Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

Summary

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

Abstract

The growing number of victims of "stem cell tourism," the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.

External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Introduction

The development of stem cell therapies for tissue regeneration has encountered a number of barriers in the past several decades, hampering efforts for efficient clinical deployment. These hurdles include poor cell retention at sites of delivery, stem cell immunogenicity, and the neoplastic potential to form teratomas¹. Tumorigenicity is of particular clinical concern as it can potentially harm stem cell transplant recipients². Accounts of tumor formation due to unregulated stem cell injections have already been reported in multiple clinical settings^3,4,5. The potential for teratoma formation is the most frequently cited clinical concern in pluripotent stem cell (PSC) development and has resulted in delays and cancellations of multiple high-profile embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) trials^6,7,8,9. Thus, there is a pressing need for a translational investigation dedicated toward providing appropriate treatment, should these iatrogenic tumors arise.

To date, most strategies to control stem cell misbehavior have focused on reducing the number of PSCs with tumorigenic potential^2,10. Unfortunately, only a small number of residual cells (e.g., 1 x 10⁴ to 1 x 10⁵ cells¹¹) is required for teratoma formation, which is far below the detection limit quoted by currently available assays^12,13. Other limitations of using these preseparation methods include low efficiency and high expense, reliance on single-cell suspensions that may not be appropriate for newer tissue-engineering approaches, and the potential impairment of cell survival and engraftment.

Few studies have addressed treatment options following teratoma formation. Perhaps the most well-studied strategy is the incorporation of "suicide" genes into stem cells^14,15. This method involves genetically manipulating the stem cells to incorporate an inducible apoptosis-activating gene that can be activated by pharmacological stimulation postinjection, thereby providing a rescue approach if injected cells produce teratomas. This approach, however, suffers from significant drawbacks, including off-target effects of genetic modifications of PSCs and the potential for a gradual development of drug resistance¹⁶. A similar approach utilizes small molecules to induce selective cell death of PSCs via the inhibition of anti-apoptotic pathways¹⁷. Other groups have targeted cell death of PSCs using antibodies against pluripotency surface markers, such as podocalyxin-like protein-1 (PODXL)¹⁸. The timing of small-molecule or antibody delivery stands to have a significant impact on the therapeutic potential of PSCs if delivered too early and may lack therapeutic efficacy if delivered too late. In addition, the systemic effects of small molecules and antibodies used in this fashion have not been studied.

An alternative approach to treating these tumors relies on using external beam radiation therapy (EBRT). EBRT is one of the primary modalities currently employed in the treatment of solid tumors¹⁹. Innovations in EBRT, including the development of the proton beam and stereotactic radiosurgery, have enabled the enhanced targeting of pathological structures while avoiding damage to normal tissue, making conformal EBRT ideal for addressing teratoma formation in anatomically sensitive structures²⁰. Additionally, this method avoids the genetic manipulation or viral transduction of stem cells, which are both fraught with additional clinical safety and efficacy concerns¹⁵. Finally, advances in micro-irradiators have enabled the application of EBRT in rodents²¹.

In this article, we demonstrate how to create a small-animal model of teratoma formation by injecting human iPSCs in mice. We then show how to apply EBRT to selectively eradicate these tumors in vivo with minimal damage to surrounding tissue. This approach provides a targeted therapy for PSC-derived teratomas while avoiding the off-target effects of the systemic delivery of biological molecules and peptides and the genetic manipulation of the PSCs. For investigational purposes, we offer an optional step to transduce stem cells with reporter genes to track tumor response to radiation therapy via bioluminescence imaging (BLI).

Protocol

This animal experiment was approved and performed under the Institutional Review Board and the Administrative Panel on Laboratory Animal Care at Stanford University.

