scholarly journals Ascorbic acid can promote the generation and expansion of neuroepithelial-like stem cells derived from hiPS/ES cells under chemically defined conditions through promoting collagen synthesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rui Bai ◽  
Yun Chang ◽  
Amina Saleem ◽  
Fujian Wu ◽  
Lei Tian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture System ◽  
Collagen Synthesis ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Induction System ◽  
Defined Culture

Abstract Introduction Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs), which hold promise in the treatment of SCI by transplantation. In our study, we aimed to establish a chemically defined culture system using serum-free medium and ascorbic acid (AA) to generate and expand long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably. Methods We induced human embryonic stem cells (hESCs)/induced PSCs (iPSCs) to neurospheres using a newly established in vitro induction system. Moreover, lt-NES cells were derived from hESC/iPSC-neurospheres using two induction systems, i.e., conventional N2 medium with gelatin-coated plates (coated) and N2+AA medium without pre-coated plates (AA), and were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons. A microelectrode array (MEA) recording system was used to evaluate the functionality of the neurons differentiated from lt-NES cells. Finally, the mechanism underlying the induction of lt-NES cells by AA was explored through RNA-seq and the use of inhibitors. Results HESCs/iPSCs were efficiently induced to neurospheres using a newly established induction system in vitro. lt-NES cells derived from hESC/iPSC-neurospheres using the two induction systems (coated vs. AA) both expressed the neural pluripotency-associated genes PAX6, NESTIN, SOX1, and SOX2. After long-term cultivation, we found that they both exhibited long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retained the ability to differentiate into general functional neurons that express β-tubulin at high levels. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathway. Conclusions This new chemically defined culture system was stable and effective regarding the generation and culture of lt-NES cells induced from hESCs/iPSCs using serum-free medium combined with AA. The lt-NES cells induced under this culture system maintained their long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons. Graphical abstract

scholarly journals Ascorbic Acid Can Promote the Generation and Expansion of Neuroepithelial-Like Stem Cells Derived from HiPS/ES Cells Under Chemically Defined Conditions Through Promoting Collagen Synthesis

2020 ◽  
Author(s):  
Rui Bai ◽  
Yun Chang ◽  
Amina Saleem ◽  
Fujian Wu ◽  
Lei Tian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ascorbic Acid ◽  
Pluripotent Stem Cells ◽  
Culture System ◽  
Serum Free Medium ◽  
Cord Injury ◽  
Induction System ◽  
Defined Culture

Abstract Introduction: Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs) which hold promise in therapy for SCI by transplantation. In our study, we aimed to establish a chemically defined culture system by using serum-free medium and ascorbic acid (AA) to generation and expansion of long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably. Methods: We induce hESC/iPSC to neurospheres by using a newly established induction system in vitro in our study. And lt-NES cells derived from hESCs/iPSCs-neurospheres using two induction systems, including conventional N2 medium with gelatin-coated (coated) and N2+AA medium without pre-coated (AA) were characterized by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons and the microelectrode array (MEA) recording system was used to evaluate the functionality of neurons differentiated from lt-NES cells. Moreover, the mechanism of AA-induced lt-NES cells was explored through RNA-seq and the use of inhibitors. Results: HESCs/iPSCs were efficiently induced to neurospheres by using a newly established induction system in vitro. And lt-NES cells derived from hESCs/iPSCs-neurospheres using two induction system (coated vs AA) both expressed neural pluripotency-associated genes PAX6, NESTIN, SOX1, SOX2. After long-term cultivation, we found that they both can maintain the long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retain the ability to differentiate into general functional neurons that highly express β-tubulin. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathways. Conclusions: Taken together, this new chemically defined culture system is stable and effective to generate and culture the lt-NES cells induced by hESCs/iPSCs using serum-free medium combined with ascorbic acid (AA). The lt-NES cells under this culture system can maintain the long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons. Keywords: Spinal cord injury, Neurospheres, Ascorbic acid, lt-NES cells, Human pluripotent stem cells.

A Serum-Free in vitro Culture System for Crayfish Organs

1986 ◽  
Vol 41 (4) ◽  
pp. 472-476 ◽  
Author(s):  
Gerd Gellissen ◽  
Marco Traub ◽  
Klaus-Dieter Spindler
Keyword(s):  
Culture System ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Serum Free Medium ◽  
Free Medium ◽  
Astacus Leptodactylus ◽  
Serum Free ◽  
In Vitro Culture System

Midgut gland and hypodermis of the crayfish Astacus leptodactylus have been cultured in a serum-free medium for several days. The medium consists of 1 part of van Harreveld solution and 1 part of an amino acid mixture supplemented with 1.2 mᴍ Na2HP04, 12 mᴍ Hepes and 80 mᴍ glucose. The antibiotics penicillin (15 mg/l) and streptomycin (25 mg/1) were added for long term culturing. This medium, called TG medium, allows the maintainance of the tissues for more than 100 h without any loss of their viability with respect to protein synthesis and secretion.

