Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Podsumowanie

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Streszczenie

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Wprowadzenie

Since the first derivation of hiPS cell lines by Takahashi et al.^1,2, hiPS cells have provided a useful tool for disease modeling, drug discovery and as source material for generating cell therapies in regenerative medicine³. hiPS cell culture has long been dependent on co-culture with fibroblast feeder cells^4,5 or on Matrigel⁶ and with media formulations containing fetal bovine serum (FBS). Batch-to-batch variances are a common consequence of the undefined nature of these culture conditions, resulting in unpredictable variations, which is a major contributor to the unreliability of these protocols⁷. The development of defined medium such as Essential 8 (E8)⁸ and defined cell culture matrices for instance LN-521⁹, allows for the establishment of highly reproducible protocols and aid in the robust generation and maintenance of homogenous hiPS cells^7,8,9,10.

Development of integration free reprogramming techniques have been a leap forward. Originally, reprogramming depended on retroviral vectors which randomly integrated into the genome with disruptive effects on genomic integrity¹¹. Advances in reprogramming methodologies includes the development of RNA based vectors. RNA vectors have an advantage over the DNA based reprogramming method as unintended integration through genomic recombination is not possible¹². SeV vectors provide high and transient expression of exogenous factors through single-stranded RNA without a DNA-phase¹¹. The reprogramming vectors delivered by the SeV are diluted throughout cell expansion and eventually shed from culture providing a foot-print free way of reprogramming. Thereafter, maintenance of pluripotency is dependent on endogenous expression of the pluripotency genes².

As pioneering hiPS cell based therapies are beginning to move into clinical trials, the demands for standardized batches, reproducibility, and safety are essential issues to address¹³. Therefore, products of animal origin should be avoided. For instance, the use of xenogeneic products has been associated with risk of nonhuman pathogen contamination. Also, cells cultured in the presence of animal derived culture components have been shown to incorporate nonhuman siliac acids into cell membranes which threatens to render derived cells immunogenic¹⁴. Hence, the need to eliminate xenogeneic products is necessary to any future clinical pursuits. This protocol applies xeno-free and defined culture in the maintenance of hiPS cells moving cells closer to clinical compliance.

This protocol describes a consistent, highly reproducible and easy-to-use method that generates standardized hiPS cells from fibroblasts. It also offers a user-friendly culture system for the maintenance of established hiPS cells. This protocol has been used to derive more than 300 hiPS cell lines in the Swedish national human iPS Core facility at Karolinska Institutet of which some lines have previously been described^15,16.

Protokół

The collection of patient material and derivation of hiPS cells is approved by the Ethics Review Board, Stockholm, March 28, 2012, Registration number: 2012/208-31/3. Cell culture steps should be performed in biosafety cabinets unless otherwise mentioned. Always practice sterile handling techniques when working with cells. Allow media, plates and reagents to reach room temperature before starting. Incubate cells at 37 ᵒC, 5% CO₂ in high humidity.

