Pub Date : 2021-11-01DOI: 10.17303/jscr.2021.3.104
Sidhu Ks
{"title":"Isolation and Characterisation of a Dental Pulp-Derived Human Mesenchymal Stem Cell Line – CKC-Endeavour-2 and its Products Under Xeno- and Serum-Free Conditions","authors":"Sidhu Ks","doi":"10.17303/jscr.2021.3.104","DOIUrl":"https://doi.org/10.17303/jscr.2021.3.104","url":null,"abstract":"","PeriodicalId":93322,"journal":{"name":"Journal of stem cell reports","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2021-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48923249","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Background Brain ischemia leads to excessive infiltration of clusters of CD8+ T and natural killer (NK) cells in the brain, which aggravate ischemic brain injury. Acute ischemic stroke also has a negative impact on the antibacterial immune response, leading to stroke-induced immunodepression and infection. Umbilical cord mesenchymal stem cell (ucMSC) have an immunosuppressive function. Therefore, we aimed to determine whether ucMSC treatment alleviates the excessive infiltration of CD8+ T and NK cells. We also investigated significant concerns that ucMSC treatment might suppress antimicrobial immunity, leading to an increased risk of infection. Methods After middle cerebral artery occlusion, stroke and post-stroke infective mice received intravenous injection of ucMSC. We performed haematoxylin and eosin staining of organs and assessed the Modified Neurological Severity Score (mNSS),the activated state of microglia,quantity and distribution of CD8 + T and NK cells. Changes of cytokines (IL-6, TNF-α, IL-10), and blood biochemical indexes were also detected.We then assessed autophagy and apoptosis of platelets, as well as mitochondrial membrane potential (MMP) and ATP levels.In vitro ucMSC was co-cultured with platelet and Escherichia coli, followed by detection of the E. coli growth curve. Results ucMSC treatment ameliorated the infiltration of CD8+ T and NK cells in the brain, reduced levels of proinflammatory cytokines, and increased anti-inflammatory cytokines.ucMSC treatment limit post-stroke infection and reduce the inflmamatory injury of various organs induced by post-stroke infection,as well as ucMSC inhibit the growth of Escherichia coli in vivo and vitro.ucMSC treatment maintained autophagy, MMP, and the production of ATP, while inhibiting apoptosis of platelets in vivo. Conclusions Based on these findings, ucMSC may represent a potential and safe therapeutic option for stroke treatment by inhibiting brain injury and limiting post-stroke infection.
{"title":"Umbilical cord mesenchymal stem cells limit post-stroke infection","authors":"Jianbang Han, Yuanlong Xie, Z. Feng, Haitao Sun, Feng Li, Q. Ouyang, Zhongfei Zhang, Xiaoxiong Zou, Ying‐qian Cai, Yu-xi Zou, Y. Tang, Xiaodan Jiang","doi":"10.21203/rs.3.rs-218526/v1","DOIUrl":"https://doi.org/10.21203/rs.3.rs-218526/v1","url":null,"abstract":"\u0000 Background\u0000\u0000Brain ischemia leads to excessive infiltration of clusters of CD8+ T and natural killer (NK) cells in the brain, which aggravate ischemic brain injury. Acute ischemic stroke also has a negative impact on the antibacterial immune response, leading to stroke-induced immunodepression and infection. Umbilical cord mesenchymal stem cell (ucMSC) have an immunosuppressive function. Therefore, we aimed to determine whether ucMSC treatment alleviates the excessive infiltration of CD8+ T and NK cells. We also investigated significant concerns that ucMSC treatment might suppress antimicrobial immunity, leading to an increased risk of infection.