Advertisement

Detection of Calcium Transients in Embryonic Stem Cells and Their Differentiated Progeny

Abstract

A central issue in stem cell biology is the determination of function and activity of differentiated stem cells, features that define the true phenotype of mature cell types. Commonly, physiological mechanisms are used to determine the functionality of mature cell types, including those of the nervous system. Calcium imaging provides an indirect method of determining the physiological activities of a mature cell. Camgaroos are variants of yellow fluorescent protein that act as intracellular calcium sensors in transfected cells. We expressed one version of the camgaroos, Camgaroo-2, in mouse embryonic stem (ES) cells under the control of the CAG promoter system. Under the control of this promoter, Camgaroo-2 fluorescence was ubiquitously expressed in all cell types derived from the ES cells that were tested. In response to pharmacological stimulation, the fluorescence levels in transfected cells correlated with cellular depolarization and hyperpolarization. These changes were observed in both undifferentiated ES cells as well as ES cells that had been neurally induced, including putative neurons that were differentiated from transfected ES cells. The results presented here indicate that Camgaroo-2 may be used like traditional fluorescent proteins to track cells as well as to study the functionality of stem cells and their progeny.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Anderson DJ, Gage FH, Weissman IL (2001) Can stem cells cross lineage boundaries? Nat Med 7(4):393–395. doi:10.1038/86439

  2. Arenas E (2002) Stem cells in the treatment of Parkinson’s disease. Brain Res Bull 57(6):795–808. doi:10.1016/S0361-9230(01)00772-9

  3. Astradsson A, Cooper O, Vinuela A, Isacson O (2008) Recent advances in cell-based therapy for Parkinson disease. Neurosurg Focus 24(3-4):E6. doi:10.3171/FOC/2008/24/3-4/E5

  4. Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI (1995) Embryonic stem cells express neuronal properties in vitro. Dev Biol 168(2):342–357. doi:10.1006/dbio.1995.1085

  5. Baird GS, Zacharias DA, Tsien RY (1999) Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci USA 96(20):11241–11246. doi:10.1073/pnas.96.20.11241

  6. Barth AL (2007) Visualizing circuits and systems using transgenic reporters of neural activity. Curr Opin Neurobiol 17(5):567–571. doi:10.1016/j.conb.2007.10.003

  7. Bauwens CL, Peerani R, Niebruegge S, Woodhouse KA, Kumacheva E, Husain M, Zandstra PW (2008) Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells 26(9):2300–2310. doi:10.1634/stemcells.2008-0183

  8. Benninger F, Beck H, Wernig M, Tucker KL, Brustle O, Scheffler B (2003) Functional integration of embryonic stem cell-derived neurons in hippocampal slice cultures. J Neurosci 23(18):7075–7083

  9. Bjorklund LM, Sanchez-Pernaute R, Chung S, Andersson T, Chen IY, McNaught KS, Brownell AL, Jenkins BG, Wahlestedt C, Kim KS, Isacson O (2002) Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci USA 99(4):2344–2349. doi:10.1073/pnas.022438099

  10. Black IB, Woodbury D (2001) Adult rat and human bone marrow stromal stem cells differentiate into neurons. Blood Cells Mol Dis 27(3):632–636. doi:10.1006/bcmd.2001.0423

  11. Brazelton TR, Rossi FM, Keshet GI, Blau HM (2000) From marrow to brain: expression of neuronal phenotypes in adult mice. Science 290(5497):1775–1779. doi:10.1126/science.290.5497.1775

  12. Brownlee C (2000) Cellular calcium imaging: so, what’s new? Trends Cell Biol 10(10):451–457. doi:10.1016/S0962-8924(00)01799-2

  13. Castro RF, Jackson KA, Goodell MA, Robertson CS, Liu H, Shine HD (2002) Failure of bone marrow cells to transdifferentiate into neural cells in vivo. Science 297(5585):1299. doi:10.1126/science.297.5585.1299

  14. Cedervall J, Ahrlund-Richter L, Svensson B, Forsgren K, Maurer FH, Vidovska D, Hertegard S (2007) Injection of embryonic stem cells into scarred rabbit vocal folds enhances healing and improves viscoelasticity: short-term results. Laryngoscope 117(11):2075–2081. doi:10.1097/MLG.0b013e3181379c7c

  15. Chou CY, Horng LS, Tsai HJ (2001) Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation. Transgenic Res 10(4):303–315. doi:10.1023/A:1016671513425

