Snake Venom Gland Organoids

Yorick Post; Jens Puschhof; Joep Beumer; Harald M. Kerkkamp; Merijn A. G. de Bakker; Julien Slagboom; Buys de Barbanson; Nienke R. Wevers; Xandor M. Spijkers; Thomas Olivier; Taline D. Kazandjian; Stuart Ainsworth; Carmen Lopez Iglesias; Willine J. van de Wetering; Maria C. Heinz; Ravian L. van Ineveld; Regina G. D. M. van Kleef; Harry Begthel; Jeroen Korving; Yotam E. Bar-Ephraim; Walter Getreuer; Anne C. Rios; Remco H. S. Westerink; Hugo J. G. Snippert; Alexander van Oudenaarden; Peter J. Peters; Freek J. Vonk; Jeroen Kool; Michael K. Richardson; Nicholas R. Casewell; Hans Clevers

doi:10.1016/j.cell.2019.11.038

Snake Venom Gland Organoids

Yorick Post, Jens Puschhof, Joep Beumer, Harald M. Kerkkamp, Merijn A. G. de Bakker, Julien Slagboom, Buys de Barbanson, Nienke R. Wevers, Xandor M. Spijkers, Thomas Olivier, Taline D. Kazandjian, Stuart Ainsworth, Carmen Lopez Iglesias, Willine J. van de Wetering, Maria C. Heinz, Ravian L. van Ineveld, Regina G. D. M. van Kleef, Harry Begthel, Jeroen Korving, Yotam E. Bar-EphraimWalter Getreuer, Anne C. Rios, Remco H. S. Westerink, Hugo J. G. Snippert, Alexander van Oudenaarden, Peter J. Peters, Freek J. Vonk, Jeroen Kool, Michael K. Richardson, Nicholas R. Casewell, Hans Clevers^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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Abstract

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.

Original language	English
Pages (from-to)	233-247.e21
Number of pages	36
Journal	Cell
Volume	180
Issue number	2
DOIs	https://doi.org/10.1016/j.cell.2019.11.038
Publication status	Published - 23 Jan 2020

Keywords

TERM PRIMARY CULTURE
IN-VITRO
SECRETORY-CELLS
STEM-CELLS
EVOLUTION
NEUROTOXINS
PROTEINS
LGR5
IDENTIFICATION
RECEPTORS

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Cite this

Post, Y., Puschhof, J., Beumer, J., Kerkkamp, H. M., de Bakker, M. A. G., Slagboom, J., de Barbanson, B., Wevers, N. R., Spijkers, X. M., Olivier, T., Kazandjian, T. D., Ainsworth, S., Iglesias, C. L., van de Wetering, W. J., Heinz, M. C., van Ineveld, R. L., van Kleef, R. G. D. M., Begthel, H., Korving, J., ... Clevers, H. (2020). Snake Venom Gland Organoids. Cell, 180(2), 233-247.e21. https://doi.org/10.1016/j.cell.2019.11.038

@article{ce43626e492f4ee79bef88ea197e964e,

title = "Snake Venom Gland Organoids",

abstract = "Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.",

keywords = "TERM PRIMARY CULTURE, IN-VITRO, SECRETORY-CELLS, STEM-CELLS, EVOLUTION, NEUROTOXINS, PROTEINS, LGR5, IDENTIFICATION, RECEPTORS",

author = "Yorick Post and Jens Puschhof and Joep Beumer and Kerkkamp, {Harald M.} and {de Bakker}, {Merijn A. G.} and Julien Slagboom and {de Barbanson}, Buys and Wevers, {Nienke R.} and Spijkers, {Xandor M.} and Thomas Olivier and Kazandjian, {Taline D.} and Stuart Ainsworth and Iglesias, {Carmen Lopez} and {van de Wetering}, {Willine J.} and Heinz, {Maria C.} and {van Ineveld}, {Ravian L.} and {van Kleef}, {Regina G. D. M.} and Harry Begthel and Jeroen Korving and Bar-Ephraim, {Yotam E.} and Walter Getreuer and Rios, {Anne C.} and Westerink, {Remco H. S.} and Snippert, {Hugo J. G.} and {van Oudenaarden}, Alexander and Peters, {Peter J.} and Vonk, {Freek J.} and Jeroen Kool and Richardson, {Michael K.} and Casewell, {Nicholas R.} and Hans Clevers",

