The Experts below are selected from a list of 282 Experts worldwide ranked by ideXlab platform
Heli Skottman - One of the best experts on this subject based on the ideXlab platform.
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Xeno- and Feeder-free differentiation of human pluripotent stem Cells to two distinct ocular epithelial Cell types using simple modifications of one method
Stem Cell Research & Therapy, 2017Co-Authors: Heidi Hongisto, Tanja Ilmarinen, Meri Vattulainen, Alexandra Mikhailova, Heli SkottmanAbstract:BackgroundHuman pluripotent stem Cells (hPSCs) provide a promising Cell source for ocular Cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and Feeder Cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target Cells: retinal pigment epithelial (RPE) Cells and corneal limbal epithelial stem Cells (LESCs).MethodsMultiple genetically distinct hPSC lines were adapted to a defined, xeno-, and Feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial Cells were established utilizing xeno-free media and a combination of extraCellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired Cell lineages. The resulting RPE Cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation.ResultsThe defined, xeno-, and Feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, Cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to Cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE Cells, and hPSC-LESCs, and demonstrated successful recovery after thawing.ConclusionsWe propose methods for efficient and scalable, directed differentiation of high-quality RPE Cells and LESCs. The two clinically relevant Cell types are generated with simple inductive modification of the same basal method, followed by adherent culture, passaging, and cryobanking.
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xeno and Feeder free differentiation of human pluripotent stem Cells to two distinct ocular epithelial Cell types using simple modifications of one method
Stem Cell Research & Therapy, 2017Co-Authors: Heidi Hongisto, Tanja Ilmarinen, Meri Vattulainen, Alexandra Mikhailova, Heli SkottmanAbstract:Human pluripotent stem Cells (hPSCs) provide a promising Cell source for ocular Cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and Feeder Cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target Cells: retinal pigment epithelial (RPE) Cells and corneal limbal epithelial stem Cells (LESCs). Multiple genetically distinct hPSC lines were adapted to a defined, xeno-, and Feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial Cells were established utilizing xeno-free media and a combination of extraCellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired Cell lineages. The resulting RPE Cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation. The defined, xeno-, and Feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, Cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to Cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE Cells, and hPSC-LESCs, and demonstrated successful recovery after thawing. We propose methods for efficient and scalable, directed differentiation of high-quality RPE Cells and LESCs. The two clinically relevant Cell types are generated with simple inductive modification of the same basal method, followed by adherent culture, passaging, and cryobanking.
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laminin 511 expression is associated with the functionality of Feeder Cells in human embryonic stem Cell culture
Stem Cell Research, 2012Co-Authors: Heidi Hongisto, Alexandra Mikhailova, Sanna Vuoristo, Riitta Suuronen, Ismo Virtanen, Timo Otonkoski, Heli SkottmanAbstract:Abstract Fibroblast Feeder Cells play an important role in supporting the derivation and long term culture of undifferentiated, pluripotent human embryonic stem Cells (hESCs). The Feeder Cells secrete various growth factors and extraCellular matrix (ECM) proteins into extraCellular milieu. However, the roles of the Feeder Cell-secreted factors are largely unclear. Animal Feeder Cells and use of animal serum also make current Feeder Cell culture conditions unsuitable for derivation of clinical grade hESCs. We established xeno-free Feeder Cell lines using human serum (HS) and studied their function in hESC culture. While human foreskin fibroblast (hFF) Feeder Cells were clearly hESC supportive, none of the established xeno-free human dermal fibroblast (hDF) Feeder Cells were able to maintain undifferentiated hESC growth. The two fibroblast types were compared for their ECM protein synthesis, integrin receptor expression profiles and key growth factor secretion. We show that hESC supportive Feeder Cells produce laminin-511 and express laminin-binding integrins α3s1, α6s1 and α7s1. These results indicate specific laminin isoforms and integrins in maintenance of hESC pluripotency in Feeder-dependent cultures. In addition, several genes with a known or possible role for hESC pluripotency were differentially expressed in distinct Feeder Cells.
Heidi Hongisto - One of the best experts on this subject based on the ideXlab platform.
