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Single cell biology beyond the era of antibodies: relevance, challenges, and promises in biomedical research

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Abstract

Research of the past two decades has proved the relevance of single cell biology in basic research and translational medicine. Successful detection and isolation of specific subsets is the key to understand their functional heterogeneity. Antibodies are conventionally used for this purpose, but their relevance in certain contexts is limited. In this review, we discuss some of these contexts, posing bottle neck for different fields of biology including biomedical research. With the advancement of chemistry, several methods have been introduced to overcome these problems. Even though microfluidics and microraft array are newer techniques exploited for single cell biology, fluorescence-activated cell sorting (FACS) remains the gold standard technique for isolation of cells for many biomedical applications, like stem cell therapy. Here, we present a comprehensive and comparative account of some of the probes that are useful in FACS. Further, we illustrate how these techniques could be applied in biomedical research. It is postulated that intracellular molecular markers like nucleostemin (GNL3), alkaline phosphatase (ALPL) and HIRA can be used for improving the outcome of cardiac as well as bone regeneration. Another field that could utilize intracellular markers is diagnostics, and we propose the use of specific peptide nucleic acid probes (PNPs) against certain miRNAs for cancer surgical margin prediction. The newer techniques for single cell biology, based on intracellular molecules, will immensely enhance the repertoire of possible markers for the isolation of cell types useful in biomedical research.

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References

  1. Ward EJ, Shcherbata HR, Reynolds SH, Fischer KA, Hatfield SD, Ruohola-Baker H (2006) Stem cells signal to the niche through the Notch pathway in the Drosophila ovary. Curr Biol 16(23):2352–2358

    Article  CAS  PubMed  Google Scholar 

  2. Tsioris K, Torres AJ, Douce TB, Love JC (2014) A new toolbox for assessing single cells. Annu Rev Chem Biomol Eng 5:455–477

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Mazutis L, Gilbert J, Ung WL, Weitz DA, Griffiths AD, Heyman JA (2013) Single-cell analysis and sorting using droplet-based microfluidics. Nat Protoc 8(5):870–891

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. He JL, Chen AT, Lee JH, Fan SK (2015) Digital microfluidics for manipulation and analysis of a single cell. Int J Mol Sci 16(9):22319–22332

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Zhang H, Liu L, Fu X, Zhu Z (2013) Microfluidic beads-based immunosensor for sensitive detection of cancer biomarker proteins using multienzyme-nanoparticle amplification and quantum dots labels. Biosens Bioelectron 42:23–30

    Article  PubMed  Google Scholar 

  6. Welch JD, Williams LA, DiSalvo M, Brandt AT, Marayati R, Sims CE, Allbritton NL, Prins JF, Yeh JJ, Jones CD (2016) Selective single cell isolation for genomics using microraft arrays. Nucleic Acids Res. doi:10.1093/nar/gkw700

    Google Scholar 

  7. Gach PC, Wang Y, Phillips C, Sims CE, Allbritton NL (2011) Isolation and manipulation of living adherent cells by micromolded magnetic rafts. Biomicrofluidics 5(3):32002–3200212

    Article  PubMed  Google Scholar 

  8. Andreu Z, Khan MA, Gonzalez-Gomez P, Negueruela S, Hortiguela R, San Emeterio J, Ferron SR, Martinez G, Vidal A, Farinas I, Lie DC, Mira H (2015) The cyclin-dependent kinase inhibitor p27 kip1 regulates radial stem cell quiescence and neurogenesis in the adult hippocampus. Stem Cells 33(1):219–229

    Article  CAS  PubMed  Google Scholar 

  9. Pauklin S, Vallier L (2013) The cell-cycle state of stem cells determines cell fate propensity. Cell 155(1):135–147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Singh AM (2015) Cell cycle-driven heterogeneity: on the road to demystifying the transitions between “poised” and “restricted” pluripotent cell states. Stem Cells Int 2015:219514

    Article  PubMed  PubMed Central  Google Scholar 

  11. Dhawan J, Laxman S (2015) Decoding the stem cell quiescence cycle—lessons from yeast for regenerative biology. J Cell Sci 128(24):4467–4474

    Article  CAS  PubMed  Google Scholar 

  12. Zou P, Yoshihara H, Hosokawa K, Tai I, Shinmyozu K, Tsukahara F, Maru Y, Nakayama K, Nakayama KI, Suda T (2011) p57(Kip2) and p27(Kip1) cooperate to maintain hematopoietic stem cell quiescence through interactions with Hsc70. Cell Stem Cell 9(3):247–261

