Kadiyala S, Young R G, Thiede M A, Bruder S P
Osiris Therapeutics, Inc., Baltimore, MD 21231, USA.
Cell Transplant. 1997 Mar-Apr;6(2):125-34. doi: 10.1177/096368979700600206.
Mesenchymal Stem Cells (MSCs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes, myoblasts, and adipocytes have been previously isolated from the marrow and periosteum of human, murine, lapine, and avian species. This study documents the existence of similar multipotential stem cells in canine marrow. The cells were isolated from marrow aspirates using a modification of techniques previously established for human MSCs (hMSCs), and found to possess similar growth and morphological characteristics, as well as osteochondrogenic potential in vivo and in vitro. On the basis of these results, the multipotential cells that were isolated and culture expanded are considered to be canine MSCs (cMSCs). The occurrence of cMSCs in the marrow was determined to be one per 2.5 x 10(4) nucleated cells. After enrichment of the cMSCs by centrifugation on a Percoll cushion, the cells were cultivated in selected lots of serum. Like the hMSCs, cMSCs grew as colonies in primary culture and on replating, grew as a monolayer culture with very uniform spindle morphology. The population doubling time for these cMSCs was approximately 2 days. The morphology and the growth kinetics of the cMSCs were retained following repeated passaging. The osteogenic phenotype could be induced in the cMSC cultures by the addition of a synthetic glucocorticoid, dexamethasone. In these osteogenic cultures, alkaline phosphatase activity was elevated up to 10-fold, and mineralized matrix production was evident. When cMSCs were loaded onto porous ceramics and implanted in autologous canine or athymic murine hosts, copious amounts of bone and cartilage were formed in the pores of the implants. The MSC-mediated osteogenesis obtained by the implantation of the various MSC-loaded matrix combinations is the first evidence of osteogenesis in a canine model by implantation of culture expanded autologous stem cells. The identification and isolation of cMSCs now makes it feasible to pursue preclinical models of bone and cartilage regeneration in canine hosts.
间充质干细胞(MSCs)具有分化为多种细胞类型的能力,如成骨细胞、软骨细胞、成肌细胞和脂肪细胞,此前已从人、鼠、兔和禽类的骨髓和骨膜中分离出来。本研究记录了犬骨髓中存在类似的多能干细胞。这些细胞是通过对先前用于人MSCs(hMSCs)的技术进行改进,从骨髓抽吸物中分离出来的,发现它们具有相似的生长和形态特征,以及体内和体外的骨软骨生成潜力。基于这些结果,分离并经培养扩增的多能细胞被认为是犬间充质干细胞(cMSCs)。经测定,骨髓中cMSCs的出现频率为每2.5×10⁴个有核细胞中有一个。通过在Percoll垫层上离心富集cMSCs后,将细胞培养于选定批次的血清中。与hMSCs一样,cMSCs在原代培养中以集落形式生长,传代培养时以单层培养形式生长,具有非常均匀的纺锤形形态。这些cMSCs的群体倍增时间约为2天。重复传代后,cMSCs的形态和生长动力学得以保留。通过添加合成糖皮质激素地塞米松,可在cMSC培养物中诱导成骨表型。在这些成骨培养物中,碱性磷酸酶活性升高至10倍,并且明显有矿化基质生成。当将cMSCs加载到多孔陶瓷上并植入自体犬或无胸腺小鼠宿主中时,植入物的孔隙中形成了大量的骨和软骨。通过植入各种负载MSC的基质组合获得的MSC介导的骨生成是通过植入培养扩增的自体干细胞在犬模型中实现骨生成的首个证据。cMSCs的鉴定和分离现在使得在犬宿主中进行骨和软骨再生的临床前模型研究成为可能。