Transduction of Human Fetal Liver Hematopoietic CD34+ Stem and Progenitor Cells into a Cell Line by Enhancing Telomerase Activity
Human Fetal Liver Hematopoietic Stem Cell Line
-
Rashmi Bhardwaj
,
Lalit Kumar
,
Deepika Chhabra
,
Atul Sharma
,
Sujata Mohanty
,
Narinder Mehra
,
Vinod Kochupillai
Abstract
Background: Human fetal liver hematopoietic stem cells have proven potential as therapeutics but lack extensive research due to their limited supply. Even in vitro expanded fetal liver hematopoietic stem cells enter senescence or lose their self-renewal capacity after a few days in culture. The present study aimed to obtain a homogeneous and persistent supply of hematopoietic stem cells from the fetal liver by establishing a cell line through immortalization of cells by enhancing telomerase activity.
Materials and Methods: Human fetal liver hematopoietic CD34+ stem and progenitor cells were transformed and immortalized using retroviruses carrying the human telomerase (hTERT) gene. Following transduction, telomerase activity was assessed using the TRAP assay and telomere length was examined by Southern blotting in transduced cells. Their characterization was conducted using flowcytometry to analyze the CD34+ population of hematopoietic stem cells and their colony forming potential using colony forming unit (CFU) assay.
Results: After transduction with hTERT, the life span of human fetal liver hematopoietic CD34+ stem and progenitor cells was extended to 80 population doublings, without any change in cell morphology or population doubling times. Constitutive hTERT expression enhanced the replicative capacity and prevented terminal differentiation of CD34+ fetal liver hematopoietic stem and progenitor cells (FLHSPCs). Moreover, hTERT-transduced stem cells maintained their telomere length and telomerase activity.
Conclusion: By introducing telomerase activity into hematopoietic stem and progenitor cells, their lifespan can be extended while maintaining stemness. These modified cells hold promise for in vitro research focused on studying hematopoietic stem cells derived from fetal liver.
1. Hou S, Liu C, Yao Y, et al. Hematopoietic Stem Cell Development in Mammalian Embryos. Adv Exp Med Biol. 2023;1442:1-16.
2. Kansal V, Sood SK, Batra AK, et al. Fetal Liver Transplantation in Aplastic Anemia. Acta Haematol 1979;62(3):128–36.
3. Kaushansky K. Thrombopoietin: the primary regulator of megakaryocyte and platelet production. Thromb Haemost. 1995;74(1):521–5.
4. Kochupillai V, Sharma S, Francis S, et al. Bone marrow reconstitution following human fetal liver infusion (FLI) in sixteen severe aplastic anemia patients. Prog Clin Biol Res. 1985;193:251–65.
5. Izzi T, Polchi P, Galimberti M, et al. Fetal liver transplant in aplastic anemia and acute leukemia. Prog Clin Biol Res. 1985;193:237–49.
6. Gale RP. Fetal liver transplantation in aplastic anemia and leukemia. Thymus. 1987;10(1-2):89–94.
7. Kochupillai V, Sharma S, Francis S, et al. Fetal liver infusion in aplastic anaemia.Thymus.1987;10(1-2):95-102.
8. Kochupillai V, Sharma S, Sundaram KR, et al. Hemopoietic improvement following fetal liver infusion in aplastic anemia. Eur J Haematol. 1991;47(5):319–25.
9. Bhardwaj R, Kumar L, Chhabra D, et al. In vitro expansion of fetal liver hematopoietic stem cells. Sci Rep. 2021;11(1):11879.
10. Allain JE, Dagher I, Mahieu-Caputo D, et al. Immortalization of a primate bipotent epithelial liver stem cell. Proc Natl Acad Sci U S A. 2002;99(6):3639–44.
11. Blackburn EH, Greider CW, Henderson E, et al. Recognition and elongation of telomeres by telomerase. Genome. 1989;31(2):553–60.
12. Stampfer MR, Bartley JC. Induction of transformation and continuous cell lines from normal human mammary epithelial cells after exposure to benzo[a]pyrene. Proc Natl Acad Sci U S A. 1985;82(8):2394–8.