    Article  CAS  PubMed  Google Scholar 

  13. Paczkowska E, Kawa M, Klos P, Staniszewska M, Sienko J, Dabkowska E (2011) Aldehyde dehydrogenase (ALDH)—a promising new candidate for use in preclinical and clinical selection of pluripotent very small embryonic-like stem cells (VSEL SCs) of high long-term repopulating hematopoietic potential. Ann Transplant 16(3):59–71

    Article  CAS  PubMed  Google Scholar 

  14. Wang X, Liu Y, Zhou K, Zhang G, Wang F, Ren J (2015) Isolation and characterization of CD105+/CD90+ subpopulation in breast cancer MDA-MB-231 cell line. Int J Clin Exp Pathol 8(5):5105–5112

    PubMed  PubMed Central  Google Scholar 

  15. Yang JH, Song Y, Seol JH, Park JY, Yang YJ, Han JW, Youn HD, Cho EJ (2011) Myogenic transcriptional activation of MyoD mediated by replication-independent histone deposition. Proc Natl Acad Sci USA 108(1):85–90

    Article  PubMed  Google Scholar 

  16. Song TY, Yang JH, Park JY, Song Y, Han JW, Youn HD, Cho EJ (2012) The role of histone chaperones in osteoblastic differentiation of C2C12 myoblasts. Biochem Biophys Res Commun 423(4):726–732

    Article  CAS  PubMed  Google Scholar 

  17. Zimmerer RM, Korn P, Demougin P, Kampmann A, Kokemuller H, Eckardt AM, Gellrich NC, Tavassol F (2013) Functional features of cancer stem cells in melanoma cell lines. Cancer Cell Int 13(1):78

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kemmerling R, Alinger B, Dietze O, Bosmuller HC, Ocker M, Wolkersdorfer GW, Berr F, Neureiter D, Kiesslich T (2012) Association of stem cell marker expression pattern and survival in human biliary tract cancer. Int J Oncol 41(2):511–522

    CAS  PubMed  Google Scholar 

  19. He QZ, Luo XZ, Wang K, Zhou Q, Ao H, Yang Y, Li SX, Li Y, Zhu HT, Duan T (2014) Isolation and characterization of cancer stem cells from high-grade serous ovarian carcinomas. Cell Physiol Biochem 33(1):173–184

    Article  CAS  PubMed  Google Scholar 

  20. Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, Viale A, Olshen AB, Gerald WL, Massague J (2005) Genes that mediate breast cancer metastasis to lung. Nature 436(7050):518–524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Shin VY, Ng EK, Chan VW, Kwong A, Chu KM (2015) A three-miRNA signature as promising non-invasive diagnostic marker for gastric cancer. Mol Cancer 14(1):202

    Article  PubMed  PubMed Central  Google Scholar 

  22. Skrha P, Hajer J, Andel M, Horinek A, Korabecna M (2015) miRNA as a new marker of diabetes mellitus and pancreatic carcinoma progression. Cas Lek Cesk 154(3):122–126

    PubMed  Google Scholar 

  23. Hsu CM, Lin PM, Wang YM, Chen ZJ, Lin SF, Yang MY (2012) Circulating miRNA is a novel marker for head and neck squamous cell carcinoma. Tumour Biol 33(6):1933–1942

    Article  CAS  PubMed  Google Scholar 

  24. Luo X, Burwinkel B, Tao S, Brenner H (2011) MicroRNA signatures: novel biomarker for colorectal cancer? Cancer Epidemiol Biomarkers Prev 20(7):1272–1286

    Article  CAS  PubMed  Google Scholar 

  25. Li X, Yang W, Lou L, Chen Y, Wu S, Ding G (2014) microRNA: a promising diagnostic biomarker and therapeutic target for hepatocellular carcinoma. Dig Dis Sci 59(6):1099–1107

    Article  CAS  PubMed  Google Scholar 

  26. Kishore A, Borucka J, Petrkova J, Petrek M (2014) Novel insights into miRNA in lung and heart inflammatory diseases. Mediators Inflamm 2014:259131

    Article  PubMed  PubMed Central  Google Scholar 

  27. Shell S, Park SM, Radjabi AR, Schickel R, Kistner EO, Jewell DA, Feig C, Lengyel E, Peter ME (2007) Let-7 expression defines two differentiation stages of cancer. Proc Natl Acad Sci USA 104(27):11400–11405

