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Cluster of differentiation (CD) 9-positive mouse pituitary cells are adult stem/progenitor cells

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Abstract

SOX2-positive cells are stem/progenitor cells that supply hormone-producing cells; they are found in the anterior lobe of the rodent pituitary gland. However, they are likely composed of several subpopulations. In rats, a SOX2-positive cell populations can be distinguished by the presence of S100β. We identified the novel markers cluster of differentiation (CD) CD9 and CD81, members of the tetraspanin superfamily, for the identification of S100β/SOX2-positive cells. Recently, CD9/CD81 double-knockout mice were generated. Although they grew normally until 3 weeks after birth, they exhibited atrophy of the pituitary gland. These findings suggested that CD9/CD81/S100β/SOX2-positive cells in the mouse pituitary are adult stem/progenitor cells. To substantiate this hypothesis, we examined CD9 and CD81 expression in the adult and developing anterior lobe. Immunohistochemistry showed that CD9/CD81-positive cells began appearing from postnatal day 0 and settled in the stem cell niches (marginal cell layer and parenchyma) of the adult anterior lobe while expressing S100β. We next isolated CD9 -positive cells from the adult anterior lobe, using the anti-CD9 antibody for cell characterisation. The cells in culture formed free-floating three-dimensional clusters (pituispheres); moreover, induction into all types of hormone-producing cells was successful. Furthermore, reduction of CD9 and CD81 mRNAs by siRNAs inhibited cell proliferation. These findings indicate that CD9/CD81/S100β/SOX2-positive cells may play a role as adult stem/progenitor cells in SOX2-positive subpopulations, thus supplying hormone-producing cells in the postnatal anterior lobe. Furthermore, CD9 and CD81 are implicated in cell proliferation. The current findings provide novel insights into adult pituitary stem/progenitor cells.

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Abbreviations

ACTH:

Adrenocorticotropic hormone

AMCA:

Dipeptide-β-Ala-Lys-Nε- 7-amino-4-methylcoumarin-3-acetic acid

BIO:

6-Bromoindirubin-3′-oxime

CD:

Cluster of differentiation

CD9/CD81/S100β/SOX2-positive:

CD9-, CD81-, S100β-, and SOX2-quadruple positive

DAPI:

4,6-Diamidino-2-phenylindole

DKO:

Double-knockout

ECM:

Extracellular matrix

E10.5:

Embryonic day 10.5

FBS:

Foetal bovine serum

GH:

Growth hormone

HE:

Haematoxylin–eosin

KSR:

KnockOut serum replacement

LH:

Luteinising hormone

MCL:

Marginal cell layer

POMC:

Proopiomelanocortin

PB:

Phosphate buffer

PBS:

Phosphate buffered saline

PRL:

Prolactin

P0:

Postnatal day 0

siRNA:

Small interfering RNA

SOX2:

Sex-determining region Y-box 2

S100β:

S100β protein

TSH:

Thyroid-stimulating hormone

WT:

Wild type

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Acknowledgements

We would like to thank Dr. A. F. Parlow, Scientific Director, National Hormone & Peptide Program for kindly supplying the mouse GH, PRL, TSHβ, LHβ, and ACTH antibodies. We are grateful to Dr. T. Kato and Y. Kato (Institute for Reproduction and Endocrinology, Meiji University) for helpful discussions. We would like to thank Editage (www.editage.jp) for English language editing.

Funding

This work was supported by JSPS KAKENHI Grants (no. 16K08475 and 19K 07255 to K.H., no. 17K08517 to K.F.).

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Authors and Affiliations

  1. Laboratory of Anatomy and Cell Biology, Department of Health Sciences, Kyorin University, 5-4-1 Shimorenjaku, Mitaka, Tokyo, 181-8612, Japan

    Kotaro Horiguchi, Rumi Hasegawa, Shu Takigami & Shunji Ohsako

  2. Department of Biochemistry, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo, 105-8461, Japan

    Saishu Yoshida

  3. Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan

    Takehiro Tsukada

  4. Department of Biological Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa, 259-1293, Japan

    Ken Fujiwara

  5. Department of Anatomy, Graduate School of Medicine, Teikyo University, 2-11-1 Kaga, Itabashi, Tokyo, 173-8605, Japan

    Takashi Nakakura

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  1. Kotaro Horiguchi
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Correspondence to Kotaro Horiguchi.

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The current study was approved by the Committee on Animal Experiments of Kyorin University based on the NIH Guidelines for the Care and Use of Laboratory Animals. This article does not contain any studies with human participants. This article does not contain any studies with human participants or animals performed by any of the authors.

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418_2020_1943_MOESM1_ESM.tif

Supplementary file1 (TIF 5792 KB) Supplementary figure 1. Determination of the anti-mouse CD9 antibody specificity. (a) In situ hybridization for Cd9 (red) and immunohistochemistry for CD9 (green). (b) In situ hybridization for Cd9 (red) and immunohistochemistry for CD9 using the pre-absorbed antibody (green). Right panels are merged images of Cd9, CD9 and DAPI staining. Lower panels are the high magnifications of the boxed areas in the upper panels. Scale bars, 200μm (Upper panel) and 10μm (Lower panel)

418_2020_1943_MOESM2_ESM.tif

Supplementary file2 (TIF 1779 KB) Supplementary figure 2. Uptake of AMCA (blue) and immunocytochemistry for CD9 (red) in primary mouse anterior lobe cells cultured for 72 h. The left image is the bright field (BF) view, while the middle and right images indicate AMCA and CD9 staining, respectively

418_2020_1943_MOESM3_ESM.tif

Supplementary file3 (TIF 1856 KB) Supplementary figure 2. Uptake of AMCA (blue) and immunocytochemistry for CD9 (red) in primary mouse anterior lobe cells cultured for 72 h. The left image is the bright field (BF) view, while the middle and right images indicate AMCA and CD9 staining, respectively

418_2020_1943_MOESM4_ESM.tif

Supplementary file4 (TIF 1623 KB) Supplementary figure 4. Differentiation of hormone-producing cells using CD9-negative pituispheres. Merged images of DAPI (blue), double-immunofluorescence against CD9 (red), and GH (green), PRL (green), S100β (green), TSH (green), LH (green), or ACTH (green) after induction are shown. Scale bars, 50 μm

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Horiguchi, K., Yoshida, S., Tsukada, T. et al. Cluster of differentiation (CD) 9-positive mouse pituitary cells are adult stem/progenitor cells. Histochem Cell Biol 155, 391–404 (2021). https://doi.org/10.1007/s00418-020-01943-0

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  • DOI: https://doi.org/10.1007/s00418-020-01943-0

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