13. Suemori H. Establishment and therapeutic use of human embryonic stem cell lines. Hum Cell. 2006;19(2):65–70.
14. Sullivan CS, Pipas JM. T Antigens of Simian Virus 40: Molecular Chaperones for Viral Replication and Tumorigenesis. Microbiol Mol Biol Rev. 2002;66(2):179-202.
15. Toouli CD, Huschtscha LI, Neumann AA, et al. Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit. Oncogene. 2002;21(1):128-39.
16. de Lange T. How Telomeres Solve the End-Protection Problem. Science. 2009;326(5955):948-52.
17. Griffith JD, Comeau L, Rosenfield S, et al. Mammalian Telomeres End in a Large Duplex Loop. Cell. 1999;97(4):503–14.
18. Allsopp RC, Weissman IL. Replicative senescence of hematopoietic stem cells during serial transplantation: does telomere shortening play a role? Oncogene. 2002;21(21):3270–3.
19. Harley CB. Telomerase and cancer therapeutics. Nat Rev Cancer. 2008;8(3):167–79.
20. Egorov EE, Terekhov SM, Vishniakova KS, et al. Telomerization as a method of obtaining immortal human cells preserving normal properties. Ontogenez. 2003;34(3):183–92.
21. Wege H, Le HT, Chui MS, et al. Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential. Gastroenterology. 2003;124(2):432–44.
22. Liu TM, Ng WM, Tan HS, et al. Molecular Basis of Immortalization of Human Mesenchymal Stem Cells by Combination of p53 Knockdown and Human Telomerase Reverse Transcriptase Overexpression. Stem Cells Dev. 2013;22(2):268–78.
23. Weinert TA, Hartwell LH. The RAD9 Gene Controls the Cell Cycle Response to DNA Damage in Saccharomyces cerevisiae. Science. 1988;241(4863):317–22.
24. Li S, Ferguson MJ, Hawkins CJ, et al. Human telomerase reverse transcriptase protects hematopoietic progenitor TF-1 cells from death and quiescence induced by cytokine withdrawal. Leukemia. 2006;20(7):1270–8.
25. Lynch CD, Zhang J. The research implications of the selection of a gestational age estimation method. Paediatr Perinat Epidemiol. 2007;21 Suppl 2:86–96.
26. Larijani B, Aghayan H-R, Goodarzi P, et al. GMP-grade human fetal liver-derived mesenchymal stem cells for clinical transplantation. Methods Mol Biol. 2015;1283:123–36.
27. Fathi E, Mesbah-Namin SA, Vietor I, et al. Mesenchymal stem cells cause induction of granulocyte differentiation of rat bone marrow C-kit+ hematopoietic stem cells through JAK3/STAT3, ERK, and PI3K signaling pathways. Iran J Basic Med Sci. 2022;25(10):1222–7.
28. Rigg RJ, Chen J, Dando JS, et al. A Novel Human Amphotropic Packaging Cell Line: High Titer, Complement Resistance, and Improved Safety. Virology. 1996;218(1):290–5.
29. Qu J, Yang Z. Protocol to produce high-titer retrovirus for transduction of mouse bone marrow cells. STAR Protoc. 2021;2(2):100459.
30. Islas-Lugo F, Vega-Estrada J, Alvis CA, et al. Developing strategies to increase plasmid DNA production in Escherichia coli DH5α using batch culture. J Biotechnol. 2016;233:66–73.
31. Zhang S, Cahalan MD. Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit. J Vis Exp. 2007; (6):247.
32. Peng L, Xiong W, Cai Y, et al. A simple, rapid method for evaluation of transfection efficiency based on fluorescent dye. Bioengineered. 2017;8(3):225–31.
33. Lai SR, Andrews LG, Tollefsbol TO. hTERT Knockdown in Human Embryonic Kidney Cells Using Double-Stranded RNA. Methods Mol Biol. 2007:405:23-9.
