Immortalization Reversibility in the Context of Cell Therapy Biosafety
Abstract
:1. Introduction
2. Immortalization of Cells
3. Reversible Immortalization: Approaches and Tools
3.1. Conditional Immortalization
3.1.1. Temperature-Dependent Reversible Immortalization
3.1.2. Tamoxifen-Dependent Reversible Immortalization
3.1.3. Tetracycline-Dependent Reversible Immortalization
3.2. Site-Specific Recombination
3.3. Small Interfering RNA-Mediated Immortalizing Gene Silence
4. Reversible Immortalization: Possible Risks and Ways to Avoid Them
4.1. De-Immortalization Efficiency: Ways to Increase and Control
4.1.1. Choosing a De-Immortalization Approach
4.1.2. Tamoxifen-Mediated Self-Recombination: De-immortalization Efficiency Increase
4.1.3. Gene Excision Control
4.2. Genetic Instability in Reversibly Immortalized Cells: Reasons and Potential Solutions
4.2.1. Process-Associated Genetic Instability
- Genetic instability associated with the immortalization–de-mmortalization process
- Genetic instability associated with immortalizing genes effects
- Genetic instability associated with culture conditions
4.2.2. Reduction of Genetic Instability
- The choice of immortalization–de-immortalization strategy and immortalizing agents
- Modification of culture conditions
5. The Need for a Tool Guaranteeing the Biosafety of Cells after Transplantation and a Promising Solution
6. Conclusions
Funding
Conflicts of Interest
References
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---|---|---|---|---|---|
1 | Human neural stem cells | hCTX0E03 (CTX) | Current clinical trial in patients with chronic ischemic stroke (NCT01151124, NCT03629275) | Positive therapeutic effect (upper extremity functionality enhancement) in stroke recovery | [16,17] |
Mice model, MCAo rat model | Treatment with CTX0E03 promotes angiogenesis | [18] | |||
Quinolinic acid-lesioned rat model of Huntington’s disease | CTX0E03 differentiate into MSNs and GABAergic neurons in the striatum; increase endogenous neurogenesis | [19] | |||
2 | Neural stem cells from human fetal spinal cord tissue | SPC-01 | Rat model, balloon-induced SCI rat model | Functional recovery after spinal cord injury: differentiate into relevant ventral neuronal subtypes, reduce inflammation, reduce glial scar formation | [20,21,22] |
3 | Hippocampal mouse cells | MHP36 | Rat model: old animals, induced brain ischemic damage | Transplanted cells repopulate hippocampal fields and areas of the cortex, differentiate into both neurons and astrocytes. Recovery of the cognitive function | [23,24,25] |
4 | Primary human olfactory ensheathing glia (OEG) cells | hTL4, Ts14 | Rat model, induced spinal cord injury | Neuroregenerative effect: axonal regeneration of adult retinal ganglion neurons | [26,27,28] |
5 | Murine astrocytes (modified, GABA producing) | BASlin65 | Rat model of parkinsonian tremor | Parkinson’s disease treatment. Reduce tremulous movements | [29,30,31] |
6 | Embryonic auditory neuroblasts | US ⁄ VOT-N33 | Rat model | Replacement of auditory sensory neurons. Auditory brainstem response increase | [32,33] |
7 | Human fetal retinal progenitor cells | GuRt09 | Neonatal hooded Lister rats, RCS dystrophic rats | Therapeutic potential in degenerative retinal diseases | [34] |
8 | Murine muscle progenitors (modified: transfer of human artificial chromosomes with dystrophin locus) | – | NSG mice | Duchenne muscular dystrophy gene therapy | [35] |
9 | Primary myoblasts | H2K 2B4 | Immunodeficient dystrophin-deficient mice | Cells differentiate forming myotubes with a myogenic protein profile, regenerate host muscle | [36] |
10 | Primary neonatal rat cardiomyocytes | – | in vitro | Potential strategy for providing cardiomyocytes for cell therapy | [37] |
11 | Human pancreatic beta cells | NAKT-15 | Streptozotocin-induced diabetic severe combined immunodeficient mice | Usage as a component of an implantable bioartificial pancreas. Control of blood glucose | [38,39] |
12 | Canine fetal pancreatic beta cells | ACT-164 | Streptozotocin-induced diabetic SCID mice | Glucose dependent insulin production. Reversed chemically induced diabetes in SCID mice model | [40] |