\u0000Methods\u0000\u0000After middle cerebral artery occlusion, stroke and post-stroke infective mice received intravenous injection of ucMSC. We performed haematoxylin and eosin staining of organs and assessed the Modified Neurological Severity Score (mNSS),the activated state of microglia,quantity and distribution of CD8 + T and NK cells. Changes of cytokines (IL-6, TNF-α, IL-10), and blood biochemical indexes were also detected.We then assessed autophagy and apoptosis of platelets, as well as mitochondrial membrane potential (MMP) and ATP levels.In vitro ucMSC was co-cultured with platelet and Escherichia coli, followed by detection of the E. coli growth curve.\u0000Results\u0000\u0000ucMSC treatment ameliorated the infiltration of CD8+ T and NK cells in the brain, reduced levels of proinflammatory cytokines, and increased anti-inflammatory cytokines.ucMSC treatment limit post-stroke infection and reduce the inflmamatory injury of various organs induced by post-stroke infection,as well as ucMSC inhibit the growth of Escherichia coli in vivo and vitro.ucMSC treatment maintained autophagy, MMP, and the production of ATP, while inhibiting apoptosis of platelets in vivo.\u0000Conclusions\u0000\u0000Based on these findings, ucMSC may represent a potential and safe therapeutic option for stroke treatment by inhibiting brain injury and limiting post-stroke infection.","PeriodicalId":93322,"journal":{"name":"Journal of stem cell reports","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2021-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46813297","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Activation of the transcription factor P53 within cancer cells is a well-characterized pathway, whereas the effects of P53 activation during development remain largely unexplored. Previous research has indicated that increased levels of P53 protein during key murine developmental stages cause defects in multiple embryonic tissues, including the heart. These findings were confirmed in several different mouse models of congenital heart defects, but P53 activation in a human system of cardiovascular development is not available. Utilizing human induced pluripotent stem cells (hiPSCs), we characterized the normal levels of P53 during cardiac differentiation and showed that levels of P53 are high in hiPSCs and decrease upon cardiac lineage commitment. We also observed P53 localization changed from mainly cytoplasmic in iPS colonies to the nucleus in the Nkx2-5 + cardiac progenitor stage. Pharmacological-mediated increase of P53 protein levels with the Mdm2 inhibitor Nutlin-3a during early (mesoderm to cardiac mesoderm) stages of cardiogenesis resulted in a sizeable loss of cardiomyocytes due to increased apoptosis and cell cycle arrest. Interestingly, increasing P53 levels did not result in apoptosis at later (cardiac progenitor to beating cardiomyocytes) stages of the cardiac differentiation. These results illustrate the temporal sensitivity to increased P53 levels during cardiogenesis. We conducted RNA-Seq on these cells with or without Nutlin-3a to ascertain transcriptional differences due to increased P53 at the different stages during the differentiation. Our results from the RNA-Seq revealed up-regulation of Sestrins after Nutlin-3a treatment suggesting a new role for P53 in the metabolism of cardiac regeneration.
{"title":"Activation of P53 Via Nutlin-3a Reveals Role for P53 In ROS Signaling During Cardiac Differentiation of hiPSCs.","authors":"Emma B Brandt, Xing Li, Timothy J Nelson","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Activation of the transcription factor P53 within cancer cells is a well-characterized pathway, whereas the effects of P53 activation during development remain largely unexplored. Previous research has indicated that increased levels of P53 protein during key murine developmental stages cause defects in multiple embryonic tissues, including the heart. These findings were