34. Ichim C v, Wells RA. Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation. J Transl Med. 2011;9:137.
35. El-Badawy A, Ghoneim NI, Nasr MA, et al. Telomerase reverse transcriptase coordinates with the epithelial-to-mesenchymal transition through a feedback loop to define properties of breast cancer stem cells. Biol Open. 2018; 7(7): bio034181.
36. Bhardwaj R, Kumar L, Chhabra D, et al. Effect of fetal liver condition media derived cytokines(IL-6 and Flt-3) on human bone marrow stem cells colony formation. Cytokine. 2022;153:155863.
37. Pamphilon D, Selogie E, McKenna D, et al. Current practices and prospects for standardization of the hematopoietic colony-forming unit assay: a report by the cellular therapy team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative. Cytotherapy. 2013;15(3):255–62.
38. Mariani E. Different rates of telomere shortening and telomerase activity reduction in CD8 T and CD16 NK lymphocytes with ageing. Exp Gerontol. 2003;38(6):653–9.
39. Mender I, Shay J. Telomerase Repeated Amplification Protocol (TRAP). Bio Protoc. 2015;5(22): e1657.
40. Herbert BS, Hochreiter AE, Wright WE, et al. Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol. Nat Protoc. 2006;1(3):1583–90.
41. Ropio J, Chebly A, Ferrer J, et al. Reliable blood cancer cells’ telomere length evaluation by qPCR. Cancer Med. 2020;9(9):3153–62.
42. Wang F, Pan X, Kalmbach K, et al. Robust measurement of telomere length in single cells. Proc Natl Acad Sci U S A. 2013;110(21):E1906-12.
43. Barczak W, Rozwadowska N, Romaniuk A, et al. Telomere length assessment in leukocytes presents potential diagnostic value in patients with breast cancer. Oncol Lett. 2016;11(3):2305–9.
44. Gilboa E. Retroviral gene transfer: applications to human therapy. Prog Clin Biol Res. 1990:352:301-11.
45. Clapp D, Dumenco L, Hatzoglou M, et al. Fetal liver hematopoietic stem cells as a target for in utero retroviral gene transfer. Blood. 1991;78(4):1132–9.
46. Miller AD, Rosman GJ. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989;7(9):980–2, 984-6, 989-90.
47. Goldman FD, Cowan KR, Tawfik DS, et al. Transduction of hTR into Hematopoietic Stem Cells Increases Telomerase Activity. Blood. 2008;112(11):4624.
48. Kim JH, Choi SC, Park CY, et al. Transplantation of Immortalized CD34+ and CD34- Adipose-Derived Stem Cells Improve Cardiac Function and Mitigate Systemic Pro-Inflammatory Responses. PLoS One. 2016;11(2):e0147853.
49. Iacomi DM, Rosca AM, Tutuianu R, et al. Generation of an Immortalized Human Adipose-Derived Mesenchymal Stromal Cell Line Suitable for Wound Healing Therapy. Int J Mol Sci. 2022;23(16):8925.
50. Holt SE, Wright WE, Shay JW. Regulation of telomerase activity in immortal cell lines. Mol Cell Biol. 1996;16(6):2932–9.
51. Engelhardt M, Kumar R, Albanell J, et al. Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells. Blood. 1997;90(1):182–93.
52. Yadav P, Vats R, Wadhwa S, et al. Enhancing Proliferation of Stem Cells from Human Exfoliated Deciduous Teeth (SHED) through hTERT Expression while Preserving Stemness and Multipotency. Stem Cell Rev Rep. 2024. Online ahead of print.
53. Zhao Q, Wang XY, Yu XX, et al. Expression of human telomerase reverse transcriptase mediates the senescence of mesenchymal stem cells through the PI3K/AKT signaling pathway. Int J Mol Med. 2015;36(3):857–64.
54. Raval A, Behbehani GK, Nguyen LXT, et al. Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice. PLoS One. 2015;10(7):e0131722.
55. Chiu C, Dragowska W, Kim NW, et al. Differential Expression of Telomerase Activity in Hematopoietic Progenitors from Adult Human Bone Marrow. Stem Cells. 1996;14(2):239–48.