scholarly journals Quantitation of the quiescent fraction of long-term culture-initiating cells in normal human blood and marrow and the kinetics of their growth factor-stimulated entry into S-phase in vitro

Blood ◽  
1995 ◽  
Vol 86 (9) ◽  
pp. 3314-3321 ◽  
Author(s):  
L Ponchio ◽  
E Conneally ◽  
C Eaves
Keyword(s):  
Specific Activity ◽  
Hematopoietic Cells ◽  
High Specific Activity ◽  
S Phase ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Long Term Culture

A method for quantitating the proportion of cycling long-term culture- initiating cells (LTC-IC) in heterogeneous populations of human hematopoietic cells is described. This procedure involves incubating the cells of interest for 16 to 24 hours in a serum-free medium containing 100 ng/mL Steel factor (SF), 20 ng/mL interleukin-3 (IL-3), and 20 ng/mL granulocyte-colony-stimulating factor (G-CSF), with or without 20 microCi/mL of high specific activity 3H-thymidine (3H-Tdr) before plating the recovered cells in standard LTC-IC assays. The details of this procedure are based in part on the finding that the number of LTC-IC (regardless of their cycling status) remains constant for at least 24 hours under these culture conditions, as long as 3H-Tdr is not present. In addition, we have determined that a 16-hour period of exposure to the 3H-Tdr is sufficient to maximize the discrimination of cycling LTC-IC but not long enough to allow a detectable redistribution of LTC-IC between noncycling and cycling compartments. Finally, any isotope reutilization that may occur is not sufficient to affect the LTC-IC 3H-Tdr suicide values measured. Application of this methodology to normally circulating LTC-IC showed these to be a primarily quiescent population. However, within 72 hours of incubation in a serum-free medium containing SF, IL-3, and G-CSF, most had entered S-phase, although there was no net change in their numbers. This suggests that, under certain conditions in vitro, self-renewal divisions of LTC-IC can occur and, at least initially, balance any losses of these cells due to their differentiation or death. In contrast, many of the LTC-IC in freshly aspirated samples of normal marrow were found to be proliferating, although those that were initially quiescent could also be recruited into S-phase within 72 hours in vitro when incubated under the same conditions used to stimulate circulating LTC-IC. This modified 3H-Tdr suicide procedure should facilitate further investigation of the mechanisms regulating the turnover of the most primitive compartments of human hematopoietic cells and how these may be altered in disease states or exploited for a variety of therapeutic applications.

scholarly journals Evaluation of growth, stemness, and angiogenic properties of dental pulp stem cells cultured in cGMP xeno-/serum-free medium

2019 ◽  
Vol 380 (1) ◽  
pp. 93-105 ◽  
Author(s):  
Chengjuan Qu ◽  
Maria Brohlin ◽  
Paul J Kingham ◽  
Peyman Kelk
Keyword(s):  
Stem Cells ◽  
Human Serum ◽  
Dental Pulp ◽  
Dental Pulp Stem Cells ◽  
In Vitro Assays ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Cells Cultured

AbstractThis study was aimed to investigate the effects of cGMP xeno-/serum-free medium (XSF, Irvine Scientific) on the properties of human dental pulp stem cells (DPSCs). DPSCs, from passage 2, were cultured in XSF or fetal bovine serum (FBS)-supplemented medium, and sub-cultured up to passage 8. Cumulative population doublings (PDs) and the number of colony-forming-units (CFUs) were determined. qRT-PCR, ELISA, and in vitro assays were used to assess angiogenic capacity. Flow cytometry was used to measure CD73, CD90, and CD105 expression. Differentiation into osteo-, adipo-, and chondrogenic cell lineages was performed. DPSCs showed more elongated morphology, a reduced rate of proliferation at later passages, and lower CFU counts in XSF compared with FBS. Expression of angiogenic factors at the gene and protein levels varied in the two media and with passage number, but cells grown in XSF had more in vitro angiogenic activity. The majority of early and late passage DPSCs cultured in XSF expressed CD73 and CD90. In contrast, the percentage of CD105 positive DPSCs in XSF medium was significantly lower with increased passage whereas the majority of cells cultured in FBS were CD105 positive. Switching XSF-cultured DPSCs to medium supplemented with human serum restored the expression of CD105. The tri-lineage differentiation of DPSCs cultured under XSF and FBS conditions was similar. We showed that despite reduced CD105 expression levels, DPSCs expanded in XSF medium maintained a functional MSC phenotype. Furthermore, restoration of CD105 expression is likely to occur upon in vivo transplantation, when cells are exposed to human serum.

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