scholarly journals Clever Experimental Designs: Shortcuts for Better iPSC Differentiation

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3540
Author(s):  
Ryota Yasui ◽  
Keisuke Sekine ◽  
Hideki Taniguchi
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Chinese Hamster ◽  
Cell Lineage ◽  
Statistical Design ◽  
Industrial Applications ◽  
Culture Conditions ◽  
Statistical Design Of Experiments ◽  
Hematopoietic Stem ◽  
Screening Design

For practical use of pluripotent stem cells (PSCs) for disease modelling, drug screening, and regenerative medicine, the cell differentiation process needs to be properly refined to generate end products with consistent and high quality. To construct and optimize a robust cell-induction process, a myriad of cell culture conditions should be considered. In contrast to inefficient brute-force screening, statistical design of experiments (DOE) approaches, such as factorial design, orthogonal array design, response surface methodology (RSM), definitive screening design (DSD), and mixture design, enable efficient and strategic screening of conditions in smaller experimental runs through multifactorial screening and/or quantitative modeling. Although DOE has become routinely utilized in the bioengineering and pharmaceutical fields, the imminent need of more detailed cell-lineage specification, complex organoid construction, and a stable supply of qualified cell-derived material requires expedition of DOE utilization in stem cell bioprocessing. This review summarizes DOE-based cell culture optimizations of PSCs, mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), and Chinese hamster ovary (CHO) cells, which guide effective research and development of PSC-derived materials for academic and industrial applications.

scholarly journals A subset of mobilized human hematopoietic stem cells express germ layer lineage genes which can be modulated by culture conditions

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Tapas Makar ◽  
Vamshi K. Nimmagadda ◽  
Poornachander R. Guda ◽  
Brian Hampton ◽  
Weiliang Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Culture Conditions ◽  
Germ Layer ◽  
Hematopoietic Stem

scholarly journals Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences.

1986 ◽  
Vol 6 (6) ◽  
pp. 2262-2266 ◽  
Author(s):  
J A Lewis ◽  
D A Matkovich
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Thymidine Kinase ◽  
Growth Phase ◽  
Chinese Hamster ◽  
Culture Conditions ◽  
Kinase Gene ◽  
Simplex Virus ◽  
Adenovirus 5

We have constructed a chimeric thymidine kinase (TK) minigene, pHe delta 6Ha, which combines the complete coding and 3' noncoding regions of a Chinese hamster TK cDNA with the promoter region and 5' untranslated region of the TK gene of herpes simplex virus type 1. We have transformed rat 4 cells to Tk+ with this gene and analyzed the pattern of TK gene expression in these transformants under various conditions of in vitro cell culture. We find that TK gene expression in these Tk+ transformants is growth phase dependent, responsive to adenovirus 5 infection, and indistinguishable in character under a variety of cell culture conditions from the pattern of TK gene expression in rat 4 cells transformed to Tk+ with the genomic Chinese hamster TK gene clone lambda HaTK.5. We are led to the conclusion that the genetic elements which mediate growth phase-dependent TK gene expression are contained entirely within the sequences of the mature cytoplasmic hamster TK mRNA.

Expression of 4-Integrin Defines the Earliest Precursor of Hematopoietic Cell Lineage Diverged From Endothelial Cells

Blood ◽  
1999 ◽  
Vol 93 (4) ◽  
pp. 1168-1177 ◽  
Author(s):  
Minetaro Ogawa ◽  
Masami Kizumoto ◽  
Satomi Nishikawa ◽  
Tetsuhiro Fujimoto ◽  
Hiroaki Kodama ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Cell Population ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Hematopoietic Cell ◽  
Hematopoietic Stem ◽  
Hematopoietic Cell Lineage ◽  
E Cadherin

Abstract Embryonic stem cells can differentiate in vitro into hematopoietic cells through two intermediate stages; the first being FLK1+ E-cadherin− proximal lateral mesoderm and the second being CD45− VE-cadherin+endothelial cells. To further dissect the CD45−VE-cadherin+ cells, we have examined distribution of 4-integrin on this cell population, because 4-integrin is the molecule expressed on hematopoietic stem cells. During culture of FLK1+ E-cadherin− cells, CD45− VE-cadherin+4-integrin− cells differentiate first, followed by 4-integrin+ cells appearing in both CD45− VE-cadherin+ and CD45−VE-cadherin− cell populations. In the CD45−VE-cadherin+ cell population, 4-integrin+ subset but not 4-integrin− subset had the potential to differentiate to hematopoietic lineage cells, whereas endothelial cell progenitors were present in both subsets. The CD45−VE-cadherin− 4-integrin+ cells also showed hematopoietic potential. Reverse transcription-polymerase chain reaction analyses showed that differential expression of the Gata2 and Myb genes correlated with the potential of the 4-integrin+ cells to give rise to hematopoietic cell differentiation. Hematopoietic CD45−VE-cadherin+ 4-integrin+ cells were also present in the yolk sac and embryonic body proper of 9.5 day postcoitum mouse embryos. Our results suggest that the expression of 4-integrin is a marker of the earliest precursor of hematopoietic cell lineage that was diverged from endothelial progenitors.

scholarly journals Silk fibroin induces chondrogenic differentiation of canine adipose–derived multipotent mesenchymal stromal cells/mesenchymal stem cells

2019 ◽  
Vol 10 ◽  
pp. 204173141983505 ◽  
Author(s):  
Metka Voga ◽  
Natasa Drnovsek ◽  
Sasa Novak ◽  
Gregor Majdic
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Culture Medium ◽  
Collagen Type ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Culture Conditions ◽  
Standard Cell ◽  
Cell Culture Medium