confirmed in several different mouse models of congenital heart defects, but P53 activation in a human system of cardiovascular development is not available. Utilizing human induced pluripotent stem cells (hiPSCs), we characterized the normal levels of P53 during cardiac differentiation and showed that levels of P53 are high in hiPSCs and decrease upon cardiac lineage commitment. We also observed P53 localization changed from mainly cytoplasmic in iPS colonies to the nucleus in the Nkx2-5 + cardiac progenitor stage. Pharmacological-mediated increase of P53 protein levels with the Mdm2 inhibitor Nutlin-3a during early (mesoderm to cardiac mesoderm) stages of cardiogenesis resulted in a sizeable loss of cardiomyocytes due to increased apoptosis and cell cycle arrest. Interestingly, increasing P53 levels did not result in apoptosis at later (cardiac progenitor to beating cardiomyocytes) stages of the cardiac differentiation. These results illustrate the temporal sensitivity to increased P53 levels during cardiogenesis. We conducted RNA-Seq on these cells with or without Nutlin-3a to ascertain transcriptional differences due to increased P53 at the different stages during the differentiation. Our results from the RNA-Seq revealed up-regulation of Sestrins after Nutlin-3a treatment suggesting a new role for P53 in the metabolism of cardiac regeneration.</p>","PeriodicalId":93322,"journal":{"name":"Journal of stem cell reports","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8415805/pdf/nihms-1732780.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39382286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-12-17DOI: 10.17303/jscr.2019.1.105
J. Murray, B. Mynampati, J. Brant, DO AbbySheffield, Marie Crandall, E. Scott
for Abstract As regenerative scaffolds exhibit varying pore sizes, producing adipose-derived stem/stromal cell (ASC) spheroids of selective sizes to populate these pores may be helpful in autologous tissue engineering. Herein we present two protocols for the initial magnetic sorting of ASCs and the subsequent size selective assembly of ASC spheroids; as the critical quality attribute of ASC identity has been shown previously, only spheroid morphology will be studied. Paramagnetic microbeads (Pro-tein G ligand Dynabeads®, Thermo Fisher Scientific) were used to create single-core paramagnetic immunobeads (scPIBs) and dual core paramagnetic immunobeads (dcPIBs). The scPIBs were created by conjugating the paramagnetic microbeads to ASC-selective primary antibodies (mouse antihuman CD 44, CD73, CD90, CD105, BD Biosciences). The dcPIBs were created by initially conjugating the paramagnetic microbeads to secondary antibodies (mouse IgG) and then conjugating the secondary antibodies to the same ASC-selective primary antibodies. The scPIBs and dcPIBs were then admixed within 15 ml of fresh lipoaspirate respectively. The ASC-scPIBs and ASC-dcPIBs were then magnetically precipitated and subsequently cultured in low adherent conditions for five days. Within twenty minutes, scPIBs isolated an average of 1.2 million putative ASCs (8 x 104 cells per ml of lipoaspirate processed) and dcPIBs isolated an average of 1.3 million putative ASCs (8.7 x 104 cells per ml of lipoaspirate processed). Spheroids comprised of ASC-scPIBs were 19.3 µm (average, +/- 5 µm) and spheroids of the ASC-dcPIBs were 216.7 µm (average, +/- 25 µm). ASCs were magnetically precipitated from fresh lipoaspirate in twenty minutes and subsequently underwent self-assembly of small (approximately 20 µm) or large (approximately 220 µm) ASC spheroids over five days. These protocols may be useful in the rapid development of size-selectable ASC spheroids, which may be particularly useful for autologous toxicology, pharmacology, disease modeling, and tissue regeneration.
