uroepithelial progenitor and pericyte markers such as CD44, CD73, and CD146 (97.37%, 97.09%, and 97.3% respectively; Figure 1A and 1B). These cells also expressed other mesenchymal markers such as alpha-smooth muscle actin and vimentin (Figure 1B). RT-PCR analysis gives evidence of a mixed population of cells in the cultures in that there is weak expression of Cytokeratin-7 (CK-7) and Uroplakin (UP)-Ia &...

Discussion

Modeling cardiovascular diseases using iPSCs is becoming a common approach to understand the genetic contribution^4-6. Some unanticipated difficulties of obtaining cell samples from patients, especially young children, can be avoided by offering the option of a non-invasive approach such as a urine collection. In this young patient population, it is often difficult to collect a volume of urine sufficient to yield enough urine cells for reprogramming. Coaching the young patients to drink fluids 30 min prior to t...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
4 Oz. Specimen Cup with Lid	STL Medical Supply	M9AMSAS340	For Urine Sample Collection
15 ml BD-Falcon Tubes	Fisher Scientific	352097
50 ml BD-Falcon Tubes	Fisher Scientific	352098
Round Glass Coverslips	Fisher Scientific	12-545-81
CytoTune-iPS Sendai Reprogramming Kit	Life Technologies	A1378-001
PBS, pH 7.4	Life Technologies	10010-023
Pen-Strep w/o Glutamine	Life Technologies	15140-122	Warm in 37 °C water bath before use
RPMI Medium 1640	Life Technologies	11875-093	Warm in 37 °C water bath before use
B-27 Supplement w/Insulin (50x)	Life Technologies	17504-044	Warm in 37 °C water bath before use
B-27 Supplement w/o Insulin (50x)	Life Technologies	0050129SA	Warm in 37 °C water bath before use
DMEM/F12 (1:1)	Life Technologies	11330-032	Warm in 37 °C water bath before use
Versene, 1:5,000	Life Technologies	15040066	Warm in 37 °C water bath before use
bFGF (10 µg)	Life Technologies	13256-029	Warm in 37 °C water bath before use
Cell Counter Cartridges	Life Technologies	C10228
Knockout Serum (500 ml Bottle)	Life Technologies	10828-028	Warm in 37 °C water bath before use
Recovery Cell Culture Freezing Medium	Life Technologies	12648-010
Keratinocyte-SFM (1x), Liquid	Life Technologies	17005-042	Warm in 37 °C water bath before use
DMEM, High Glucose, Glutamax	Life Technologies	10566-016	Warm in 37 °C water bath before use
Ham's F-12 Nutrient Mix	Life Technologies	11765-054	Warm in 37 °C water bath before use
EGF Recombinant Human Protein, Liquid Form	Life Technologies	PHG0311L	Warm in 37 °C water bath before use
Insulin, Human Recombinant, Zinc Soln	Life Technologies	12585-014	Warm in 37 °C water bath before use
TeSR-E8	Stem Cell Technologies	5940	Warm in 37 °C water bath before use
ROCK Inhibitor (Y-27632)	Selleck	S1049	Warm in 37 °C water bath before use
Matrigel	BD Biosciences	354277	hESC Qualified Matrix, LVED Free
RNeasy Mini Kit	Qiagen	74104
iScript cDNA Synthesis Kit	Bio-Rad	170-8890
DreamTaq Green PCR Master Mix (2x)	Thermo-Scientific	K1081
FBS-Qualified	Sigma Aldrich	F6178	Warm in 37 °C water bath before use
Adenine Bioreagent	Sigma Aldrich	A2786-5G	Warm in 37 °C water bath before use
Cholera Toxin from Vibrio cholerae	Sigma Aldrich	C8052-.5MG	Warm in 37 °C water bath before use
Hydrocortisone	Sigma Aldrich	H0888-1G	Warm in 37 °C water bath before use
Holo-transferrin from human	Sigma Aldrich	T0665-50MG	Warm in 37 °C water bath before use
3,3',5-Triiodo-L-thyronine sodium salt	Sigma Aldrich	T6397-100MG	Warm in 37 °C water bath before use
DAPI	Santa Cruz Biotechnology	SC-3598
Fluoromount Aquous mounting medium	Sigma Aldrich	F4680
16% Paraformaldehyde	Alfa-Aesar	43368
Antibodies
Mouse anti CD44 - labeled with FITC	BD Biosciences	560977
Mouse anti CD146 - labeled with PE	BD Biosciences	561013
Mouse anti CD73 - labeled with PE	BD Biosciences	561014
Mouse anti-α-smooth muscle actin	Sigma Aldrich	A2547
Mouse anti-Vimentin	Abcam	ab8978-100
Mouse Anti - TRA-1-81	Life Technologies	411100
Rabbit anti Oct 3/4	Santa Cruz Biotechnology	SC-9081
Mouse Anti Dystrophin	Leica Biosystems	NCL-DYSB