56. Vaziri H, Benchimol S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr Biol. 1998; 8(5):279–82.
57. Zhu Y, Liu X, Ding X, et al. Telomere and its role in the aging pathways: telomere shortening, cell senescence and mitochondria dysfunction. Biogerontology. 2019;20(1):1–16.
58. Kong CM, Lee XW, Wang X. Telomere shortening in human diseases. FEBS J. 2013;280(14):3180–93.
59. Masutomi K, Possemato R, Wong JMY, et al. The telomerase reverse transcriptase regulates chromatin state and DNA damage responses. Proc Natl Acad Sci U S A. 2005;102(23):8222-7.
2. Kansal V, Sood SK, Batra AK, et al. Fetal Liver Transplantation in Aplastic Anemia. Acta Haematol 1979;62(3):128–36.
3. Kaushansky K. Thrombopoietin: the primary regulator of megakaryocyte and platelet production. Thromb Haemost. 1995;74(1):521–5.
4. Kochupillai V, Sharma S, Francis S, et al. Bone marrow reconstitution following human fetal liver infusion (FLI) in sixteen severe aplastic anemia patients. Prog Clin Biol Res. 1985;193:251–65.
5. Izzi T, Polchi P, Galimberti M, et al. Fetal liver transplant in aplastic anemia and acute leukemia. Prog Clin Biol Res. 1985;193:237–49.
6. Gale RP. Fetal liver transplantation in aplastic anemia and leukemia. Thymus. 1987;10(1-2):89–94.
7. Kochupillai V, Sharma S, Francis S, et al. Fetal liver infusion in aplastic anaemia.Thymus.1987;10(1-2):95-102.
8. Kochupillai V, Sharma S, Sundaram KR, et al. Hemopoietic improvement following fetal liver infusion in aplastic anemia. Eur J Haematol. 1991;47(5):319–25.
9. Bhardwaj R, Kumar L, Chhabra D, et al. In vitro expansion of fetal liver hematopoietic stem cells. Sci Rep. 2021;11(1):11879.
10. Allain JE, Dagher I, Mahieu-Caputo D, et al. Immortalization of a primate bipotent epithelial liver stem cell. Proc Natl Acad Sci U S A. 2002;99(6):3639–44.
11. Blackburn EH, Greider CW, Henderson E, et al. Recognition and elongation of telomeres by telomerase. Genome. 1989;31(2):553–60.
12. Stampfer MR, Bartley JC. Induction of transformation and continuous cell lines from normal human mammary epithelial cells after exposure to benzo[a]pyrene. Proc Natl Acad Sci U S A. 1985;82(8):2394–8.
13. Suemori H. Establishment and therapeutic use of human embryonic stem cell lines. Hum Cell. 2006;19(2):65–70.
14. Sullivan CS, Pipas JM. T Antigens of Simian Virus 40: Molecular Chaperones for Viral Replication and Tumorigenesis. Microbiol Mol Biol Rev. 2002;66(2):179-202.
15. Toouli CD, Huschtscha LI, Neumann AA, et al. Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit. Oncogene. 2002;21(1):128-39.
16. de Lange T. How Telomeres Solve the End-Protection Problem. Science. 2009;326(5955):948-52.
17. Griffith JD, Comeau L, Rosenfield S, et al. Mammalian Telomeres End in a Large Duplex Loop. Cell. 1999;97(4):503–14.
18. Allsopp RC, Weissman IL. Replicative senescence of hematopoietic stem cells during serial transplantation: does telomere shortening play a role? Oncogene. 2002;21(21):3270–3.
19. Harley CB. Telomerase and cancer therapeutics. Nat Rev Cancer. 2008;8(3):167–79.
20. Egorov EE, Terekhov SM, Vishniakova KS, et al. Telomerization as a method of obtaining immortal human cells preserving normal properties. Ontogenez. 2003;34(3):183–92.