Under appropriate culture conditions, mesenchymal stem cells (MSC), also called more properly multipotent mesenchymal stromal cells (MMSC), can be induced toward differentiation into different cell lineages. In order to guide stem cell fate within an environment resembling the stem cell niche, different biomaterials are being developed. In the present study, we used silk fibroin (SF) as a biomaterial supporting the growth of MMSC and studied its effect on chondrogenesis of canine adipose–derived MMSC (cADMMSC). Adipose tissue was collected from nine privately owned dogs. MMSC were cultured on SF films and SF scaffolds in a standard cell culture medium. Cell morphology was evaluated by scanning electron microscopy (SEM). Chondrogenic differentiation was evaluated by alcian blue staining and mRNA expression of collagen type 1, collagen type 2, Sox9, and Aggrecan genes. cADMMSC cultured on SF films and SF scaffolds stained positive using alcian blue. SEM images revealed nodule-like structures with matrix vesicles and fibers resembling chondrogenic nodules. Gene expression of chondrogenic markers Sox9 and Aggrecan were statistically significantly upregulated in cADMMSC cultured on SF films in comparison to negative control cADMMSC. This result suggests that chondrogenesis of cADMMSC could occur when cells were grown on SF films in a standard cell culture medium without specific culture conditions, which were previously considered necessary for induction of chondrogenic differentiation.

scholarly journals Stem cell culture on polyvinyl alcohol hydrogels having different elasticity and immobilized with ECM-derived oligopeptides

2017 ◽  
Vol 37 (7) ◽  
pp. 647-660 ◽  
Author(s):  
Saradaprasan Muduli ◽  
Li-Hua Chen ◽  
Meng-Pei Li ◽  
Zhao-wen Heish ◽  
Cheng-Hui Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Poly Vinyl Alcohol ◽  
Hematopoietic Stem ◽  
Stem Cell Culture

Abstract The physical characteristics of cell culture materials, such as their elasticity, affect stem cell fate with respect to cell proliferation and differentiation. We systematically investigated the morphologies and characteristics of several stem cell types, including human amniotic-derived stem cells, human hematopoietic stem cells, human induced pluripotent stem (iPS) cells, and embryonic stem (ES) cells on poly(vinyl alcohol) (PVA) hydrogels immobilized with and without extracellular matrix-derived oligopeptide. Human ES cells did not adhere well to soft PVA hydrogels immobilized with oligovitronectin, whereas they did adhere well to PVA hydrogel dishes with elasticities greater than 15 kPa. These results indicate that biomaterials such as PVA hydrogels should be designed to possess minimum elasticity to facilitate human ES cell attachment. PVA hydrogels immobilized with and without extracellular matrix-derived oligopeptides are excellent candidates of cell culture biomaterials for investigations into how cell culture biomaterial elasticity affects stem cell culture and differentiation.

Evidence for Killing of Mesenchymal Stem Cells (MSC) by Autologous Natural Killer Lymphocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1290-1290 ◽  
Author(s):  
Alessandro Poggi ◽  
Anna-Maria Massaro ◽  
Simone Negrini ◽  
Ivana Pierri ◽  
Manuela Balocco ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
T Lymphocytes ◽  
Nk Cells ◽  
Natural Killer ◽  
Viral Infections ◽  
Nk Cell ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Inhibitory Receptors

Abstract In this study, Mesenchymal Stem Cells (MSC) were obtained from bone marrow of 10 patients suffering from acute myeloid leukemia (AML), six M0/1 two M2, and two M5 (according to the FAB classification), 8 out of 10 in post-chemotherapy complete remission. These cells differentiated into adipocytes or osteoblasts under appropriate culture conditions. MSC were CD44+, CD73a+ CD73b+ CD105+, beta1 integrin+, ICAM1+, HLA-I+, HLA-II+ (variable proportions), CD45−, CD31−, CD34− and they constitutively expressed the stress-inducible MHC-related molecules MIC-A and the UL16 (induced at the surface of cells infected by cytomegalovirus) binding protein ULBP3. These molecules are reported ligands for the NKG2D receptor expressed by natural killer (NK) and CD8+ T lymphocytes, effector cells that are thought to play a role in host defence against tumors. NK cells have also been shown to regulate normal differentiation of hemopoietic precursor into the myeloid or lymphoid cell lineage. Moreover, it has been stated that NK cells are not able to damage autologous cells, as they receive negative signals through inhibitory receptors, including killer Ig-like receptors (KIR) or C-type lectin inhibitory receptors (CLIR), which bind to HLA-I discrete alleles. Surprisingly, we found that autologous IL2-activated, but not freshly isolated, NK cells lysed MSC, while T lymphocytes did not kill self or non-self MSC. Binding of ICAM-1 expressed by MSC to its receptor, the integrin LFA-1, expressed by NK cells plays a key role in MSC/NK interaction. More importantly, NKG2D/MICA and/or NKG2D/ULBP3 engagement is responsible for the delivery of lethal hit. Conversely, it appears that HLA-I molecules do not protect MSC from NK cell-mediated injury. Taken together, these data suggest that NK cells, when activated as it may occur during the first response to viral infections, are able to eliminate MSC, thus altering the normal interactions with hemopoietic precursors and possibly affecting their differentiation. This mechanism might also contribute to the development of aberrant precursors as observed in acute leukaemias.

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