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Xeno- and Feeder-free differentiation of human pluripotent stem Cells to two distinct ocular epithelial Cell types using simple modifications of one method
Stem Cell Research & Therapy, 2017Co-Authors: Heidi Hongisto, Tanja Ilmarinen, Meri Vattulainen, Alexandra Mikhailova, Heli SkottmanAbstract:BackgroundHuman pluripotent stem Cells (hPSCs) provide a promising Cell source for ocular Cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and Feeder Cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target Cells: retinal pigment epithelial (RPE) Cells and corneal limbal epithelial stem Cells (LESCs).MethodsMultiple genetically distinct hPSC lines were adapted to a defined, xeno-, and Feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial Cells were established utilizing xeno-free media and a combination of extraCellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired Cell lineages. The resulting RPE Cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation.ResultsThe defined, xeno-, and Feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, Cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to Cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE Cells, and hPSC-LESCs, and demonstrated successful recovery after thawing.ConclusionsWe propose methods for efficient and scalable, directed differentiation of high-quality RPE Cells and LESCs. The two clinically relevant Cell types are generated with simple inductive modification of the same basal method, followed by adherent culture, passaging, and cryobanking.
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xeno and Feeder free differentiation of human pluripotent stem Cells to two distinct ocular epithelial Cell types using simple modifications of one method
Stem Cell Research & Therapy, 2017Co-Authors: Heidi Hongisto, Tanja Ilmarinen, Meri Vattulainen, Alexandra Mikhailova, Heli SkottmanAbstract:Human pluripotent stem Cells (hPSCs) provide a promising Cell source for ocular Cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and Feeder Cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target Cells: retinal pigment epithelial (RPE) Cells and corneal limbal epithelial stem Cells (LESCs). Multiple genetically distinct hPSC lines were adapted to a defined, xeno-, and Feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial Cells were established utilizing xeno-free media and a combination of extraCellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired Cell lineages. The resulting RPE Cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation. The defined, xeno-, and Feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, Cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to Cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE Cells, and hPSC-LESCs, and demonstrated successful recovery after thawing. We propose methods for efficient and scalable, directed differentiation of high-quality RPE Cells and LESCs. The two clinically relevant Cell types are generated with simple inductive modification of the same basal method, followed by adherent culture, passaging, and cryobanking.
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laminin 511 expression is associated with the functionality of Feeder Cells in human embryonic stem Cell culture
Stem Cell Research, 2012Co-Authors: Heidi Hongisto, Alexandra Mikhailova, Sanna Vuoristo, Riitta Suuronen, Ismo Virtanen, Timo Otonkoski, Heli SkottmanAbstract:Abstract Fibroblast Feeder Cells play an important role in supporting the derivation and long term culture of undifferentiated, pluripotent human embryonic stem Cells (hESCs). The Feeder Cells secrete various growth factors and extraCellular matrix (ECM) proteins into extraCellular milieu. However, the roles of the Feeder Cell-secreted factors are largely unclear. Animal Feeder Cells and use of animal serum also make current Feeder Cell culture conditions unsuitable for derivation of clinical grade hESCs. We established xeno-free Feeder Cell lines using human serum (HS) and studied their function in hESC culture. While human foreskin fibroblast (hFF) Feeder Cells were clearly hESC supportive, none of the established xeno-free human dermal fibroblast (hDF) Feeder Cells were able to maintain undifferentiated hESC growth. The two fibroblast types were compared for their ECM protein synthesis, integrin receptor expression profiles and key growth factor secretion. We show that hESC supportive Feeder Cells produce laminin-511 and express laminin-binding integrins α3s1, α6s1 and α7s1. These results indicate specific laminin isoforms and integrins in maintenance of hESC pluripotency in Feeder-dependent cultures. In addition, several genes with a known or possible role for hESC pluripotency were differentially expressed in distinct Feeder Cells.
Jennifer A Foltz - One of the best experts on this subject based on the ideXlab platform.