  16. Chung S, Andersson T, Sonntag KC, Bjorklund L, Isacson O, Kim KS (2002) Analysis of different promoter systems for efficient transgene expression in mouse embryonic stem cell lines. Stem Cells 20(2):139–145. doi:10.1634/stemcells.20-2-139

  17. Coutts M, Keirstead HS (2008) Stem cells for the treatment of spinal cord injury. Exp Neurol 209(2):368–377. doi:10.1016/j.expneurol.2007.09.002

  18. Daley GQ, Goodell MA, Snyder EY (2003) Realistic prospects for stem cell therapeutics. Hematology (Am Soc Hematol Educ Program) 1:398–418. doi:10.1182/asheducation-2003.1.398

  19. Fu Y, Wang Y, Evans SM (1998) Viral sequences enable efficient and tissue-specific expression of transgenes in Xenopus. Nat Biotechnol 16(3):253–257. doi:10.1038/nbt0398-253

  20. Gage FH (2000) Mammalian neural stem cells. Science 287(5457):1433–1438. doi:10.1126/science.287.5457.1433

  21. Garaschuk O, Griesbeck O, Konnerth A (2007) Troponin C-based biosensors: a new family of genetically encoded indicators for in vivo calcium imaging in the nervous system. Cell Calcium 42(4–5):351–361. doi:10.1016/j.ceca.2007.02.011

  22. Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY (2001) Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem 276(31):29188–29194. doi:10.1074/jbc.M102815200

  23. Harkany T, Andang M, Kingma HJ, Gorcs TJ, Holmgren CD, Zilberter Y, Ernfors P (2004) Region-specific generation of functional neurons from naive embryonic stem cells in adult brain. J Neurochem 88(5):1229–1239. doi:10.1046/j.1471-4159.2003.02243.x

  24. Heim N, Garaschuk O, Friedrich MW, Mank M, Milos RI, Kovalchuk Y, Konnerth A, Griesbeck O (2007) Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor. Nat Methods 4(2):127–129. doi:10.1038/nmeth1009

  25. Hsiao CD, Hsieh FJ, Tsai HJ (2001) Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish. Dev Dyn 220(4):323–336. doi:10.1002/dvdy.1113

  26. Hua J, Sidhu K (2008) Recent advances in the derivation of germ cells from the embryonic stem cells. Stem Cells Dev 17(3):399–411. doi:10.1089/scd.2007.0225

  27. Jiang Y, Henderson D, Blackstad M, Chen A, Miller RF, Verfaillie CM (2003) Neuroectodermal differentiation from mouse multipotent adult progenitor cells. Proc Natl Acad Sci USA 100(Suppl 1):11854–11860

  28. Kim BJ, Seo JH, Bubien JK, Oh YS (2002) Differentiation of adult bone marrow stem cells into neuroprogenitor cells in vitro. Neuroreport 13(9):1185–1188. doi:10.1097/00001756-200207020-00023

  29. Klassen H, Sakaguchi DS, Young MJ (2004) Stem cells and retinal repair. Prog Retin Eye Res 23(2):149–181. doi:10.1016/j.preteyeres.2004.01.002

  30. Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD (2000) Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 18(6):675–679. doi:10.1038/76536

  31. Levy YS, Merims D, Panet H, Barhum Y, Melamed E, Offen D (2003) Induction of neuron-specific enolase promoter and neuronal markers in differentiated mouse bone marrow stromal cells. J Mol Neurosci 21(2):121–132. doi:10.1385/JMN:21:2:121

  32. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R (2001) Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 292(5520):1389–1394. doi:10.1126/science.1058866

  33. Mayginnes JP, Reed SE, Berg HG, Staley EM, Pintel DJ, Tullis GE (2006) Quantitation of encapsidated recombinant adeno-associated virus DNA in crude cell lysates and tissue culture medium by quantitative, real-time PCR. J Virol Methods 137(2):193–204. doi:10.1016/j.jviromet.2006.06.011

  34. McDonald JW, Howard MJ (2002) Repairing the damaged spinal cord: a summary of our early success with embryonic stem cell transplantation and remyelination. Prog Brain Res 137:299–309

  35. McDonald JW, Liu XZ, Qu Y, Liu S, Mickey SK, Turetsky D, Gottlieb DI, Choi DW (1999) Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med 5(12):1410–1412. doi:10.1038/70986

