PDF(Pubmed)
Abstract：
BACKGROUND: • Neural stem/progenitor cells (NSPCs) are multipotent self-renewing cells that can be isolated from the brain or spinal cord. As they need to be isolated from neural tissues, it is difficult to study human NSPCs. To facilitate NSPC research, we attempted to isolate NSPCs from dogs, as dogs share the environment and having many similar diseases with humans. We collected and established primary cultures of ependymal and subependymal cells from the central canal of the cervical spinal cord of adult dogs. To isolate pure NSPCs, we employed the monolayer culture and selective medium culture methods. We further tested the ability of the NSPCs to form neurospheres (using the suspension culture method) and evaluated their differentiation potential.
RESULTS: • The cells had the ability to grow as cultures for up to 10 passages; the growth curves of the cells at the 3rd, 6th, and 9th passages showed similar patterns. The NSPCs were able to grow as neurospheres as well as monolayers, and immunostaining at the 3rd, 6th, and 9th passages showed that these cells expressed NSPC markers such as nestin and SOX2 (immunofluorescent staining). Monolayer cultures of NSPCs at the 3rd, 6th, and 9th passages were cultured for approximately 14 days using a differentiation medium and were observed to successfully differentiate into neural lineage and glial cells (astrocytes, neurons, and oligodendrocytes) at all the three passages tested.
CONCLUSIONS: • It is feasible to isolate and propagate (up to at least 10 passages) canine cervical spinal cord-derived NSPCs with the capacity to differentiate into neuronal and glial cells. To the best of our knowledge this is the first study to successfully isolate, propagate, and differentiate canine NSPCs derived from cervical spinal cord in the adult canine, and we believe that these cells will contribute to the field of spinal cord regeneration in veterinary and comparative medicine.
RESULTS: • The cells had the ability to grow as cultures for up to 10 passages; the growth curves of the cells at the 3rd, 6th, and 9th passages showed similar patterns. The NSPCs were able to grow as neurospheres as well as monolayers, and immunostaining at the 3rd, 6th, and 9th passages showed that these cells expressed NSPC markers such as nestin and SOX2 (immunofluorescent staining). Monolayer cultures of NSPCs at the 3rd, 6th, and 9th passages were cultured for approximately 14 days using a differentiation medium and were observed to successfully differentiate into neural lineage and glial cells (astrocytes, neurons, and oligodendrocytes) at all the three passages tested.
CONCLUSIONS: • It is feasible to isolate and propagate (up to at least 10 passages) canine cervical spinal cord-derived NSPCs with the capacity to differentiate into neuronal and glial cells. To the best of our knowledge this is the first study to successfully isolate, propagate, and differentiate canine NSPCs derived from cervical spinal cord in the adult canine, and we believe that these cells will contribute to the field of spinal cord regeneration in veterinary and comparative medicine.
摘要：
背景：•神经干/祖细胞（NSPC）是可以从脑或脊髓分离的多能自我更新细胞。因为它们需要从神经组织中分离出来，研究人类NSPCs是困难的。为了促进NSPC研究，我们试图从狗身上分离出NSPCs，因为狗与人类共享环境并患有许多类似的疾病。我们从成年犬的颈脊髓中央管收集并建立了室管膜和室管膜下细胞的原代培养物。要分离纯NSPC，我们采用单层培养和选择性培养基培养方法。我们进一步测试了NSPC形成神经球的能力（使用悬浮培养方法）并评估了它们的分化潜力。
结果：•细胞具有培养多达10代的能力；细胞在第3代的生长曲线，6th,第9段显示出类似的模式。NSPCs能够生长为神经球和单层，3号免疫染色，6th,和第9代显示这些细胞表达NSPC标记,例如巢蛋白和SOX2(免疫荧光染色)。NSPC的单层培养在第三，6th,和第9代使用分化培养基培养约14天，并观察到成功分化为神经谱系和神经胶质细胞（星形胶质细胞，神经元，和少突胶质细胞）在测试的所有三个传代中。
结论：•分离和繁殖（至少10代）犬颈脊髓来源的NSPCs具有分化为神经元和神经胶质细胞的能力是可行的。据我们所知，这是第一个成功分离的研究，传播,并区分成年犬的颈脊髓来源的犬NSPCs，我们相信这些细胞将有助于兽医学和比较医学的脊髓再生领域。
结果：•细胞具有培养多达10代的能力；细胞在第3代的生长曲线，6th,第9段显示出类似的模式。NSPCs能够生长为神经球和单层，3号免疫染色，6th,和第9代显示这些细胞表达NSPC标记,例如巢蛋白和SOX2(免疫荧光染色)。NSPC的单层培养在第三，6th,和第9代使用分化培养基培养约14天，并观察到成功分化为神经谱系和神经胶质细胞（星形胶质细胞，神经元，和少突胶质细胞）在测试的所有三个传代中。
结论：•分离和繁殖（至少10代）犬颈脊髓来源的NSPCs具有分化为神经元和神经胶质细胞的能力是可行的。据我们所知，这是第一个成功分离的研究，传播,并区分成年犬的颈脊髓来源的犬NSPCs，我们相信这些细胞将有助于兽医学和比较医学的脊髓再生领域。