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evaluation of serum free media formulations in Feeder Cell stimulated expansion of natural killer Cells
Cytotherapy, 2020Co-Authors: Jena E Moseman, Jennifer A Foltz, Kinnari Sorathia, Erica L HeipertzAbstract:Abstract Background Optimal expansion of therapeutic natural killer (NK) Cell products has required media supplementation with human or fetal bovine serum, which raises safety and regulatory concerns for clinical manufacturing. Serum-free media (SFM) have been optimized for T-Cell expansion, but few SFM systems have been developed for NK Cells. Here, we compare six commercial clinical-grade SFM with our standard fetal bovine serum–containing medium for their ability to support NK Cell expansion and function. Methods Human peripheral blood NK Cells were expanded in selected media by recursive weekly stimulation with K562-based Feeder Cells expressing membrane-bound interleukin-21 and CD137L. Expansion was the primary readout, and the best-performing SFM was then compared with standard medium for cytotoxicity, phenotype, degranulation and cytokine secretion. Multiple lots were compared for consistency, and media was analyzed throughout for nutrient consumption and metabolic byproducts. Results TexMACS, OpTmizer, SCGM, ABS-001 and StemXVivo demonstrated equal or inferior NK Cell expansion kinetics compared with standard medium, but expansion was markedly superior with AIM V + 5% Immune Cell Serum Replacement (ICSR; mean 5448 vs. 2621-fold expansion in 14 days). Surprisingly, NK Cells expanded in AIM V + ICSR also showed increased cytotoxicity, tumor necrosis factor α secretion and DNAM-1, NKG2D, NKp30, FasL, granzyme B and perforin expression. Lot-to-lot variability was minimal. Glucose and glutamine consumption were inversely related to lactate and ammonia production. Discussion The AIM V + ICSR SFM system supports exCellent ex vivo expansion of clinical-grade NK Cells with the phenotype and function needed for adoptive immunotherapy.
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evaluation of serum free media formulations in Feeder Cell expansion of natural killer Cells
Cytotherapy, 2019Co-Authors: Jena E Moseman, Jennifer A FoltzAbstract:Background & Aim Natural Killer (NK) Cells are rapidly evolving as a Cellular therapy due to their innate ability to kill a wide range of cancerous Cells without prior sensitization and broad safety profile without concerns for causing graft-versus host disease. However, the clinical grade production of NK Cell products has historically required the addition of fetal bovine or human serum, which have safety, ethical, and regulatory concerns. Serum-free media have been established for T Cell activation and expansion, but little is known about serum-free media in manufacturing NK Cells. Here, we sought to assess a broad range of serum-free media in supporting NK Cell expansion with K562-based Feeder Cells for use in adoptive immunotherapy. Methods, Results & Conclusion Cryopreserved expanded human peripheral blood NK Cells (ePBNK) were re-stimulated at 1:1 ratio with irradiated K562 expressing mbIL21 and 4-1BBL (CSTX002) and propagated for 7 days in 6 different clinical-grade serum-free media. The three serum-free media that yielded the highest Cell numbers (TexMACS, ABS011, and AIM-V/ICSR) were further compared for support of 2-week NK Cell expansion starting from freshly-isolated PBNK, and the resulting ePBNK were assessed for phenotype, cytotoxicity, degranulation, and cytokine production. NK Cells did not have acceptable expansion kinetics with OpTmizer, SCGM, or StemXVivo. Surprisingly, NK Cells had significantly improved expansion with AIM-V/ICSR (5448-fold) compared to RPMI + FBS control (2621-fold). Additionally, the AIM-V/ICSR expanded NK Cells showed increased anti-tumor cytotoxicity and TNFa secretion, and had increased expression of several NK Cell activating receptors and effector molecules such as DNAM-1, NKG2D, NKp30, FasL, granzyme B, and perforin. These results indicate that the serum-free AIM-V/ICSR expansion medium can support ex vivo expansion of NK Cells with high effector function and phenotype compared to the standard RPMI + FBS medium and is an effective alternative for use in adoptive NK Cell therapy.
Makoto Senoo - One of the best experts on this subject based on the ideXlab platform.