  36. McKay R (2000) Stem cells–hype and hope. Nature 406(6794):361–364. doi:10.1038/35019186

  37. Meyer JS, Katz ML, Maruniak JA, Kirk MD (2004) Neural differentiation of mouse embryonic stem cells in vitro and after transplantation into eyes of mutant mice with rapid retinal degeneration. Brain Res 1014(1–2):131–144. doi:10.1016/j.brainres.2004.04.019

  38. Meyer JS, Katz ML, Maruniak JA, Kirk MD (2006) Embryonic stem cell-derived neural progenitors incorporate into degenerating retina and enhance survival of host photoreceptors. Stem Cells 24(2):274–283. doi:10.1634/stemcells.2005-0059

  39. Miyawaki A, Griesbeck O, Heim R, Tsien RY (1999) Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci USA 96(5):2135–2140. doi:10.1073/pnas.96.5.2135

  40. Nagai T, Sawano A, Park ES, Miyawaki A (2001) Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci USA 98(6):3197–3202. doi:10.1073/pnas.051636098

  41. Nakai J, Ohkura M, Imoto K (2001) A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nat Biotechnol 19(2):137–141. doi:10.1038/84397

  42. Neuhuber B, Gallo G, Howard L, Kostura L, Mackay A, Fischer I (2004) Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype. J Neurosci Res 77(2):192–204. doi:10.1002/jnr.20147

  43. Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108(2):193–199. doi:10.1016/0378-1119(91)90434-D

  44. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 407(3):313–319. doi:10.1016/S0014-5793(97)00313-X

  45. Orlovskaya I, Schraufstatter I, Loring J, Khaldoyanidi S (2008) Hematopoietic differentiation of embryonic stem cells. Methods 45(2):159–167. doi:10.1016/j.ymeth.2008.03.002

  46. Park KI, Ourednik J, Ourednik V, Taylor RM, Aboody KS, Auguste KI, Lachyankar MB, Redmond DE, Snyder EY (2002) Global gene and cell replacement strategies via stem cells. Gene Ther 9(10):613–624. doi:10.1038/sj.gt.3301721

  47. Parker MA, Corliss DA, Gray B, Anderson JK, Bobbin RP, Snyder EY, Cotanche DA (2007) Neural stem cells injected into the sound-damaged cochlea migrate throughout the cochlea and express markers of hair cells, supporting cells, and spiral ganglion cells. Hear Res 232(1–2):29–43. doi:10.1016/j.heares.2007.06.007

  48. Pierret C, Spears K, Maruniak JA, Kirk MD (2006) Neural crest as the source of adult stem cells. Stem Cells Dev 15(2):286–291. doi:10.1089/scd.2006.15.286

  49. Pierret C, Spears K, Morrison JA, Maruniak JA, Katz ML, Kirk MD (2007) Elements of a neural stem cell niche derived from embryonic stem cells. Stem Cells Dev 16(6):1017–1026. doi:10.1089/scd.2007.0012

  50. Pologruto TA, Yasuda R, Svoboda K (2004) Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators. J Neurosci 24(43):9572–9579. doi:10.1523/JNEUROSCI.2854-04.2004

  51. Prockop DJ (2003) Further proof of the plasticity of adult stem cells and their role in tissue repair. J Cell Biol 160(6):807–809. doi:10.1083/jcb.200302117

  52. Puceat M (2008) Protocols for cardiac differentiation of embryonic stem cells. Methods 45(2):168–171. doi:10.1016/j.ymeth.2008.03.003

  53. Reed SE, Staley EM, Mayginnes JP, Pintel DJ, Tullis GE (2006) Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J Virol Methods 138(1–2):85–98. doi:10.1016/j.jviromet.2006.07.024

  54. Robertson EJ (1997) Derivation and maintenance of embryonic stem cell cultures. Methods Mol Biol 75:173–184

  55. Roy NS, Cleren C, Singh SK, Yang L, Beal MF, Goldman SA (2006) Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat Med 12(11):1259–1268. doi:10.1038/nm1495

  56. Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336:688–690

  57. Soria B, Roche E, Berna G, Leon-Quinto T, Reig JA, Martin F (2000) Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49(2):157–162. doi:10.2337/diabetes.49.2.157

  58. Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37(2):173–182. doi:10.1016/0165-0270(91)90128-M

  59. Takahashi M, Palmer TD, Takahashi J, Gage FH (1998) Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina. Mol Cell Neurosci 12(6):340–348. doi:10.1006/mcne.1998.0721

  60. Takahashi A, Camacho P, Lechleiter JD, Herman B (1999) Measurement of intracellular calcium. Physiol Rev 79(4):1089–1125