note = "Funding Information: We thank Reinier van der Linden for flow cytometry assistance; Benedetta Artegiani and Delilah Hendriks for generation of CRISPR-HOT technology and assistance in applying it to snake venom gland organoids; Anko de Graaff and the Hubrecht Imaging Centre (HIC) for microscopy assistance; BaseClear B.V. for bulk mRNA sequencing and de novo transcriptome assembly; Bas Ponsioen for providing the lentiviral H2B-RFP construct; Single Cell Discoveries for the provided single-cell sequencing service and support; Jeremie Tai-A-Pin and Harold van der Ploeg for donating venom gland material to this study; and Sebastiaan Voskuil, Edwin Boel, Marc Bonten, and Frank Driehuis for advice regarding antibiotic treatments. X.M.S. was supported by ALS Foundation Netherlands . N.R.C. was supported by a Sir Henry Dale Fellowship ( 200517/Z/16/Z ) jointly funded by the Wellcome Trust and the Royal Society . Funding Information: We thank Reinier van der Linden for flow cytometry assistance; Benedetta Artegiani and Delilah Hendriks for generation of CRISPR-HOT technology and assistance in applying it to snake venom gland organoids; Anko de Graaff and the Hubrecht Imaging Centre (HIC) for microscopy assistance; BaseClear B.V. for bulk mRNA sequencing and de novo transcriptome assembly; Bas Ponsioen for providing the lentiviral H2B-RFP construct; Single Cell Discoveries for the provided single-cell sequencing service and support; Jeremie Tai-A-Pin and Harold van der Ploeg for donating venom gland material to this study; and Sebastiaan Voskuil, Edwin Boel, Marc Bonten, and Frank Driehuis for advice regarding antibiotic treatments. X.M.S. was supported by ALS Foundation Netherlands. N.R.C. was supported by a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by the Wellcome Trust and the Royal Society. Y.P. J.P. J.B. and H.C. conceptualized the project, designed the experiments, interpreted the results, and wrote the manuscript. H.B. and J. Korving performed immunohistochemistry experiments. J.P. B.d.B. A.v.O. M.C.H. and H.J.G.S. performed bulk and single-cell mRNA sequencing analysis on de novo transcriptome. Y.E.B.-E. assisted with FACS experiments. C.L.I. W.J.v.d.W. and P.J.P. performed transmission electron microscopy. M.A.G.d.B. performed in situ hybridization experiments. R.L.v.I. and A.C.R. generated immunofluorescent images. J.S. and J. Kool performed mass spectrometry analysis. S.A. T.D.K. and N.R.C. performed toxin gene expression analysis on bulk RNA sequencing data. N.R.W. X.M.S. and T.O. performed and analyzed toxicity tests on Mimetas OrganoPlate. H.M.K. F.J.V. and M.K.R. assisted in initializing the project and provided access to venom gland tissue. R.H.S.W. and R.G.D.M.v.K performed and analyzed toxicity tests on rat cortical neurons in MEA plates. W.G. and N.R.C. provided venom gland tissue. H.C. is inventor on several patents related to organoid technology; his full disclosure is given at https://www.uu.nl/staff/JCClevers/. N.R.W. and T.O. are employees of MIMETAS BV, the Netherlands, which is marketing the OrganoPlate. OrganoPlate is a registered trademark of MIMETAS. Publisher Copyright: {\textcopyright} 2019 Elsevier Inc.",

year = "2020",

month = jan,

day = "23",

doi = "10.1016/j.cell.2019.11.038",

language = "English",

volume = "180",

pages = "233--247.e21",

journal = "Cell",

issn = "0092-8674",

publisher = "Cell Press",

number = "2",

}

Post, Y, Puschhof, J, Beumer, J, Kerkkamp, HM, de Bakker, MAG, Slagboom, J, de Barbanson, B, Wevers, NR, Spijkers, XM, Olivier, T, Kazandjian, TD, Ainsworth, S, Iglesias, CL, van de Wetering, WJ, Heinz, MC, van Ineveld, RL, van Kleef, RGDM, Begthel, H, Korving, J, Bar-Ephraim, YE, Getreuer, W, Rios, AC, Westerink, RHS, Snippert, HJG, van Oudenaarden, A, Peters, PJ, Vonk, FJ, Kool, J, Richardson, MK, Casewell, NR & Clevers, H 2020, 'Snake Venom Gland Organoids', Cell, vol. 180, no. 2, pp. 233-247.e21. https://doi.org/10.1016/j.cell.2019.11.038

TY - JOUR

T1 - Snake Venom Gland Organoids

AU - Post, Yorick

AU - Puschhof, Jens

AU - Beumer, Joep

AU - Kerkkamp, Harald M.

AU - de Bakker, Merijn A. G.

AU - Slagboom, Julien

AU - de Barbanson, Buys

AU - Wevers, Nienke R.

AU - Spijkers, Xandor M.

AU - Olivier, Thomas

AU - Kazandjian, Taline D.

AU - Ainsworth, Stuart

AU - Iglesias, Carmen Lopez

AU - van de Wetering, Willine J.