1. Isolation of Human Fibroblasts from Dermal Biopsy

1. Preparations of culture medium, digestive enzymes, coating of dishes and dissecting tools.
   1. Prepare Fibroblast medium: 44.5 mL Iscove's Modified Dulbecco's Medium (IMDM), 5 mL Fetal bovine serum (FBS) and 500 µL penicillin/streptomycin (PEST) (see Table 1), store at 4 °C.
   2. Prepare digestive enzymes at a working concentration of 1 mg/mL: weigh aliquots of 4 mg of dispase and collagenase type I powder in separate 15-mL tubes and dissolve in 4 mL of DMEM/F12 + 1% PEST. Sterilize the enzymatic solution by passing it through a 0.22 µm strainer. Prepare fresh enzyme solutions every time.
   3. Coat one well in a 6-well tissue culture plate (or 35-mm tissue culture dish) per biopsy dissected with 1 mL of 0.1% gelatin in phosphate buffered saline (DPBS). Incubate at room temperature for 30 min.
   4. Autoclave one set of surgical scissors and forceps per biopsy for dissection.
2. Biopsy handling
   NOTE: Process biopsies as fast as possible after being taken. Store in PBS + 1% PEST for up to 48 h at 4 °C. Biopsy should be 2 - 4 mm in diameter in size and in accordance with ethical permits.
   1. Transfer the biopsy into a 35-mm dish. Move the biopsy to a new 35-mm dish and submerge the biopsy in 2 mL of 70% ethanol for 30 s.
   2. Move the biopsy to a third 35-mm dish and wash with 1 mL of sterile Hank's Balanced Salt Solution (HBSS) supplemented with 1% PEST.
   3. Transfer the biopsy to a fourth 35-mm dish, add 1 mL of freshly prepared 0.1% dispase solution.
   4. Cut the skin biopsy into 1 - 2 mm³ pieces in the dish using surgical scissors and forceps and then transfer biopsy pieces including the dispase solution into a 15-mL tube. Add an additional 2 mL of dispase.
   5. Rinse the 35-mm dish with a 1 mL dispase solution and transfer to the 15-mL tube.
   6. Incubate the biopsy at 4 °C overnight.
   7. Add 4 mL of 0.1% collagenase I to the tube and incubate at 37 °C for 4 h.
   8. Centrifuge the digested cells 300 x g for 3 min, aspirate the supernatant and resuspend the digest in 2 mL of fibroblast medium.
   9. Remove the gelatin solution from the 6-well tissue culture plate. Transfer the cell suspension including any remaining pieces to 6-well tissue culture plate (or 35-mm tissue culture dish) precoated with 0.1% gelatin in DPBS.
   10. Change medium every third day.
3. Expansion of human fibroblasts
   NOTE: Fibroblasts are ready to be passaged to a T25 (25 cm²) tissue culture flask when 80% confluent (Figure 2B). Fibroblasts typically reach 80% confluency within 7 days. However, time required can vary greatly but should be no longer than 4 weeks.
   1. Aspirate medium and wash once with 2.5 mL of DPBS.
   2. Remove DPBS and add 1 mL of Trypsin-EDTA (0.05%) and incubate at 37 °C for 5 min or until cells display rounded edges and start to detach.
   3. Add 2 mL of fibroblast medium, if necessary rinse the cells ensuring proper disassociation of cells. Transfer the cell solution to a 15-mL tube and centrifuge at 300 x g for 3 min.
   4. Discard supernatant and resuspend the cell pellet in 5 mL of fibroblast medium and plate fibroblasts in a non-coated cell tissue culture T25 flask. When expanding fibroblasts after this step, dissociate as described above and seed 12 x 10³ cells/cm².
   5. Once confluent cells are ready to be frozen, expand or plate for reprogramming. Freeze down two rounds of fibroblast before any reprogramming is started (refer to next section for freezing procedure). Reprogramming efficiency is generally higher for lower passage fibroblasts, cells at passage 2 - 4 is optimal. However, successful reprogramming of fibroblasts up to passage 10 has been conducted using this protocol.
4. Freezing and thawing of fibroblasts
   1. Prepare fresh fibroblast freezing medium: 90% FBS + 10% dimethylsulfoxide (DMSO). Keep at 4 °C (Table 1). Once confluent, the T25 tissue culture flask can be frozen into three cryovials. Prepare 500 µL of fibroblast freezing medium per vial frozen.
   2. To freeze cells, dissociate cells as described in Step 1.3, count cells using a hemocytometer and transfer portions of 3 x 10⁵ cells to 15-mL tubes. Spin tubes at 300 x g for 3 min. Aspirate supernatant.
   3. Resuspend cell pellets in 500 µL of 4 ˚C fibroblast freezing medium and transfer into cryovials. Place the vials in a freezing container and incubate cells at -80 °C for 24 h before transferring them to liquid nitrogen for long term storage.
   4. To thaw the cells, submerge the bottom part of the cryovial in 37 °C water. Once liquefied, transfer the cell-suspension to a 15-mL tube and add 5 mL of fibroblast medium. Spin cells 300 x g for 3 min.
   5. Remove the supernatant and resuspend the cell pellet in 5 mL of fibroblast medium. Transfer cells to a non-coated T25 tissue culture flask and incubate in 37 °C.