1. Cell Culture of iPSCs

1. Grow human iPSCs derived by lentiviral reprogramming on 6-well plates coated with basement membrane matrix (e.g., matrigel, referred to as matrix hereon).
2. Daily change the media of the iPSCs with enriched culture medium (see Table of Materials) incubating at 37 °C and 5% CO₂.
3. Once the cells reach 80–90% confluence (approximately every 4 days), add 1 mL of recombinant cell-dissociation enzyme (see Table of Materials) per well and incubate at 37 °C for 5 min.
4. After 5 min, dissociate the cells from the well by pipetting, transfer them to a 15 mL tube, and centrifuge at 300 x g.
5. After the centrifugation, aspirate the supernatant and resuspend the cell pellet into enriched culture medium (see Table of Materials) enriched with Y27632 inhibitor at a 1:1,000 dilution.
6. Perform a cell count of the dissociated cells and replate the cells on matrix-coated 6-well plates at a density of 2 x 10⁵ to 4 x 10⁵.

2. Transduction of iPSCs with a Double-fusion Reporter Gene

1. Passage iPSCs in 6-well plates as per routine and add enriched culture medium (see Table of Materials) containing 6 µg/mL hexadimethrine bromide.
   NOTE: The ideal colony size is 200–400 cells/colony to yield the highest transduction efficiency.
2. Concentrate self-inactivating lentivirus carrying firefly luciferase and green fluorescent protein (FLuc-eGFP) driven by human ubiquitin promotor-C by sediment centrifugation with an SW-29 rotor at 50,000 x g for 2 h at 4 °C.
3. Add the viral concentrate to the iPSCs in a 6-well plate at a multiplicity of infection (MOI) of 10 and incubate overnight at 37 °C at 5% CO₂.
   NOTE: The multiplicity of infection was determined by the expression of monomeric fluorescence protein analyzed by a fluorescence-activated cell sorting (FACS) scan.
4. The following day, remove the virus by centrifugation of the iPSC 6-well plates at 300 x g for 6 min at room temperature.
5. Change the media daily with enriched culture medium (see Table of Materials) and passage as per protocol. Utilize a fluorescence microscope to determine the approximate transduction efficiency for eGFP.
6. An efficiency of 30–40% is sufficient for FACS sorting. Proceed to the FACS of hiPSCs expressing eGFP if at least 30–40% of the cells express eGFP.
7. To confirm FLuc activity ex vivo, plate the cells expressing GFP sorted by FACS at a density of 5,000 cells per well.
8. Incubate the transduced cells and non-transduced cells (which will serve as the negative control) with the bioluminescence reporter probe D-luciferin (100 μmol/L) for 6 h. Measure the bioluminescence with a microplate spectrofluorometer.

3. Transplantation of PSCs in the Dorsal Flank for Teratoma Formation in Immunodeficient Mice

1. Add 1 mL of recombinant cell-dissociation enzyme mix per 6-well plate containing human iPSCs transduced with a double-fusion reporter gene (FLuc-GFP) in culture (see section 2) and incubate for 5 min.
2. After the incubation period, disperse the cells by pipette aspiration and expression. Add an equal volume of culture medium and then centrifuge at 250 x g for 4 min.
3. After centrifugation is complete, aspirate the supernatant solution, resuspend the cell pellet in 30 µL of matrix, and place it on ice to preserve its viability prior to injection. Confirm a harvest of 1 x 10⁶ cells using a hemocytometer.
4. If utilizing double-fusion reporter-gene-transfected cells, suspend the double-positive FACS cells (from section 2) in 30 µL of matrix.
5. Induce anesthesia in 8-10 week athymic nude mice using 2% isoflurance with 1L/min oxygen and then use maintenance 2% isoflurane with 1L/min of oxygen.
6. Using a 28.5 G syringe, inject cell/matrix mixture (see Table of Materials) suspension into the subcutaneous space at the flank, aiming for an injection of in total 5 x 10³ to 5 x 10⁶ cells (approximately 100 ul per injection).
7. Consider a more caudal injection site if planning to maintain mice for prolonged period and anticipating large tumor growth. 
8. Anesthetized animals are allowed to recover on a heated pad until ambulatory (typically <1h) with monitoring of respiratory rate, skin color of toes until ambulatory. 

4. Bioluminescence Imaging (BLI) of Transplanted Cells to Assess Cell Survival and Teratoma Growth

1. At the desired timepoints after inoculation, perform an intraperitoneal (IP) injection of 375 mg/kg of the reporter probe D-Luciferin into the mice.
2. 10 min after an IP injection, image the bioluminescence signal in the anesthetized animals (performed as described in step 3.5) for 30 min using 1 min acquisition windows at 5-min intervals.
   NOTE: Weekly image acquisitions are recommended. Anesthesia is maintained during imaging by delivering inhaled isoflurane via a nose cone.
3. For data analysis, draw a region of interest (ROI) over the BLI signal and, then, normalize for the acquisition time to quantify emissions in units of maximum photons per second per square centimeter per steradian (photons/s/cm²/sr).