21. Wege H, Le HT, Chui MS, et al. Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential. Gastroenterology. 2003;124(2):432–44.
22. Liu TM, Ng WM, Tan HS, et al. Molecular Basis of Immortalization of Human Mesenchymal Stem Cells by Combination of p53 Knockdown and Human Telomerase Reverse Transcriptase Overexpression. Stem Cells Dev. 2013;22(2):268–78.
23. Weinert TA, Hartwell LH. The RAD9 Gene Controls the Cell Cycle Response to DNA Damage in Saccharomyces cerevisiae. Science. 1988;241(4863):317–22.
24. Li S, Ferguson MJ, Hawkins CJ, et al. Human telomerase reverse transcriptase protects hematopoietic progenitor TF-1 cells from death and quiescence induced by cytokine withdrawal. Leukemia. 2006;20(7):1270–8.
25. Lynch CD, Zhang J. The research implications of the selection of a gestational age estimation method. Paediatr Perinat Epidemiol. 2007;21 Suppl 2:86–96.
26. Larijani B, Aghayan H-R, Goodarzi P, et al. GMP-grade human fetal liver-derived mesenchymal stem cells for clinical transplantation. Methods Mol Biol. 2015;1283:123–36.
27. Fathi E, Mesbah-Namin SA, Vietor I, et al. Mesenchymal stem cells cause induction of granulocyte differentiation of rat bone marrow C-kit+ hematopoietic stem cells through JAK3/STAT3, ERK, and PI3K signaling pathways. Iran J Basic Med Sci. 2022;25(10):1222–7.
28. Rigg RJ, Chen J, Dando JS, et al. A Novel Human Amphotropic Packaging Cell Line: High Titer, Complement Resistance, and Improved Safety. Virology. 1996;218(1):290–5.
29. Qu J, Yang Z. Protocol to produce high-titer retrovirus for transduction of mouse bone marrow cells. STAR Protoc. 2021;2(2):100459.
30. Islas-Lugo F, Vega-Estrada J, Alvis CA, et al. Developing strategies to increase plasmid DNA production in Escherichia coli DH5α using batch culture. J Biotechnol. 2016;233:66–73.
31. Zhang S, Cahalan MD. Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit. J Vis Exp. 2007; (6):247.
32. Peng L, Xiong W, Cai Y, et al. A simple, rapid method for evaluation of transfection efficiency based on fluorescent dye. Bioengineered. 2017;8(3):225–31.
33. Lai SR, Andrews LG, Tollefsbol TO. hTERT Knockdown in Human Embryonic Kidney Cells Using Double-Stranded RNA. Methods Mol Biol. 2007:405:23-9.
34. Ichim C v, Wells RA. Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation. J Transl Med. 2011;9:137.
35. El-Badawy A, Ghoneim NI, Nasr MA, et al. Telomerase reverse transcriptase coordinates with the epithelial-to-mesenchymal transition through a feedback loop to define properties of breast cancer stem cells. Biol Open. 2018; 7(7): bio034181.
36. Bhardwaj R, Kumar L, Chhabra D, et al. Effect of fetal liver condition media derived cytokines(IL-6 and Flt-3) on human bone marrow stem cells colony formation. Cytokine. 2022;153:155863.
37. Pamphilon D, Selogie E, McKenna D, et al. Current practices and prospects for standardization of the hematopoietic colony-forming unit assay: a report by the cellular therapy team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative. Cytotherapy. 2013;15(3):255–62.
38. Mariani E. Different rates of telomere shortening and telomerase activity reduction in CD8 T and CD16 NK lymphocytes with ageing. Exp Gerontol. 2003;38(6):653–9.
39. Mender I, Shay J. Telomerase Repeated Amplification Protocol (TRAP). Bio Protoc. 2015;5(22): e1657.
40. Herbert BS, Hochreiter AE, Wright WE, et al. Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol. Nat Protoc. 2006;1(3):1583–90.
41. Ropio J, Chebly A, Ferrer J, et al. Reliable blood cancer cells’ telomere length evaluation by qPCR. Cancer Med. 2020;9(9):3153–62.