13 | Murine pancreatic beta cells | – | Streptozotocin-induced diabetic mice model | Cells produce and secrete high amounts of insulin. Restore and maintain euglycemia | [41] |
14 | Human hepatocytes (modified, insulin-producing) | YOCK-13 | Immunosuppressed totally pancreatectomized diabetic pig | Glucose dependent insulin production. Reversed chemically induced diabetes in SCID mice model | [42] |
15 | Human hepatocytes | 16T-3 | Pig model of acute liver failure | Survival prolongation | [43] |
16 | Human hepatocytes | NKNT-3 | Rat model of acute liver failure | Usage as a component of the biohybrid artificial liver or for transplantation into the spleen. Improvement of biochemical parameters, survival increase | [44,45,46] |
17 | Murine fetal hepatic progenitor cells | iHPC | Recellularization the decellularized liver scaffolds in vitro | Cells efficiently recellularize decellularized liver scaffolds | [47] |
18 | Murine embryonic hepatic progenitors | HP14-19-CD | Nude mice, CCl4-induced acute liver injury model | Repair liver biochemical index and structure | [8] |
19 | Embryonic rat adrenal cells Bovine adrenal cells | RAD5.2 BADA.20 | In vitro | Potential application in the treatment of chronic pain | [48] |
20 | Human renal proximal tubule epithelial cells | ciPTECs | In vitro, athymic nude rats | Albumin and phosphate reabsorption. Potential application as a cell component of bio-artificial kidney or in bioengineered renal tubules | [49,50,51] |
21 | Murine keratinocytes | iKera | Mouse skin injury model | Using cells embedded in citrate-based scaffold. Cutaneous wound reepithelialization and healing | [52] |
22 | Murine epidermal melanocytes | iMC | Athymic mice | Melanin production in vivo | [53] |
23 | Murine bone marrow stromal stem cells | imBMSCs | In vitro; nude mice, subcutaneous imBMSCs implantation | [54] | |
24 | Human infrapatellar fat pad-derived stem cells | – | In vitro | Increase of proliferative, chondrogenic, and adipogenic abilities of IPFSCs during serial passaging | [12] |
25 | Murine embryonic fibroblasts (mesenchymal stem cells, MSCs) | piMEF | In vitro; nude mice, subcutaneous MSCs implantation | Osteogenic, chondrogenic, and adipogenic differentiation in vitro and in vivo, matrix mineralization in vitro | [55] |
26 | Primary mouse Achilles tenocytes | iMAT | In vitro | Potential application for tendon repair | [56] |
27 | Primary fibroblasts from adult mice | – | In vitro, Injection of iPSCs into SCID mice | Retaining reprogramming (into iPSCs) potential upon serial passaging | [57] |
No. | De-Immortalization Approach | Strategy | Cell Types and Immortogenes | Clinical Applications Listed in: | References |
---|---|---|---|---|---|
Immortalization reversal mediated by changing conditions | |||||
1 | Temperature-dependent immortalization reversal | tsA58 SV40T immortogene (temperature-sensitive mutant allele) inactivation at body-specific temperature 37 °C (active at 33 °C cultural conditions) | tsA58 SV40T-transducted cells: | Table 1, No. 5, 7, 19, 20 | [29,30,31,34,48,49,50,51] |
human fetal retinal progenitor cells | |||||
murine astrocytic transgenic cell line BAS8.1 | |||||
embryonic rat adrenal cells | |||||
bovine adrenal cells | |||||
human renal proximal tubule epithelial cells | |||||
Cells from H-2Kb-tsA58 transgenic mouse | Table 1, No. 3, 6, 9 | [23,24,25,32,33,36,80,81,82] | |||
hippocampal cells | |||||
embryonic auditory neuroblasts | |||||
myoblasts | |||||
alveolar type II cells | |||||
Kupfer cells | |||||
cardiac endothelial cells | |||||
2 | Tamoxifen-dependent immortalization reversal | c-MycER (or v-MycER conjugates of c-Myc, v-Myc oncoproteins and a modified mouse estradiol receptor) inactivation at 4-OHT absence | c-MycERTAM-immortalized: | Table 1, No. 1, 2 | [16,17,18,19,20,21,22,83,84,85] |
human neural stem cells | |||||
neural stem cells from human fetal spinal cord tissue | |||||
neural stem cells from human fetal spinal cord tissue | |||||