AU - Heinz, Maria C.

AU - van Ineveld, Ravian L.

AU - van Kleef, Regina G. D. M.

AU - Begthel, Harry

AU - Korving, Jeroen

AU - Bar-Ephraim, Yotam E.

AU - Getreuer, Walter

AU - Rios, Anne C.

AU - Westerink, Remco H. S.

AU - Snippert, Hugo J. G.

AU - van Oudenaarden, Alexander

AU - Peters, Peter J.

AU - Vonk, Freek J.

AU - Kool, Jeroen

AU - Richardson, Michael K.

AU - Casewell, Nicholas R.

AU - Clevers, Hans

N1 - Funding Information: We thank Reinier van der Linden for flow cytometry assistance; Benedetta Artegiani and Delilah Hendriks for generation of CRISPR-HOT technology and assistance in applying it to snake venom gland organoids; Anko de Graaff and the Hubrecht Imaging Centre (HIC) for microscopy assistance; BaseClear B.V. for bulk mRNA sequencing and de novo transcriptome assembly; Bas Ponsioen for providing the lentiviral H2B-RFP construct; Single Cell Discoveries for the provided single-cell sequencing service and support; Jeremie Tai-A-Pin and Harold van der Ploeg for donating venom gland material to this study; and Sebastiaan Voskuil, Edwin Boel, Marc Bonten, and Frank Driehuis for advice regarding antibiotic treatments. X.M.S. was supported by ALS Foundation Netherlands . N.R.C. was supported by a Sir Henry Dale Fellowship ( 200517/Z/16/Z ) jointly funded by the Wellcome Trust and the Royal Society . Funding Information: We thank Reinier van der Linden for flow cytometry assistance; Benedetta Artegiani and Delilah Hendriks for generation of CRISPR-HOT technology and assistance in applying it to snake venom gland organoids; Anko de Graaff and the Hubrecht Imaging Centre (HIC) for microscopy assistance; BaseClear B.V. for bulk mRNA sequencing and de novo transcriptome assembly; Bas Ponsioen for providing the lentiviral H2B-RFP construct; Single Cell Discoveries for the provided single-cell sequencing service and support; Jeremie Tai-A-Pin and Harold van der Ploeg for donating venom gland material to this study; and Sebastiaan Voskuil, Edwin Boel, Marc Bonten, and Frank Driehuis for advice regarding antibiotic treatments. X.M.S. was supported by ALS Foundation Netherlands. N.R.C. was supported by a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by the Wellcome Trust and the Royal Society. Y.P. J.P. J.B. and H.C. conceptualized the project, designed the experiments, interpreted the results, and wrote the manuscript. H.B. and J. Korving performed immunohistochemistry experiments. J.P. B.d.B. A.v.O. M.C.H. and H.J.G.S. performed bulk and single-cell mRNA sequencing analysis on de novo transcriptome. Y.E.B.-E. assisted with FACS experiments. C.L.I. W.J.v.d.W. and P.J.P. performed transmission electron microscopy. M.A.G.d.B. performed in situ hybridization experiments. R.L.v.I. and A.C.R. generated immunofluorescent images. J.S. and J. Kool performed mass spectrometry analysis. S.A. T.D.K. and N.R.C. performed toxin gene expression analysis on bulk RNA sequencing data. N.R.W. X.M.S. and T.O. performed and analyzed toxicity tests on Mimetas OrganoPlate. H.M.K. F.J.V. and M.K.R. assisted in initializing the project and provided access to venom gland tissue. R.H.S.W. and R.G.D.M.v.K performed and analyzed toxicity tests on rat cortical neurons in MEA plates. W.G. and N.R.C. provided venom gland tissue. H.C. is inventor on several patents related to organoid technology; his full disclosure is given at https://www.uu.nl/staff/JCClevers/. N.R.W. and T.O. are employees of MIMETAS BV, the Netherlands, which is marketing the OrganoPlate. OrganoPlate is a registered trademark of MIMETAS. Publisher Copyright: © 2019 Elsevier Inc.

PY - 2020/1/23

Y1 - 2020/1/23

N2 - Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.

AB - Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.

KW - TERM PRIMARY CULTURE

KW - IN-VITRO

KW - SECRETORY-CELLS

KW - STEM-CELLS

KW - EVOLUTION

KW - NEUROTOXINS

KW - PROTEINS

KW - LGR5

KW - IDENTIFICATION

KW - RECEPTORS

U2 - 10.1016/j.cell.2019.11.038

DO - 10.1016/j.cell.2019.11.038

M3 - Article

C2 - 31978343

SN - 0092-8674

VL - 180

SP - 233-247.e21

JO - Cell

JF - Cell

IS - 2

ER -