Bruder S P, Jaiswal N, Haynesworth S E
Osiris Therapeutics, Inc., Baltimore, MD 21231-2001, USA.
J Cell Biochem. 1997 Feb;64(2):278-94. doi: 10.1002/(sici)1097-4644(199702)64:2<278::aid-jcb11>3.0.co;2-f.
Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 +/- 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime.
最近的研究表明，人类骨髓中存在一部分能够沿着多个间充质谱系分化的细胞。这些间充质干细胞（MSCs）不仅具有多谱系发育潜能，而且可以在体外培养许多代而不明显表现出分化表型。本研究的目的是确定纯化的MSCs在广泛传代培养及冷冻保存后的生长动力学、自我更新能力和成骨潜能。从正常髂嵴骨髓抽吸物中建立MSCs原代培养物，取一份进行冷冻保存和解冻，然后将冷冻和未冷冻的细胞群体平行传代培养多达15代。对每一代细胞进行生长动力学分析，并检测其在含有地塞米松的成骨诱导培养基中的成骨潜能。原代培养中的纺锤形人MSCs表现出一个生长延迟期，随后是对数期，最终进入生长平台期。传代培养的细胞经历相同的阶段，然而，对数期的生长速率以及培养固定时间后的最终细胞数量会随着传代次数的增加而减少。骨髓来源的成人MSCs的群体倍增平均数确定为38±4，此时细胞最终变得非常宽大扁平，然后退化。细胞的成骨潜能在每一代中都得以保留，碱性磷酸酶（APase）活性显著增加以及矿化结节聚集体的形成证明了这一点。此外，细胞的冷冻保存和解冻过程对其生长或成骨分化均无影响。重要的是，这些研究表明MSCs的复制性衰老并非终末分化状态，因为这些细胞仍能够沿着成骨谱系进展。使用群体倍增潜能作为生物学年龄的衡量指标表明，MSCs介于胚胎组织和成人组织之间，因此，可能在成年人的一生中为间充质祖细胞提供原位来源。