human late-adherent olfactory mucosa cells | |||||
human embryonic stem cells | |||||
human hepatocytes | |||||
3 | Tetracycline-dependent reversible immortalization: | SV40T-immortalized murine pancreatic beta cells | Table 1, No. 13 | [41,86,87,88] | |
Silencing of immortogene expression at tetracycline (or doxycycline) absence | SV40T-immortalized granulosa porcine cells | ||||
TeT-On | Silencing of immortogene expression by adding of tetracycline (or doxycycline) | SV40T-immortalized human conjunctival epithelial cells | |||
hTERT-immortalized human bone mesenchymal stromal cells | [89] | ||||
TeT-Off | v-Myc-immortalized neuronal Rat progenitor cells | ||||
Immortalization reversal mediated by immortalizing gene excision | |||||
4 | FLT-FRP-mediated gene excision | Immortogene is flanked by FRP sites, site-specific recombination upon Ad-FLP transfection | SV40T (SSR#41)-immortalized murine epidermal melanocytes | Table 1, No. 22, 23, 25, 26 | [53,54,55,56] |
SV40T (SSR#41)-murine bone marrow stromal stem cells | |||||
SV40T CRISPR/Cas9-mediated immortalized murine bone marrow stromal stem cells | |||||
SV40T PiggyBac transposon-mediated immortalized murine embryonic fibroblasts (mesenchymal stem cells) | |||||
SV40T PiggyBac transposon-mediated immortalized primary mouse Achilles tenocytes | |||||
5 | Cre-LoxP-mediated gene excision | Immortogene is flanked by LoxP sites, site-specific recombination upon Ad-Cre transfection | hTERT+BMI1-, SV40T- immortalized primary human olfactory ensheathing glia | Table 1, No. 4, 10, 11, 12 | [26,27,28,37,40] |
hTERT+BMI1-, SV40T- immortalized primary neonatal rat cardiomyocytes | |||||
SV40T-immortalized canine fetal pancreatic beta cells | |||||
6 | Small interfering RNA-mediated gene silence | Knockdown the expression of immortogene in a sequence-specific way by mediating targeted mRNA degradation | SV40T-immortalized mouse keratinocytes | Table 1, No. 21 | [52,90] |
siRNA-SV40T and siRNA-hTERT | SV40T + hTERT-immortalized human pancreatic beta cells |
Process Stage | Key Recommendations |
---|---|
Immortalization | Avoid the use of SV40T, E6/E7 HPV16 as immortogenes. Avoid using non-site-specific retroviral-mediated immortogene insertion. Give preference to more selectively PiggyBac- or (the best option) CRISPR/Cas9-mediated insertion. If it is enough to get the effect, use decellularized extracellular matrix (dECM) to mediate immortalization (suitable for stem cells including MSCs). |
Cultivation | Use CO2 level selected individually for particular cell type. If it is technically possible, use dECM during culturing. |
De-immortalization | Give preference to a de-immortalization strategy involving immortogene reduction followed by selection of successfully de-immortalized cells. Use PiggiBac-FLT-FRT combination for seamless immortogene removal or use CRISPR/Cas9-mediated site-spesific immortogene insertion preliminary to reduce significance of “footprint” mutations. |
Pre-transplantation control | Pay particular attention to the presence of mutations in p16INK4a/pRb and p53 signaling pathways. |
After transplantation | Transplanted (or used in bio-artificial organs) cells must carry a suicide gene to have an opportunity to selectively kill the cells if malignant transformation is suspected. |
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Sutyagina, O.I.; Beilin, A.K.; Vorotelyak, E.A.; Vasiliev, A.V. Immortalization Reversibility in the Context of Cell Therapy Biosafety. Int. J. Mol. Sci. 2023, 24, 7738. https://doi.org/10.3390/ijms24097738
Sutyagina OI, Beilin AK, Vorotelyak EA, Vasiliev AV. Immortalization Reversibility in the Context of Cell Therapy Biosafety. International Journal of Molecular Sciences. 2023; 24(9):7738. https://doi.org/10.3390/ijms24097738
Chicago/Turabian StyleSutyagina, Oksana I., Arkadii K. Beilin, Ekaterina A. Vorotelyak, and Andrey V. Vasiliev. 2023. "Immortalization Reversibility in the Context of Cell Therapy Biosafety" International Journal of Molecular Sciences 24, no. 9: 7738. https://doi.org/10.3390/ijms24097738