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kitade Y, Akao Y (2010) MicroRNAs and their therapeutic potential for human diseases: microRNAs, miR-143 and -145, function as anti-oncomirs and the application of chemically modified miR-143 as an anti-cancer drug. J Pharmacol Sci 114(3):276–280

    Article  CAS  PubMed  Google Scholar 

  29. Pan X, Wang ZX, Wang R (2010) MicroRNA-21: a novel therapeutic target in human cancer. Cancer Biol Ther 10(12):1224–1232

    Article  CAS  PubMed  Google Scholar 

  30. MacKenzie TA, Schwartz GN, Calderone HM, Graveel CR, Winn ME, Hostetter G, Wells WA, Sempere LF (2014) Stromal expression of miR-21 identifies high-risk group in triple-negative breast cancer. Am J Pathol 184(12):3217–3225

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hedback N, Jensen DH, Specht L, Fiehn AM, Therkildsen MH, Friis-Hansen L, Dabelsteen E, von Buchwald C (2014) MiR-21 expression in the tumor stroma of oral squamous cell carcinoma: an independent biomarker of disease free survival. PLoS One 9(4):e95193

    Article  PubMed  PubMed Central  Google Scholar 

  32. Melbo-Jorgensen C, Ness N, Andersen S, Valkov A, Donnem T, Al-Saad S, Kiselev Y, Berg T, Nordby Y, Bremnes RM, Busund LT, Richardsen E (2014) Stromal expression of MiR-21 predicts biochemical failure in prostate cancer patients with Gleason score 6. PLoS One 9(11):e113039

    Article  PubMed  PubMed Central  Google Scholar 

  33. Dumas A, Le-Bury G, Marie-Anais F, Herit F, Mazzolini J, Guilbert T, Bourdoncle P, Russell DG, Benichou S, Zahraoui A, Niedergang F (2015) The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking. J Cell Biol 211(2):359–372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Smith LM, May RC (2013) Mechanisms of microbial escape from phagocyte killing. Biochem Soc Trans 41(2):475–490

    Article  CAS  PubMed  Google Scholar 

  35. Hawn TR, Matheson AI, Maley SN, Vandal O (2013) Host-directed therapeutics for tuberculosis: can we harness the host? Microbiol Mol Biol Rev 77(4):608–627

    Article  PubMed  PubMed Central  Google Scholar 

  36. Stoltenburg R, Reinemann C, Strehlitz B (2007) SELEX—a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol Eng 24(4):381–403

    Article  CAS  PubMed  Google Scholar 

  37. Champanhac C, Teng IT, Cansiz S, Zhang L, Wu X, Zhoa Z, Fu T, Tan W (2015) Development of a panel of DNA Aptamers with high affinity for pancreatic ductal adenocarcinoma. Sci Rep 5:16788

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hung LY, Wang CH, Che YJ, Fu CY, Chang HY, Wang K, Lee GB (2015) Screening of aptamers specific to colorectal cancer cells and stem cells by utilizing On-chip Cell-SELEX. Sci Rep 5:10326

    Article  PubMed  PubMed Central  Google Scholar 

  39. Liu Z, Lu Y, Pu Y, Liu J, Liu B, Yu B, Chen K, Fu T, Yang CJ, Liu H, Tan W (2015) Using aptamers to elucidate esophageal cancer clinical samples. Sci Rep 5:18516

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Sun H, Zu Y (2015) A highlight of recent advances in aptamer technology and its application. Molecules 20(7):11959–11980

    Article  CAS  PubMed  Google Scholar 

  41. Mayer G, Ahmed MS, Dolf A, Endl E, Knolle PA, Famulok M (2010) Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures. Nat Protoc 5(12):1993–2004

    Article  CAS  PubMed  Google Scholar 

  42. Meyer S, Maufort JP, Nie J, Stewart R, McIntosh BE, Conti LR, Ahmad KM, Soh HT, Thomson JA (2013) Development of an efficient targeted cell-SELEX procedure for DNA aptamer reagents. PLoS One 8(8):e71798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Xu Y, Phillips JA, Yan J, Li Q, Fan ZH, Tan W (2009) Aptamer-based microfluidic device for enrichment, sorting, and detection of multiple cancer cells. Anal Chem 81(17):7436–7442