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Promotion of Human Epidermal Keratinocyte Expansion in Feeder Cell Co-culture
Skin Tissue Engineering, 2019Co-Authors: Daisuke Suzuki, Filipa Pinto, Makoto SenooAbstract:Co-culture of human epidermal keratinocytes with mouse 3T3-J2 Feeder Cells, developed by Green and colleagues, has been used worldwide to generate skin autografts since the early 1980s. In addition, co-culture with 3T3-J2 Cells has served as a fundamental tool in skin stem Cell biology as it allows the evaluation of self-renewal capacity of epidermal stem Cells. This chapter describes a recent improvement in the Green method to promote further the expansion of human epidermal keratinocytes utilizing a small-molecule inhibitor of TGF-β signaling. This new protocol enables more rapid expansion of human epidermal keratinocytes in co-culture with not only 3T3-J2 Cells but also other Feeder Cells including human dermal fibroblasts and human preadipocytes, two major alternatives to 3T3-J2 Cells, with a long-term goal of developing customized skin autografts.
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inhibition of tgf β signaling promotes expansion of human epidermal keratinocytes in Feeder Cell co culture
Wound Repair and Regeneration, 2017Co-Authors: Daisuke Suzuki, Filipa Pinto, Makoto SenooAbstract:Cultured epidermal autografts have been used worldwide since 1981 for patients with extensive third-degree burn wounds and limited skin donor sites. Despite significant progress in techniques toward improving clinical outcome of skin grafts, the long in vitro preparation time of cultured autografts has remained a major factor limiting its widespread use. Here, we show that pharmacological inhibition of TGF-β signaling promotes the expansion of human epidermal keratinocytes (HEKs) with high proliferative potential in co-cultures with both murine 3T3-J2 Cells and human Feeder Cells, including dermal fibroblasts and preadipocytes. In contrast, TGF-β signaling inhibition does not enhance the growth of HEKs in a serum- and Feeder-free condition, an alternative approach to propagate HEKs for subsequent autograft production. Our results have important implications for the use of TGF-β signaling inhibition as a viable therapeutic strategy for improving Green's methodology and for more efficient production of customized skin autografts with human Feeder Cells. This article is protected by copyright. All rights reserved.
Afshin Raouf - One of the best experts on this subject based on the ideXlab platform.
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A robust Cell culture system for large scale Feeder Cell-free expansion of human breast epithelial progenitors
Stem Cell Research & Therapy, 2018Co-Authors: Sumanta Chatterjee, Pratima Basak, Edward Buchel, Leigh C Murphy, Afshin RaoufAbstract:BackgroundNormal human breast epithelial Cells are maintained by the proliferation and differentiation of different human breast epithelial progenitors (HBEPs). However, these progenitor subsets can only be obtained at low frequencies, limiting their further characterization. Recently, it was reported that HBEPs can be minimally expanded in Matrigel cocultures with stromal Feeder Cells. However, variability of generating healthy Feeder Cells significantly impacts the effective expansion of HBEPs.MethodsHere, we report a robust Feeder Cell-free culture system for large-scale expansion of HBEPs in two-dimensional cultures.ResultsUsing this Cell culture system HBEPs can be exponentially expanded as bulk cultures. Moreover, purified HBEP subtypes can also be separately expanded using our Cell culture system. The expanded HBEPs retain their undifferentiated phenotype and form distinct epithelial colonies in colony forming Cell assays.ConclusionsThe availability of a culture system enabling the large-scale expansion of HBEPs facilitates their application to screening platforms and other Cell-based assays.
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A robust Cell culture system for large scale Feeder Cell-free expansion of human breast epithelial progenitors
Stem Cell Research & Therapy, 2018Co-Authors: Sumanta Chatterjee, Pratima Basak, Edward Buchel, Leigh C Murphy, Afshin RaoufAbstract:Normal human breast epithelial Cells are maintained by the proliferation and differentiation of different human breast epithelial progenitors (HBEPs). However, these progenitor subsets can only be obtained at low frequencies, limiting their further characterization. Recently, it was reported that HBEPs can be minimally expanded in Matrigel cocultures with stromal Feeder Cells. However, variability of generating healthy Feeder Cells significantly impacts the effective expansion of HBEPs. Here, we report a robust Feeder Cell-free culture system for large-scale expansion of HBEPs in two-dimensional cultures. Using this Cell culture system HBEPs can be exponentially expanded as bulk cultures. Moreover, purified HBEP subtypes can also be separately expanded using our Cell culture system. The expanded HBEPs retain their undifferentiated phenotype and form distinct epithelial colonies in colony forming Cell assays. The availability of a culture system enabling the large-scale expansion of HBEPs facilitates their application to screening platforms and other Cell-based assays.