5. Teratoma Irradiation Using a Preclinical Image-guided Irradiator (Figure 1)

1. Anesthetize a mouse in a knockdown box using 2% isoflurane in 100% oxygen at a flow rate of 1 L/min. After the mouse is fully anesthetized, transfer it to the bed of an image-guided pre-clinical irradiator (see Table of Materials). Maintain anesthesia by 2% isoflurane continuously via a nose cone.
2. Acquire micro-CT images as a set of 400 projection images over 360° using a 40 kVp, 2 mA X-ray beam, and reconstruct those into volumetric images with an isotropic pixel size of 0.2 mm.
3. Plan a radiation treatment using the micro-CT images using the RT_Image software package (http://rtimage.sourceforge.net/) and perform the treatment.
   NOTE: The treatment plan used consists of two 225 kVp X-ray beams, oriented to pass through the superficial target teratoma while skirting the surface of the rest of the mouse and sparing the underlying viscera. The exposure times for the beams are adjusted based on quarterly system calibration data so that the dose at the center of the target tumor was 6 Gy.
4. Repeat the treatment process on three consecutive days to deliver a total of 18 Gy to the target tumor.
5. Maintain standard post-treatment care of the animals.

Results

Injected mice typically will demonstrate teratoma growth formation after 4–8 weeks as confirmed by BLI imaging (Figure 2). Tumors will shrink dramatically when irradiated with a cumulative dose of 18 Gy given one month after cell delivery, resulting in a significant decrease in luciferase signal (Figure 2). Importantly, normal tissues taken 5 mm from the irradiated site do not appear to have any significant damage (

Discussion

Preclinical data and anecdotal cases from victims of "stem cell tourism" confirm that the risk of developing teratomas is a serious drawback associated with PSC treatments²³. Development of careful approaches to prevent and treat the neoplastic risk associated with stem cell therapies is, therefore, an important step in facilitating the clinical translation of regenerative stem cell therapies. In this article, we described a method of therapeutic targeting of PSC-associated teratomas using...

Materials

Name	Company	Catalog Number	Comments
Induced Pluripotent Stem Cell Control Line	Stanford University	Nguyen Lab	Cell culture of iPSC
Corning matrigel basement membrane matrix 354234	Fisher Scientific	CB-40234	Cell culture of iPSC
Essential 8 culture medium	ATCC-The global bioresource center	30-2203	Cell culture of iPSC
Tryple E	Gibco	12605-036	Cell culture of iPSC
Y27632 inhibitor 2 HCL (ROCK Inhibitor)	Fisher Scientific	S104950MG	Cell culture of iPSC
Lentivirus	Cyagen	P170721-1001cjn	Transduction of iPSC with double fusion reporter gene
Polyrbrene Infection/Transfection Reagent	Millipore Sigma	TR-1003-G	Transduction of iPSC with double fusion reporter gene
Fluc-eGFP reporter gene driven by ubiquitin promoter	Stanford University	Sam Gambhir lab	Transduction of iPSC with double fusion reporter gene
D-luciferin	Perkin Elmer	122799	Transduction of iPSC with double fusion reporter gene and BLI
Flow cytometer (BD FACSARIA III)	BD Biosciences	FACSAria	Transduction of iPSC with double fusion reporter gene
microplate spectrofluorometer (Glomax Navigator System)	Promega Bio Systems, Sunnyvale, CA	GM2000	Transduction of iPSC with double fusion reporter gene
Xenogen IVIS 200	Perkin Elmer	124262	BLI
Isoflurane	Sigma-Aldrich	CDS019936	irradiation
X-Rad SmART image-guided irradiator	Precision X-ray Inc., North Branford, CT	X-Rad SmART	irradiation
RT_Image software package	Stanford University (http://rtimage.sourceforge.net/)	RT_Image v0.2β	Irradiation