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Guo KT, SchAfer R, Paul A, Gerber A, Ziemer G, Wendel HP (2006) A new technique for the isolation and surface immobilization of mesenchymal stem cells from whole bone marrow using high-specific DNA aptamers. Stem Cells 24(10):2220–2231

    Article  CAS  PubMed  Google Scholar 

  45. Bratu DP (2006) Molecular beacons: fluorescent probes for detection of endogenous mRNAs in living cells. Methods Mol Biol 319:1–14

    Article  CAS  PubMed  Google Scholar 

  46. Yeh HY, Yates MV, Mulchandani A, Chen W (2008) Visualizing the dynamics of viral replication in living cells via Tat peptide delivery of nuclease-resistant molecular beacons. Proc Natl Acad Sci USA 105(45):17522–17525

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. King FW, Liszewski W, Ritner C, Bernstein HS (2011) High-throughput tracking of pluripotent human embryonic stem cells with dual fluorescence resonance energy transfer molecular beacons. Stem Cells Dev 20(3):475–484

    Article  CAS  PubMed  Google Scholar 

  48. Marble HD, Sutermaster BA, Kanthilal M, Fonseca VC, Darling EM (2014) Gene expression-based enrichment of live cells from adipose tissue produces subpopulations with improved osteogenic potential. Stem Cell Res Ther 5(5):145

    Article  PubMed  PubMed Central  Google Scholar 

  49. Han SX, Jia X, Ma JL, Zhu Q (2013) Molecular beacons: a novel optical diagnostic tool. Arch Immunol Ther Exp (Warsz) 61(2):139–148

    Article  Google Scholar 

  50. Ryoo SR, Lee J, Yeo J, Na HK, Kim YK, Jang H, Lee JH, Han SW, Lee Y, Kim VN, Min DH (2013) Quantitative and multiplexed microRNA sensing in living cells based on peptide nucleic acid and nano graphene oxide (PANGO). ACS Nano 7(7):5882–5891

    Article  CAS  PubMed  Google Scholar 

  51. Copolovici DM, Langel K, Eriste E, Langel U (2014) Cell-penetrating peptides: design, synthesis, and applications. ACS Nano 8(3):1972–1994

    Article  CAS  PubMed  Google Scholar 

  52. Ruoslahti E (1996) RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 12:697–715

    Article  CAS  PubMed  Google Scholar 

  53. Raucher D, Ryu JS (2015) Cell-penetrating peptides: strategies for anticancer treatment. Trends Mol Med 21(9):560–570

    Article  CAS  PubMed  Google Scholar 

  54. Kersemans V, Kersemans K, Cornelissen B (2008) Cell penetrating peptides for in vivo molecular imaging applications. Curr Pharm Des 14(24):2415–2447

    Article  CAS  PubMed  Google Scholar 

  55. Kochurani KJ, Suganya AA, Nair MG, Louis JM, Majumder A, Kumar KS, Abraham P, Dutta D, Maliekal TT (2015) Live detection and purification of cells based on the expression of a histone chaperone, HIRA, using a binding peptide. Sci Rep 5:17218

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Hansen M, Kilk K, Langel U (2008) Predicting cell-penetrating peptides. Adv Drug Deliv Rev 60(4–5):572–579

    Article  CAS  PubMed  Google Scholar 

  57. Doppler SA, Deutsch MA, Lange R, Krane M (2013) Cardiac regeneration: current therapies-future concepts. J Thorac Dis 5(5):683–697

    PubMed  PubMed Central  Google Scholar 

  58. Doppler SA, Deutsch MA, Lange R, Krane M (2015) Direct reprogramming—the future of cardiac regeneration? Int J Mol Sci 16(8):17368–17393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Russo V, Yu C, Belliveau P, Hamilton A, Flynn LE (2014) Comparison of human adipose-derived stem cells isolated from subcutaneous, omental, and intrathoracic adipose tissue depots for regenerative applications. Stem Cells Transl Med 3(2):206–217

    Article  CAS  PubMed  Google Scholar 

  60. Manginas A, Goussetis E, Koutelou M, Karatasakis G, Peristeri I, Theodorakos A, Leontiadis E, Plessas N, Theodosaki M, Graphakos S, Cokkinos DV (2007) Pilot study to evaluate the safety and feasibility of intracoronary CD133(+) and CD133(−) CD34(+) cell therapy in patients with nonviable anterior myocardial infarction. Catheter Cardiovasc Interv 69(6):773–781

