Generation of osteoclasts from isolated hematopoietic progenitor cells

Noriyoshi Kurihara, T. Suda, Y. Miura, H. Nakauchi, H. Kodama, K. Hiura, Y. Hakeda, M. Kumegawa

Research output: Contribution to journalArticle

209 Citations (Scopus)

Abstract

A variety of studies have shown that osteoclasts originate from bone marrow, but their exact progenitors and differentiation pathway remain unclear. The treatment of mice with a high dose of 5-fluorouracil (5-FU) results in an enrichment for primitive hematopoietic progenitors; using this procedure, we prepared a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro. When spleen cells from mice pretreated in vivo with 5-FU were cultured in the presnece of methylcellulose medium containing recombinant interleukin-3 (rIL-3), small colonies consisting of blast cells with little sign of differentiation developed on day 7 of culture. We lifted these blast colonies, pooled them, and replated them as secondary methylcellulose cultures in the presence of rIL-3 and erythropoietin. Approximately 60% of the cells formed colonies comprising various combinations of neutrophils, macrophages, eosinophils, mast cells, megakaryocytes, and erythroblasts. We replated such blast cells into microtiter wells and cultured them in the presence of rIL-3 (100 U/mL) or recombinant granulocyte-macrophage colony stimulating factor (GM-CSF)(100 U/mL) plus 1,25(OH)2D3 (10-7 mol/L). Multinucleated cells appeared from day 14 of culture and approximately 100 giant cells per well were scored on day 21 of culture. Parathyroid hormone (1 U/mL) also induced the multinucleated cell formation. May-Grunwald-Giemsa staining revealed the large cells containing many nuclei in their cytoplasm, which is characteristic of bone-resorbing cells or osteoclasts. These cells showed a tartrate-resistant acid phosphatase (TRAP) activity. Calcitonin caused a striking shape change in these cells and suppressed the formation of multinucleated cells. Moreover, electron microscopy shows that these cells were able to resorb fetal calvariae. In the presence of r granulocyte-colony stimulating factor, r macrophage-colony stimulating factor, or r interleukin-6 plus 1,25(OH)2D3, formation of TRAP-positive multinucleated cells was lower compared with the support of rIL-3 or rGM-CSF. Mature macrophages collected from colonies did not form the multinucleated cells as described above, even in the presence of rIL-3 and 1,25(OH)2D3. Moreover, to exclude the possibility that osteoclasts generated from non-blast cells, we performed a cloning experiment from one isolated blast cell and demonstrated that single cells differentiate into osteoclasts or macrophages in the presence of rIL-3 with or without 1,25(OH)2D3. This system will provide a useful model for further analysis of osteoclast formation in vitro.

Original languageEnglish (US)
Pages (from-to)1295-1302
Number of pages8
JournalBlood
Volume74
Issue number4
StatePublished - 1989
Externally publishedYes

Fingerprint

Interleukin-3
Osteoclasts
Hematopoietic Stem Cells
Macrophages
Methylcellulose
Acid Phosphatase
Fluorouracil
Bone
Cells
Macrophage Colony-Stimulating Factor
Cloning
Calcitonin
Granulocyte Colony-Stimulating Factor
Granulocyte-Macrophage Colony-Stimulating Factor
Erythropoietin
Parathyroid Hormone
Interleukin-1
Plating
Electron microscopy
Interleukin-6

ASJC Scopus subject areas

  • Hematology

Cite this

Kurihara, N., Suda, T., Miura, Y., Nakauchi, H., Kodama, H., Hiura, K., ... Kumegawa, M. (1989). Generation of osteoclasts from isolated hematopoietic progenitor cells. Blood, 74(4), 1295-1302.

Generation of osteoclasts from isolated hematopoietic progenitor cells. / Kurihara, Noriyoshi; Suda, T.; Miura, Y.; Nakauchi, H.; Kodama, H.; Hiura, K.; Hakeda, Y.; Kumegawa, M.

In: Blood, Vol. 74, No. 4, 1989, p. 1295-1302.

Research output: Contribution to journalArticle

Kurihara, N, Suda, T, Miura, Y, Nakauchi, H, Kodama, H, Hiura, K, Hakeda, Y & Kumegawa, M 1989, 'Generation of osteoclasts from isolated hematopoietic progenitor cells', Blood, vol. 74, no. 4, pp. 1295-1302.
Kurihara N, Suda T, Miura Y, Nakauchi H, Kodama H, Hiura K et al. Generation of osteoclasts from isolated hematopoietic progenitor cells. Blood. 1989;74(4):1295-1302.
Kurihara, Noriyoshi ; Suda, T. ; Miura, Y. ; Nakauchi, H. ; Kodama, H. ; Hiura, K. ; Hakeda, Y. ; Kumegawa, M. / Generation of osteoclasts from isolated hematopoietic progenitor cells. In: Blood. 1989 ; Vol. 74, No. 4. pp. 1295-1302.
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AU - Hakeda, Y.

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N2 - A variety of studies have shown that osteoclasts originate from bone marrow, but their exact progenitors and differentiation pathway remain unclear. The treatment of mice with a high dose of 5-fluorouracil (5-FU) results in an enrichment for primitive hematopoietic progenitors; using this procedure, we prepared a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro. When spleen cells from mice pretreated in vivo with 5-FU were cultured in the presnece of methylcellulose medium containing recombinant interleukin-3 (rIL-3), small colonies consisting of blast cells with little sign of differentiation developed on day 7 of culture. We lifted these blast colonies, pooled them, and replated them as secondary methylcellulose cultures in the presence of rIL-3 and erythropoietin. Approximately 60% of the cells formed colonies comprising various combinations of neutrophils, macrophages, eosinophils, mast cells, megakaryocytes, and erythroblasts. We replated such blast cells into microtiter wells and cultured them in the presence of rIL-3 (100 U/mL) or recombinant granulocyte-macrophage colony stimulating factor (GM-CSF)(100 U/mL) plus 1,25(OH)2D3 (10-7 mol/L). Multinucleated cells appeared from day 14 of culture and approximately 100 giant cells per well were scored on day 21 of culture. Parathyroid hormone (1 U/mL) also induced the multinucleated cell formation. May-Grunwald-Giemsa staining revealed the large cells containing many nuclei in their cytoplasm, which is characteristic of bone-resorbing cells or osteoclasts. These cells showed a tartrate-resistant acid phosphatase (TRAP) activity. Calcitonin caused a striking shape change in these cells and suppressed the formation of multinucleated cells. Moreover, electron microscopy shows that these cells were able to resorb fetal calvariae. In the presence of r granulocyte-colony stimulating factor, r macrophage-colony stimulating factor, or r interleukin-6 plus 1,25(OH)2D3, formation of TRAP-positive multinucleated cells was lower compared with the support of rIL-3 or rGM-CSF. Mature macrophages collected from colonies did not form the multinucleated cells as described above, even in the presence of rIL-3 and 1,25(OH)2D3. Moreover, to exclude the possibility that osteoclasts generated from non-blast cells, we performed a cloning experiment from one isolated blast cell and demonstrated that single cells differentiate into osteoclasts or macrophages in the presence of rIL-3 with or without 1,25(OH)2D3. This system will provide a useful model for further analysis of osteoclast formation in vitro.

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