Luzuriaga Jon, Pineda Jose Ramon, Irastorza Igor, Uribe-Etxebarria Veronica, García-Gallastegui Patricia, Encinas Juan Manuel, Chamero Pablo, Unda Fernando, Ibarretxe Gaskon
Department of Cell Biology and Histology, Faculty of Medicine and Nursing, University of the Basque Country, UPV/EHU, Leioa, Spain.
Achucarro Basque Center for Neuroscience, UPV/EHU Scientific Park, Leioa, Spain,
Cell Physiol Biochem. 2019;52(6):1361-1380. doi: 10.33594/000000096.
BACKGROUND/AIMS: Human Dental Pulp Stem Cells (hDPSCs) are one of the most promising types of cells to regenerate nerve tissues. Standard DMEM+10% fetal bovine serum (FBS) culture medium allows a fast expansion of hDPSC as a surface-adherent cell monolayer. However, the use of FBS also compromises the clinical use of these protocols, and its longterm presence favors hDPSCs differentiation toward mesenchymal cell-derived lineages, at the expense of a reduced capability to generate neural cells. The objective of this work was to characterize the role of neurotrophin signaling on hDPSCs using a serum-free culture protocol, and to assess the neurogenic and gliogenic capacity of hDPSCs for future nerve tissue bioengineering and regeneration.
We compared the different expression of neurotrophin receptors by RT-PCR, Q-PCR, and IF of hDPSCs cultured with different growth media in the presence or absence of serum. Moreover, we assessed the response of hDPSCs to stimulation of neurotransmitter receptors by live cell calcium imaging under these different media. Finally, we compared the osteogenic potential of hDPSCs by Alizarin red staining, and the differentiation to gliogenic/neurogenic fates by immunostaining for Schwann lineage and neuronal lineage markers. We tested a commercial serum-free medium designed for the growth of mesenchymal stem cells: StemPro MSCTM (STP).
hDPSCs cultured in STP generated small non-adherent floating dentospheres that showed very low proliferation rates, in contrast to standard FBS-containing medium. We found that hDPSCs grown in STP conditions overexpressed neurotrophin receptor genes NTRK2 (TrkB) and NTRK3 (TrkC). Interestingly, the stimulation of these receptors by adding their respective ligands BDNF and NT-3 to STP medium enhanced the neural crest (NC) progenitor features of cultured hDPSCs. We observed a 10 to 100-fold increase of migratory NC cell markers HNK1 and P75, and a significant overexpression of pluripotency core factors SOX2, OCT4 and NANOG. Moreover, hDPSCs cultured in BDNF/NT-3 supplemented STP showed a largely increased potential to differentiate towards neuronal and Schwann glial lineage cells, assessed by positive immunostaining for DCX, NeuN and S100ß, p75 markers, respectively.
Our results demonstrate that the use of BDNF and NT-3 combined with STP induced the partial reprogramming of ectomesenchymal hDPSCs to generate early NC progenitor cells, which are far more competent for neuronal and glial differentiation than hDPSCs grown in the presence of FBS.
背景/目的：人牙髓干细胞（hDPSCs）是最有希望用于神经组织再生的细胞类型之一。标准的含10%胎牛血清（FBS）的DMEM培养基可使hDPSCs作为贴壁细胞单层快速扩增。然而，FBS的使用也影响了这些方案的临床应用，并且其长期存在有利于hDPSCs向间充质细胞衍生谱系分化，却以生成神经细胞的能力降低为代价。本研究的目的是使用无血清培养方案来表征神经营养因子信号在hDPSCs中的作用，并评估hDPSCs在未来神经组织生物工程和再生中的神经发生和胶质发生能力。
我们通过RT-PCR、Q-PCR和免疫荧光（IF）比较了在有无血清的情况下用不同生长培养基培养的hDPSCs神经营养因子受体的不同表达。此外，我们在这些不同培养基下通过活细胞钙成像评估了hDPSCs对神经递质受体刺激的反应。最后，我们通过茜素红染色比较了hDPSCs的成骨潜力，并通过对雪旺氏谱系和神经元谱系标志物进行免疫染色来比较其向胶质/神经命运的分化。我们测试了一种专为间充质干细胞生长设计的商业无血清培养基：StemPro MSC™（STP）。
与标准的含FBS培养基相比，在STP中培养的hDPSCs形成了小的非贴壁漂浮牙球，其增殖率非常低。我们发现，在STP条件下生长的hDPSCs过表达神经营养因子受体基因NTRK2（TrkB）和NTRK3（TrkC）。有趣的是，通过向STP培养基中添加各自的配体BDNF和NT-3来刺激这些受体，增强了培养的hDPSCs的神经嵴（NC）祖细胞特征。我们观察到迁移性NC细胞标志物HNK1和P75增加了10到100倍，并且多能性核心因子SOX2、OCT4和NANOG显著过表达。此外，通过分别对DCX、NeuN和S100β、p75标志物进行阳性免疫染色评估，在补充了BDNF/NT-3的STP中培养的hDPSCs向神经元和雪旺氏胶质谱系细胞分化的潜力大大增加。
我们的结果表明，BDNF和NT-3与STP联合使用可诱导外胚间充质hDPSCs部分重编程以生成早期NC祖细胞，这些祖细胞在神经元和胶质细胞分化方面比在FBS存在下生长的hDPSCs更有能力。