    Article  PubMed  Google Scholar 

  61. Patel AN, Geffner L, Vina RF, Saslavsky J, Urschel HC Jr, Kormos R, Benetti F (2005) Surgical treatment for congestive heart failure with autologous adult stem cell transplantation: a prospective randomized study. J Thorac Cardiovasc Surg 130(6):1631–1638

    Article  PubMed  Google Scholar 

  62. Stamm C, Kleine HD, Choi YH, Dunkelmann S, Lauffs JA, Lorenzen B, David A, Liebold A, Nienaber C, Zurakowski D, Freund M, Steinhoff G (2007) Intramyocardial delivery of CD133+ bone marrow cells and coronary artery bypass grafting for chronic ischemic heart disease: safety and efficacy studies. J Thorac Cardiovasc Surg 133(3):717–725

    Article  PubMed  Google Scholar 

  63. Perin EC, Silva GV, Zheng Y, Gahremanpour A, Canales J, Patel D, Fernandes MR, Keller LH, Quan X, Coulter SA, Moore WH, Herlihy JP, Willerson JT (2012) Randomized, double-blind pilot study of transendocardial injection of autologous aldehyde dehydrogenase-bright stem cells in patients with ischemic heart failure. Am Heart J 163(3):415–421

    Article  CAS  PubMed  Google Scholar 

  64. Tsai RY, McKay RD (2002) A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes Dev 16(23):2991–3003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Hariharan N, Quijada P, Mohsin S, Joyo A, Samse K, Monsanto M, De La Torre A, Avitabile D, Ormachea L, McGregor MJ, Tsai EJ, Sussman MA (2015) Nucleostemin rejuvenates cardiac progenitor cells and antagonizes myocardial aging. J Am Coll Cardiol 65(2):133–147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Cheng MQ, Wahafu T, Jiang GF, Liu W, Qiao YQ, Peng XC, Cheng T, Zhang XL, He G, Liu XY (2016) A novel open-porous magnesium scaffold with controllable microstructures and properties for bone regeneration. Sci Rep 6:24134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Lee JH, Shin YC, Lee SM, Jin OS, Kang SH, Hong SW, Jeong CM, Huh JB, Han DW (2015) Enhanced osteogenesis by reduced graphene oxide/hydroxyapatite nanocomposites. Sci Rep 5:18833

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Zhang X, Zeng D, Li N, Wen J, Jiang X, Liu C, Li Y (2016) Functionalized mesoporous bioactive glass scaffolds for enhanced bone tissue regeneration. Sci Rep 6:19361

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Dosier CR, Uhrig BA, Willett NJ, Krishnan L, Li MT, Stevens HY, Schwartz Z, Boyan BD, Guldberg RE (2015) Effect of cell origin and timing of delivery for stem cell-based bone tissue engineering using biologically functionalized hydrogels. Tissue Eng Part A 21(1–2):156–165

    Article  CAS  PubMed  Google Scholar 

  70. Yamada Y, Nakamura S, Ito K, Umemura E, Hara K, Nagasaka T, Abe A, Baba S, Furuichi Y, Izumi Y, Klein OD, Wakabayashi T (2013) Injectable bone tissue engineering using expanded mesenchymal stem cells. Stem Cells 31(3):572–580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Jakus AE, Shah RN (2016) Multi- and mixed 3D-printing of graphene-hydroxyapatite hybrid materials for complex tissue engineering. J Biomed Mater Res A. doi:10.1002/jbm.a.35684

    PubMed  Google Scholar 

  72. Correia CR, Pirraco RP, Cerqueira MT, Marques AP, Reis RL, Mano JF (2016) Semipermeable capsules wrapping a multifunctional and self-regulated co-culture microenvironment for osteogenic differentiation. Sci Rep 6:21883

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We acknowledge the funding from Department of Biotechnology, India (BT/PR14379/Med/30/536/2010). We also sincerely thank the help of Praveen Chandran and Avinash Kumar in manuscript preparation.

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  1. Cancer Research, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, 695014, India

    Parvin Abraham & Tessy Thomas Maliekal

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Abraham, P., Maliekal, T.T. Single cell biology beyond the era of antibodies: relevance, challenges, and promises in biomedical research. Cell. Mol. Life Sci. 74, 1177–1189 (2017). https://doi.org/10.1007/s00018-016-2382-z

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