摘要由于再生支架具有不同的孔径，产生选择性大小的脂肪来源干/基质细胞（ASC）球体来填充这些孔可能有助于自体组织工程。在此，我们提出了两种方案，用于ASC的初始磁性分选和随后ASC球体的尺寸选择性组装；正如ASC同一性的关键质量属性之前已经显示的那样，将只研究球体形态。顺磁性微珠（Pro tein G配体Dynabeads®，Thermo Fisher Scientific）用于制造单核顺磁性免疫珠（scPIB）和双核顺磁性免疫球（dcPIB）。scPIBs是通过将顺磁性微珠与ASC选择性一级抗体（小鼠抗人CD44、CD73、CD90、CD105、BD Biosciences）偶联而产生的。dcPIB是通过最初将顺磁性微珠与二级抗体（小鼠IgG）偶联，然后将二级抗体与相同的ASC选择性一级抗体偶联而产生的。然后将scPIBs和dcPIBs分别混合在15ml新鲜的脂肪抽吸物中。然后将ASC scPIBs和ASC dcPIBs磁性沉淀，随后在低粘附条件下培养5天。在20分钟内，scPIBs平均分离出120万个推定ASCs（每毫升处理的吸脂物8 x 104个细胞），dcPIBs则平均分离出130万个推定ASC（每毫升加工的吸脂器8.7 x 104个单元）。ASC scPIB组成的球体为19.3µm（平均值+/-5µm），ASC dcPIB的球体为216.7µm（均值+/-25µm）。ASCs在20分钟内从新鲜的吸脂器中磁性沉淀，随后在5天内经历小（约20µm）或大（约220µm）ASC球体的自组装。这些方案可能有助于快速开发可选择大小的ASC球体，这可能对自体毒理学、药理学、疾病建模和组织再生特别有用。
{"title":"Assembly of Size Selective Multicellular Spheroids of Adipose-Derived Stem/Stromal Cells for Use in Regenerative Tissue Engineering: A Methods and Morphologic Study","authors":"J. Murray, B. Mynampati, J. Brant, DO AbbySheffield, Marie Crandall, E. Scott","doi":"10.17303/jscr.2019.1.105","DOIUrl":"https://doi.org/10.17303/jscr.2019.1.105","url":null,"abstract":"for Abstract As regenerative scaffolds exhibit varying pore sizes, producing adipose-derived stem/stromal cell (ASC) spheroids of selective sizes to populate these pores may be helpful in autologous tissue engineering. Herein we present two protocols for the initial magnetic sorting of ASCs and the subsequent size selective assembly of ASC spheroids; as the critical quality attribute of ASC identity has been shown previously, only spheroid morphology will be studied. Paramagnetic microbeads (Pro-tein G ligand Dynabeads®, Thermo Fisher Scientific) were used to create single-core paramagnetic immunobeads (scPIBs) and dual core paramagnetic immunobeads (dcPIBs). The scPIBs were created by conjugating the paramagnetic microbeads to ASC-selective primary antibodies (mouse antihuman CD 44, CD73, CD90, CD105, BD Biosciences). The dcPIBs were created by initially conjugating the paramagnetic microbeads to secondary antibodies (mouse IgG) and then conjugating the secondary antibodies to the same ASC-selective primary antibodies. The scPIBs and dcPIBs were then admixed within 15 ml of fresh lipoaspirate respectively. The ASC-scPIBs and ASC-dcPIBs were then magnetically precipitated and subsequently cultured in low adherent conditions for five days. Within twenty minutes, scPIBs isolated an average of 1.2 million putative ASCs (8 x 104 cells per ml of lipoaspirate processed) and dcPIBs isolated an average of 1.3 million putative ASCs (8.7 x 104 cells per ml of lipoaspirate processed). Spheroids comprised of ASC-scPIBs were 19.3 µm (average, +/- 5 µm) and spheroids of the ASC-dcPIBs were 216.7 µm (average, +/- 25 µm). ASCs were magnetically precipitated from fresh lipoaspirate in twenty minutes and subsequently underwent self-assembly of small (approximately 20 µm) or large (approximately 220 µm) ASC spheroids over five days. These protocols may be useful in the rapid development of size-selectable ASC spheroids, which may be particularly useful for autologous toxicology, pharmacology, disease modeling, and tissue regeneration.","PeriodicalId":93322,"journal":{"name":"Journal of stem cell reports","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49385589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
J. Murray, A. Doty, Noah J. Jones, Marie Crandall, E. Scott
Adipose tissue-derived mesenchymal stem cells (ASCs) may be isolated in clinically useful quantities without in vitro expansion. The purpose of this study is to validate a novel method for the enrichment of primary ASCs using paramagnetic beads. Primary rabbit anti-mouse antibodies were bound to paramagnetic microbeads. Secondary antibodies, selective for ASCs, were then bound to the primary antibodies to construct so-called paramagnetic immunobeads (PIBs). PIBs were then added to fresh human lipoaspirate to create ASC-PIB conjugates (aPIBS) over 10 minutes. A hand-held magnet was then placed adjacent to the lipoaspirate-aPIBs mixture, and over the next 10 minutes, the aPIBs were precipitated. Live cell count per mL of lipoaspirate was 9.6 x 104. Scanning electron microscopy revealed precipitates consistent with aPIBs. Flow cytometry identified cell-bound markers for CD90 and CD105 while culture confirmed tri-lineage differentiation, all attributes diagnostic of ASCs. This study validates that functional ASCs may be isolated from lipoaspirate by magnetic enrichment in 20 minutes. As both the harvest of adipose tissue by liposuction and this ASC enrichment technique do not require electricity, fresh primary therapeutic ASCs may be isolated in any point-of-care setting, even in developing countries.