  61. Tenenbaum L, Peschanski M, Melas C, Rodesh F, Lehtonen E, Stathopoulos A, Velu T, Brotchi J, Levivier M (2004) Efficient early and sustained transduction of human fetal mesencephalon using adeno-associated virus type 2 vectors. Cell Transplant 13(5):565–571. doi:10.3727/000000004783983684

  62. Tsai RY, Kittappa R, McKay RD (2002) Plasticity, niches, and the use of stem cells. Dev Cell 2(6):707–712. doi:10.1016/S1534-5807(02)00195-8

  63. Tullis GE, Shenk T (2000) Efficient replication of adeno-associated virus type 2 vectors: a cis-acting element outside of the terminal repeats and a minimal size. J Virol 74(24):11511–11521. doi:10.1128/JVI.74.24.11511-11521.2000

  64. Van Hoffelen SJ, Young MJ, Shatos MA, Sakaguchi DS (2003) Incorporation of murine brain progenitor cells into the developing mammalian retina. Invest Ophthalmol Vis Sci 44(1):426–434. doi:10.1167/iovs.02-0269

  65. Wagers AJ, Sherwood RI, Christensen JL, Weissman IL (2002) Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 297(5590):2256–2259. doi:10.1126/science.1074807

  66. Wang XL, Jiang XD, Liang PJ (2008) Intracellular calcium concentration changes initiated by N-methyl-d-aspartic acid receptors in retinal horizontal cells. Neuroreport 19(6):675–678. doi:10.1097/WNR.0b013e3282fb7902

  67. Ward CM, Stern PL (2002) The human cytomegalovirus immediate-early promoter is transcriptionally active in undifferentiated mouse embryonic stem cells. Stem Cells 20(5):472–475. doi:10.1634/stemcells.20-5-472

  68. Wobus AM, Kaomei G, Shan J, Wellner MC, Rohwedel J, Ji G, Fleischmann B, Katus HA, Hescheler J, Franz WM (1997) Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation and enhances development of ventricular cardiomyocytes. J Mol Cell Cardiol 29(6):1525–1539. doi:10.1006/jmcc.1997.0433

  69. Woodbury D, Schwarz EJ, Prockop DJ, Black IB (2000) Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 61(4):364–370. doi:10.1002/1097-4547(20000815)61:4<364::AID-JNR2>3.0.CO;2-C

  70. Yan X, Liu Y, Han Q, Jia M, Liao L, Qi M, Zhao RC (2007) Injured microenvironment directly guides the differentiation of engrafted Flk-1(+) mesenchymal stem cell in lung. Exp Hematol 35(9):1466–1475. doi:10.1016/j.exphem.2007.05.012

  71. Young MJ, Ray J, Whiteley SJ, Klassen H, Gage FH (2000) Neuronal differentiation and morphological integration of hippocampal progenitor cells transplanted to the retina of immature and mature dystrophic rats. Mol Cell Neurosci 16(3):197–205. doi:10.1006/mcne.2000.0869

  72. Yu D, Baird GS, Tsien RY, Davis RL (2003) Detection of calcium transients in Drosophila mushroom body neurons with camgaroo reporters. J Neurosci 23(1):64–72

  73. Zeng X, Rao MS (2007) Human embryonic stem cells: long term stability, absence of senescence and a potential cell source for neural replacement. Neuroscience 145(4):1348–1358. doi:10.1016/j.neuroscience.2006.09.017

  74. Zeng X, Chen J, Sanchez JF, Coggiano M, Dillon-Carter O, Petersen J, Freed WJ (2003) Stable expression of hrGFP by mouse embryonic stem cells: promoter activity in the undifferentiated state and during dopaminergic neural differentiation. Stem Cells 21(6):647–653. doi:10.1634/stemcells.21-6-647

Download references

Acknowledgments

The authors would like to thank Elizabeth Staley for assistance in conducting some of the experiments included in this manuscript. This work was supported by NIH grants NS44494 to GT and NS045813 to MDK.

Author information

Correspondence to Jason S. Meyer.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Meyer, J.S., Tullis, G., Pierret, C. et al. Detection of Calcium Transients in Embryonic Stem Cells and Their Differentiated Progeny. Cell Mol Neurobiol 29, 1191 (2009). https://doi.org/10.1007/s10571-009-9413-3

Download citation

Keywords

  • Calcium imaging
  • Stem cells
  • Camgaroo
  • Fluorescent indicators