2. SeV Vector Reprogramming of Fibroblasts

1. Preparation of vector aliquots
   CAUTION: Handle the SeV under biosafety level 2 containment. Refer to manufacturer's protocol and local guidelines¹⁷. Virus titer varies between batches.
   1. Before preparing aliquots calculate the amount of vector needed for 5 x 10⁴ cells according to manufacturer's protocol (e.g., CytoTune 2.0). The virus titer generally varies from 8 x 10⁷ - 1.5 x 10⁸. Thaw and combine vectors 5:5:3 (KOS MOI = 5, hc-myc MOI = 5 and hKlf4 = MOI 3). Portion the amount calculated for the reprogramming of 5 x 10⁴ cells into autoclaved 1.5 mL tubes. Dilute virus aliquots with fibroblast medium to a final volume of 250 µL for reprogramming.
      NOTE: Each vial is valid for reprogramming of one well of a 24-well tissue culture plate with 5 x 10⁴ fibroblasts. Store virus aliquots at -80 °C.
2. SeV Vector transduction
   1. Aspirate cell culture medium and wash with 1 mL of DPBS. Aspirate DPBS and add 1 mL of Trypsin-EDTA (0.05%). Incubate at 37 °C for 5 min or until cells are detached.
   2. Add 2 mL of fibroblast medium and resuspend the cells and transfer to a 15-mL tube. Centrifuge 300 x g for 3 min.
   3. Aspirate the supernatant and resuspend the pellet in 2 mL of fibroblast medium.
   4. Count fibroblasts by using a hemocytometer and seed 5 x 10⁴ cells in one well of a 24-well tissue culture plate. Incubate cells at 37 °C overnight.
   5. Aspirate cell culture medium, add 250 µL of the SeV solution and incubate at 37 °C overnight.
   6. Change medium daily to fresh fibroblast medium. Allow transduced cells to grow for 7 days before being passaged to LN-521 coated plates. Prepare LN-521 plates the day before passage. Refer to 2.3.1 for coating procedure.
3. Replating of transduced fibroblasts
   1. Preparation of LN-521 coated dishes: Dilute LN-521 in DPBS to a final concentration of 0.63 µg/cm². Pipette 2 mL of the LN-521 solution per 60-mm tissue culture dish. Seal the 60-mm tissue culture dish using parafilm. Incubate overnight at 4 °C.
   2. Prepare Essential 8 medium (E8) aliquots for easier handling: 48.5 mL of E8 medium, 1 mL of E8 supplement and 500 µL of PEST (Table 1).
   3. 7 days after transduction, passage the cells to a LN-521 coated 60 mm tissue culture dish. Add 250 µL of Trypsin-EDTA (0.05%) and incubate at 37 °C for 5 min or until cells have detached. Add 2 mL of fibroblast medium and centrifuge cells for 3 min at 300 x g.
   4. Aspirate the supernatant. Resuspend pellet in 2 mL of fibroblast medium and count cells in a hemocytometer. Aspirate LN-521 solution from the precoated plate and seed 4 x 10⁴ cells in 5ml of fibroblast medium in the LN-521 precoated 60 mm dish. Add Rho-kinase inhibitor Y-27632 (ROCKi) to a final concentration of 5 µM. Incubate in 37 °C overnight.
   5. Importantly, the next day, change medium to E8 medium. Change E8 medium daily. hiPS cell colonies will emerge within 2 - 3 weeks.

3. Picking of Colonies and Expansion of hiPS Cells

NOTE: The following steps are done outside of the biosafety cabinet under a stereo microscope. The use of hairnet and procedure masks are recommended. Work carefully not to contaminate the cell culture.

1. Prepare LN-521 coated tissue culture 24-well plates the day before picking. Dilute LN-521 in DPBS 0.63 µg/cm². Pipette 250 µL of the LN-521 solution into one well of a 24-well tissue culture plate. Seal plates using parafilm. Incubate overnight at 4 °C.
   NOTE: Colonies are ready to be picked when they reach a size > 1 mm in diameter. Colonies should display sharp edges and grow in homogeneous monolayers (Figure 2D).
2. Aspirate the LN-521 solution and add 500 µL of E8 into one well of a 24-well tissue culture plate.
3. Cut colonies into smaller pieces with a scalpel in a grid like pattern under a stereomicroscope. Mechanically scrape the cell sheets from the dish and transfer the sheets using a 200 µL pipette to the prepared well (Figure 2D-2E). Pick colonies into separate wells.
4. Allow cells to attach without changing medium for 48 h. Thereafter, change E8 medium daily.
5. When the colonies have reached a size of > 5 mm, enzymatically passage cells as single cells to a new well of a LN-521 coated plate.
6. Aspirate the cell culture medium from cells and wash with 250 µL of DPBS.
7. Aspirate DPBS. Add 250 µL of dissociation reagent and incubate for 3 min at 37 °C.
8. Observe that the cells have started to round up. Detach cells from the plate by rinsing the cells with dissociation reagent a few times.
9. Add 500 µL of E8 medium to a 15-mL tube. Transfer the cells in the dissociation reagent to the tube and centrifuge for 3 min at 300 x g.
10. Aspirate the LN-521 solution from the LN-521 precoated well and add 500 µL room temperature E8 medium.
11. Aspirate the supernatant and resuspend the cell pellet in 500 µL of E8 medium.
12. Count the cells using a hemocytometer and seed 2.5 x 10⁴ - 5 x 10⁴ cells in the prepared LN-521 precoated well (1.25 x 10⁴ -  2.5 x 10⁴ cells/cm²). Cell culture format can easily be upscaled to suit the intended purpose.
13. Add ROCKi to a final concentration of 10 µM. Incubate cells at 37 °C.
14. Change E8 medium daily. Cells are usually ready to passage after 4 - 6 days. Enzymatically passage cells as single cells as described above when about 90% confluent.
    NOTE: The reprogramming vectors are progressively diluted during hiPS cell expansion and not detectable after passage 12. Cells that are impartially reprogrammed commonly cease to proliferate around passage 6 - 10 or lose their characteristic pluripotent stem cell morphology (Figure 3B).
15. Freezing and thawing of hiPS cells
    1. To freeze cells, enzymatically dissociate cells as single cells as described above, count cells in a hemocytometer and transfer 2.5 x 10⁵ cells to a 15-mL tube containing 1 mL of E8 medium. Centrifuge at 300 x g for 3 min. Aspirate the supernatant.
    2. Resuspend cell pellet in 250 µL of 4 °C PSC cryomedium and transfer to a cryovial. Place the vial in a freezing container and incubate cells at -80 °C for 24 h before transferring them to liquid nitrogen for long term storage.
    3. To thaw cells, prepare a precoated LN-521 well of a 24-well tissue culture plate by aspirating the LN-521 solution and adding 250 µL of E8 medium. Submerge the bottom part of the cryovial in 37 °C water until only a small icicle remains.
    4. Add 250 µL of E8 Medium to the cryovial and transfer the cell-suspension to a 15-mL tube and add 1 mL of E8 medium. Spin cells 300 x g for 3 min.
    5. Remove the supernatant and resuspend the cell pellet in 250 µL of room temperature E8 medium. Transfer the cell suspension to the prepared well and add of 1% cell viability supplement or 10 µM ROCKi. Incubate the tissue culture plate at 37 °C.

