Thrombopoietin is synthesized by bone marrow stromal cells
Thrombopoietin is synthesized by bone marrow stromal cells
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DOI：
10.1182/blood.v90.9.3444
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发表时间：
1997-11-01
期刊：
影响因子：
20.3
通讯作者：
Waller, EK
中科院分区：
文献类型：
--
作者：
Guerriero, A;Worford, L;Waller, EK
我们先前已经通过荧光激活细胞分选（FAGS）利用CD 34、CD 38和HLA-DR的差异表达来表征胎儿骨髓中所含的基质祖细胞，并且发现少量的基质祖细胞包含在CD 34（+）细胞部分中。在本研究中，来自胎儿和成人骨髓样本的CD 34（+）和CD 34（-）亚群中基质祖细胞的频率约为单核细胞分数的5，000分之一。使用多参数单细胞分选，20个具有CD 34（+）、CD 38（-）、HLA-DR-、CDw 90（+）表型的胎儿骨髓细胞中有1个是克隆源性基质祖细胞，而大于5个具有CD 34（-）、CD 38（-）、HLA-DR-、CBw 90（+）表型的单细胞中有1个形成基质培养物。我们发现，由骨髓细胞的CD 34（+）部分中所含的造血和基质祖细胞启动的培养物形成了混合的造血/基质细胞培养物，在不添加造血细胞因子的情况下，该混合的造血/基质细胞培养物维持了造血祖细胞的活力3周。我们采用逆转录-聚合酶链反应（RT-PCR）扩增白细胞介素-3（IL-3）、干细胞因子（SCF）、CD 34、Flt 3/Flk 2配体（FL）和血小板生成素（TPO）mRNA序列，对来源于CD 34（+）或CD 34（-）骨髓细胞的基质培养物合成的一些造血细胞因子进行了表征。我们发现TPO mRNA在大于90%的由CD 34（+）或CD 34（-）细胞启动的基质培养物中普遍表达，SCF、FL和CD 34 mRNA表达可变。特别是，SCF和CD 34 mRNA仅在CD 34（+）骨髓细胞启动的基质培养物中检测到，尽管CD 34（+）和CD 34（-）基质细胞之间的差异无统计学意义。在任何基质培养物中均未发现IL-3 mRNA。存在于培养物上清液中的可溶性SCF和TPO的酶联免疫吸附测定（ELISA）证明，一些培养的基质细胞分泌生物学上显著量的蛋白质：16份由胎儿和成人骨髓启动的基质培养物的条件培养基中有8份含有超过32 pg/ml的SCF（在ELISA的线性范围内），中值为32 pg/mL（范围，9至230），而24份条件培养基样品中有13份的TPO超过16 pg/mL（在ELISA的线性范围内），中值为37 pg/mL（范围，16至106）。我们的数据表明，由单个骨髓细胞启动的基质培养物可以产生FL、SCF和TPO。基质细胞局部产生早期作用细胞因子和TPO可能与骨髓微环境中造血干细胞自我更新和巨核细胞生成的调节有关。(C)1997年，美国血液学会。
We have previously characterized stromal progenitor cells contained in fetal bone marrow by fluorescence-activated cell sorting (FAGS) using the differential expression of CD34, CD38, and HLA-DR, and found that a small number were contained wit hin the CD34(+) cell fraction. In the present study, the frequency of stromal progenitors in both the CD34(+) and CD34(-) subpopulations from samples of fetal and adult bone marrow was approximately one in 5,000 of the mononuclear cell fraction. Using multiparameter single-cell sorting, one in 20 fetal bone marrow cells with the CD34(+), CD38(-), HLA-DR-, CDw90(+) phenotype were clonogenic stromal progenitors, whereas greater than one in five single cells with the CD34(-), CD38(-), HLA-DR-, CBw90(+) phenotype formed stromal cultures. We found that cultures initiated by hematopoietic and stromal progenitors contained within the CD34(+) fraction of bone marrow cells formed mixed hematopoietic/stromal cell cultures that maintained the viability of the hematopoietic progenitor cells for 3 weeks in the absence of added hematopoietic cytokines. We characterized some of the hematopoietic cytokines synthesized by stromal cultures derived from either CD34(+) or CD34(-) bone marrow cells using reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of interleukin-3 (IL-3), stem cell factor (SCF), CD34, Flt3/Flk2 ligand (FL), and thrombopoietin (TPO) mRNA sequences. We found ubiquitous expression of TPO mRNA in greater than 90% of stromal cultures initiated by either CD34(+) or CD34(-) cells, and variable expression of SCF, FL, and CD34 mRNA. In particular, SCF and CD34 mRNA were detected only in stromal cultures initiated by CD34(+) bone marrow cells, although the differences between CD34(+) and CD34(-) stromal cells were not statistically significant. IL-3 mRNA was not found in any stromal cultures. An enzyme-linked immunosorbent assay (ELISA) of soluble SCF and TPO present in culture supernatants demonstrated that biologically significant amounts of protein were secreted by some cultured stromal cells: eight of 16 samples of conditioned media from stromal cultures initiated by fetal and adult bone marrow contained more than 32 pg/ml SCF (in the linear range of the ELISA), with a median value of 32 pg/mL (range, 9 to 230), while 13 of 24 samples of conditioned media had more than 16 pg/mL TPO (in the linear range of the ELISA), with a median of 37 pg/mL (range, 16 to 106). Our data indicate that stromal cultures initiated by single bone marrow cells can make FL, SCF, and TPO. Local production of early-acting cytokines and TPO by stromal cells may be relevant to the regulation of hematopoietic stem cell self-renewal and megakaryocytopoiesis in the bone marrow microenvironment. (C) 1997 by The American Society of Hematology.