42. Wang F, Pan X, Kalmbach K, et al. Robust measurement of telomere length in single cells. Proc Natl Acad Sci U S A. 2013;110(21):E1906-12.
43. Barczak W, Rozwadowska N, Romaniuk A, et al. Telomere length assessment in leukocytes presents potential diagnostic value in patients with breast cancer. Oncol Lett. 2016;11(3):2305–9.
44. Gilboa E. Retroviral gene transfer: applications to human therapy. Prog Clin Biol Res. 1990:352:301-11.
45. Clapp D, Dumenco L, Hatzoglou M, et al. Fetal liver hematopoietic stem cells as a target for in utero retroviral gene transfer. Blood. 1991;78(4):1132–9.
46. Miller AD, Rosman GJ. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989;7(9):980–2, 984-6, 989-90.
47. Goldman FD, Cowan KR, Tawfik DS, et al. Transduction of hTR into Hematopoietic Stem Cells Increases Telomerase Activity. Blood. 2008;112(11):4624.
48. Kim JH, Choi SC, Park CY, et al. Transplantation of Immortalized CD34+ and CD34- Adipose-Derived Stem Cells Improve Cardiac Function and Mitigate Systemic Pro-Inflammatory Responses. PLoS One. 2016;11(2):e0147853.
49. Iacomi DM, Rosca AM, Tutuianu R, et al. Generation of an Immortalized Human Adipose-Derived Mesenchymal Stromal Cell Line Suitable for Wound Healing Therapy. Int J Mol Sci. 2022;23(16):8925.
50. Holt SE, Wright WE, Shay JW. Regulation of telomerase activity in immortal cell lines. Mol Cell Biol. 1996;16(6):2932–9.
51. Engelhardt M, Kumar R, Albanell J, et al. Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells. Blood. 1997;90(1):182–93.
52. Yadav P, Vats R, Wadhwa S, et al. Enhancing Proliferation of Stem Cells from Human Exfoliated Deciduous Teeth (SHED) through hTERT Expression while Preserving Stemness and Multipotency. Stem Cell Rev Rep. 2024. Online ahead of print.
53. Zhao Q, Wang XY, Yu XX, et al. Expression of human telomerase reverse transcriptase mediates the senescence of mesenchymal stem cells through the PI3K/AKT signaling pathway. Int J Mol Med. 2015;36(3):857–64.
54. Raval A, Behbehani GK, Nguyen LXT, et al. Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice. PLoS One. 2015;10(7):e0131722.
55. Chiu C, Dragowska W, Kim NW, et al. Differential Expression of Telomerase Activity in Hematopoietic Progenitors from Adult Human Bone Marrow. Stem Cells. 1996;14(2):239–48.
56. Vaziri H, Benchimol S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr Biol. 1998; 8(5):279–82.
57. Zhu Y, Liu X, Ding X, et al. Telomere and its role in the aging pathways: telomere shortening, cell senescence and mitochondria dysfunction. Biogerontology. 2019;20(1):1–16.
58. Kong CM, Lee XW, Wang X. Telomere shortening in human diseases. FEBS J. 2013;280(14):3180–93.
59. Masutomi K, Possemato R, Wong JMY, et al. The telomerase reverse transcriptase regulates chromatin state and DNA damage responses. Proc Natl Acad Sci U S A. 2005;102(23):8222-7.
| Files | ||
| Issue | Vol 18 No 4 (2024) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijhoscr.v18i4.16758 | |
| Keywords | ||
| Fetal liver; Hematopoietic stem cells; Immortalization; Telomerase activity, human telomerase reverse transcriptase (hTERT) | ||
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How to Cite
1.
Bhardwaj R, Kumar L, Chhabra D, Sharma A, Mohanty S, Mehra N, Kochupillai V. Transduction of Human Fetal Liver Hematopoietic CD34+ Stem and Progenitor Cells into a Cell Line by Enhancing Telomerase Activity. Int J Hematol Oncol Stem Cell Res. 2024;18(4):331-344.