Wyniki

From biopsy to hiPS cells

The entire process from biopsy to established hiPS cells, clear of reprogramming vector and ready for characterization, takes approximately 16 weeks (Figure 1). A more detailed timeline is specified in Figure 2A. Approximately 4 weeks is needed to establish and expand fibroblast cultures. The first hiPS cell colonies started to emerge about three-...

Dyskusje

The expected result of this protocol is the successful generation of several clonally derived hiPS cell lines. Importantly, the method for the maintenance of and expansion of established hiPS cells here described is reliable and can be performed with little prior experience of stem cell culture. Enzymatic single cell passaging with ROCKi together with the LN-521 matrix is known to maintain cells as karyotypically normal, pluripotent and readily able to differentiate while avoiding induced heterogeneity that colony based ...

Materiały

Name	Company	Catalog Number	Comments
Dispase	Life Technologies	171105-041	Biopsy digestion
Collagenase	Sigma	C0130	Biopsy digestion
Gelatin	Sigma	G1393	Fibroblast matrix
IMDM	Life Technologies	21980002-032	Fibroblast medium
FBS	Invitrogen	10270106	Fibroblast medium
Penicillin/Streptomycin	Life Technologies	15070-063	Antibiotic
Essential 8 media	ThermFisher Scientific	A1517001	iPS cell culture media
LN-521	Biolamina	LN521-03	iPS cell culture matrix
TrypLE select 1X	ThermoFisher Scientific	12563011	Dissociation reagent
Rho-kinase inhibitor Y27632	Millipore	SCM075	Rho-kinase inhibitor (ROCKi)
CytoTune – iPS 2.0 reprogramming kit	Life Technologies	A1377801	Sendai virus reprogramming vector
35 mm tissue culture dish	Sarstedt	83.3900.300	Cell culture
60 mm tissue culture dishes	Sarstedt	83.3901.300	Cell culture
24 well tissue culture plates	Sarstedt	83.3922.300	Cell culture
T25 tissue culture flasks	VWR	734-2311	Cell culture
15 mL tubes	Corning	430791	Centrifuge tubes
1.5 mL tube	Eppendorf	0030123.301	1.5 mL tube
CoolCell cell LX	Biocision	BCS-405	Freezing container
DMSO	Sigma	D2650-100ml	Fibroblast freezing
Cryovials 1.8 mL	VWR	479-6847	Cryovials
PSC Cryopreservation Kit	Gibco	A2644601	PSC CryomediumRevitaCell supplement
Trypsin-EDTA (0.05%)	ThermoFisherScientific	25300054	Fibroblast dissociation enzyme
DMEM/F12	Life Technologies	31331-028	Digestive enzymes dilutant
DPBS	Life Technologies	14190-094	PBS
HBSS	ThermoFIsher Scientific	14025-050	Biopsy preparation
Haemocytometer	Sigma	BRAND, 718920	Cell counting
Parafilm	VWR	291-1213	Sealing plates for storage