{"title":"A New Ball for an Old Trick: Paramagnetic Cell Sorting of Human Mesenchymal Stem Cells from Adipose Tissue","authors":"J. Murray, A. Doty, Noah J. Jones, Marie Crandall, E. Scott","doi":"10.17303/jscr.2019","DOIUrl":"https://doi.org/10.17303/jscr.2019","url":null,"abstract":"Adipose tissue-derived mesenchymal stem cells (ASCs) may be isolated in clinically useful quantities without in vitro expansion. The purpose of this study is to validate a novel method for the enrichment of primary ASCs using paramagnetic beads. Primary rabbit anti-mouse antibodies were bound to paramagnetic microbeads. Secondary antibodies, selective for ASCs, were then bound to the primary antibodies to construct so-called paramagnetic immunobeads (PIBs). PIBs were then added to fresh human lipoaspirate to create ASC-PIB conjugates (aPIBS) over 10 minutes. A hand-held magnet was then placed adjacent to the lipoaspirate-aPIBs mixture, and over the next 10 minutes, the aPIBs were precipitated. Live cell count per mL of lipoaspirate was 9.6 x 104. Scanning electron microscopy revealed precipitates consistent with aPIBs. Flow cytometry identified cell-bound markers for CD90 and CD105 while culture confirmed tri-lineage differentiation, all attributes diagnostic of ASCs. This study validates that functional ASCs may be isolated from lipoaspirate by magnetic enrichment in 20 minutes. As both the harvest of adipose tissue by liposuction and this ASC enrichment technique do not require electricity, fresh primary therapeutic ASCs may be isolated in any point-of-care setting, even in developing countries.","PeriodicalId":93322,"journal":{"name":"Journal of stem cell reports","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47790238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-06-30DOI: 10.17303/jscr.2019.1.103
{"title":"Beyond a “Pinch of this and a Handful of that:” Using Manufacturing Standards for the Isolation of Human Mesenchymal Stem Cells From Adipose Tissue- A Novel Point-of-Care Device","authors":"","doi":"10.17303/jscr.2019.1.103","DOIUrl":"https://doi.org/10.17303/jscr.2019.1.103","url":null,"abstract":"","PeriodicalId":93322,"journal":{"name":"Journal of stem cell reports","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42833661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-05-03DOI: 10.17303/jscr.2019.1.101
{"title":"The L-PRF Membrane (Fibrin Rich in Platelets and Leukocytes) And Its Derivatives Useful as A Source of Stem Cells in Wound Surgery","authors":"","doi":"10.17303/jscr.2019.1.101","DOIUrl":"https://doi.org/10.17303/jscr.2019.1.101","url":null,"abstract":"","PeriodicalId":93322,"journal":{"name":"Journal of stem cell reports","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44800491","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-05-03DOI: 10.17303/jscr.2019.1.102
{"title":"Transcriptome Analysis of Mesenchymal Stem Cells Differentiated into Insu- lin-Producing Cells Reveals Dissimilarities with Pancreatic Beta Cells in Re- sponse to Glucose","authors":"","doi":"10.17303/jscr.2019.1.102","DOIUrl":"https://doi.org/10.17303/jscr.2019.1.102","url":null,"abstract":"","PeriodicalId":93322,"journal":{"name":"Journal of stem cell reports","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47200024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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