scholarly journals Studies of human pluripotential hemopoietic stem cells (CFU-GEMM) in vitro

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 309-316 ◽  
Author(s):  
RC Ash ◽  
DA Detrick ◽  
ED Zanjani
Keyword(s):  
Stem Cells ◽  
Culture System ◽  
Cell Populations ◽  
In Vitro Growth ◽  
Limited Capacity ◽  
Self Renewal ◽  
Low Frequencies ◽  
Cell Class ◽  
Clonal Assay

Abstract An in vitro clonal assay for a class of human hemopoietic progenitors (CFU-GEMM) with several characteristics of pluripotential stem cells has been previously described. In the presence of medium conditioned by leukocytes stimulated with phytohemagglutinin (PHA-LCM) and erythropoietin (Ep), CFU-GEMM give rise to mixed hemopoietic colonies containing granulocytic, erythroid, monocyte-macrophage, and megakaryocytic elements. In initial studies we found that CFU-GEMM were present in equal but low frequencies in blood (B) and bone marrow (M) mononuclear cell populations. However, when the culture system was modified by the substitution of Iscove's modified Dulbecco's medium for alpha-MEM and the addition of mercaptoethanol, a significant enhancement of mixed colony formation occurred, and an approximately 3- 4-fold difference in the frequency of CFU-GEMM between B and M emerged. Replating studies showed the formation of secondary differentiated hemopoietic colonies and at least a limited capacity for self-renewal of CFU-GEMM. The in vitro growth of normal CFU-GEMM was highly dependent on hemopoietin(s) present in PHA-LCM. In vitro detection of CFU-GEMM, however, requires only relatively low permissive concentrations of Ep, in contrast to the high Ep requirement for optimal BFU-E growth in vitro. These and other data described demonstrate CFU-GEMM to be a distinct multipotential stem cell class whose assay may prove useful in the study of human blood dyscrasias.

scholarly journals Studies of human pluripotential hemopoietic stem cells (CFU-GEMM) in vitro

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 309-316 ◽  
Author(s):  
RC Ash ◽  
DA Detrick ◽  
ED Zanjani
Keyword(s):  
Stem Cells ◽  
Culture System ◽  
Cell Populations ◽  
In Vitro Growth ◽  
Limited Capacity ◽  
Self Renewal ◽  
Low Frequencies ◽  
Cell Class ◽  
Clonal Assay

An in vitro clonal assay for a class of human hemopoietic progenitors (CFU-GEMM) with several characteristics of pluripotential stem cells has been previously described. In the presence of medium conditioned by leukocytes stimulated with phytohemagglutinin (PHA-LCM) and erythropoietin (Ep), CFU-GEMM give rise to mixed hemopoietic colonies containing granulocytic, erythroid, monocyte-macrophage, and megakaryocytic elements. In initial studies we found that CFU-GEMM were present in equal but low frequencies in blood (B) and bone marrow (M) mononuclear cell populations. However, when the culture system was modified by the substitution of Iscove's modified Dulbecco's medium for alpha-MEM and the addition of mercaptoethanol, a significant enhancement of mixed colony formation occurred, and an approximately 3- 4-fold difference in the frequency of CFU-GEMM between B and M emerged. Replating studies showed the formation of secondary differentiated hemopoietic colonies and at least a limited capacity for self-renewal of CFU-GEMM. The in vitro growth of normal CFU-GEMM was highly dependent on hemopoietin(s) present in PHA-LCM. In vitro detection of CFU-GEMM, however, requires only relatively low permissive concentrations of Ep, in contrast to the high Ep requirement for optimal BFU-E growth in vitro. These and other data described demonstrate CFU-GEMM to be a distinct multipotential stem cell class whose assay may prove useful in the study of human blood dyscrasias.

scholarly journals Niche Laminin and IGF-1 Additively Coordinate the Maintenance of Oct-4 Through CD49f/IGF-1R-Hif-2α Feedforward Loop in Mouse Germline Stem Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Heng-Kien Au ◽  
Syue-Wei Peng ◽  
Chin-Lin Guo ◽  
Chien-Chia Lin ◽  
Yi-Lin Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture System ◽  
Cell Model ◽  
Underlying Mechanism ◽  
Laminin Receptor ◽  
Germline Stem Cells ◽  
Self Renewal ◽  
Serum Free ◽  
Feedforward Loop

The mechanism on how extracellular matrix (ECM) cooperates with niche growth factors and oxygen tension to regulate the self-renewal of embryonic germline stem cells (GSCs) still remains unclear. Lacking of an appropriate in vitro cell model dramatically hinders the progress. Herein, using a serum-free culture system, we demonstrated that ECM laminin cooperated with hypoxia and insulin-like growth factor 1 receptor (IGF-1R) to additively maintain AP activity and Oct-4 expression of AP+GSCs. We found the laminin receptor CD49f expression in d2 testicular GSCs that were surrounded by laminin. Laminin and hypoxia significantly increased the GSC stemness-related genes, including Hif-2α, Oct-4, IGF-1R, and CD49f. Cotreatment of IGF-1 and laminin additively increased the expression of IGF-IR, CD49f, Hif-2α, and Oct-4. Conversely, silencing IGF-1R and/or CD49f decreased the expression of Hif-2α and Oct-4. The underlying mechanism involved CD49f/IGF1R-(PI3K/AKT)-Hif-2α signaling loop, which in turn maintains Oct-4 expression, symmetric self-renewal, and cell migration. These findings reveal the additive niche laminin/IGF-IR network during early GSC development.

scholarly journals Establishment of a feeder and serum-free culture system for human embryonic stem cells

Zygote ◽  
2020 ◽  
Vol 28 (3) ◽  
pp. 175-182
Author(s):  
LiYun Wang ◽  
RuiNa Zhang ◽  
RongHua Ma ◽  
GongXue Jia ◽  
ShengYan Jian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Culture System ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Self Renewal ◽  
Serum Free ◽  
Genes Encoding

SummaryStem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cells in vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and α-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and α-fetoprotein were expressed in H9 cells, suggesting that the cells maintained in vitro differentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs.

scholarly journals Cancer Stem Cell Microenvironment Models with Biomaterial Scaffolds In Vitro

Processes ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 45
Author(s):  
Ghmkin Hassan ◽  
Said M. Afify ◽  
Shiro Kitano ◽  
Akimasa Seno ◽  
Hiroko Ishii ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Biology ◽  
Advanced Technology ◽  
Cancer Microenvironment ◽  
Critical Factors ◽  
Self Renewal ◽  
Cell Microenvironment ◽  
Biological Phenomena ◽  
Biomaterial Scaffolds

Defined by its potential for self-renewal, differentiation and tumorigenicity, cancer stem cells (CSCs) are considered responsible for drug resistance and relapse. To understand the behavior of CSC, the effects of the microenvironment in each tissue are a matter of great concerns for scientists in cancer biology. However, there are many complicated obstacles in the mimicking the microenvironment of CSCs even with current advanced technology. In this context, novel biomaterials have widely been assessed as in vitro platforms for their ability to mimic cancer microenvironment. These efforts should be successful to identify and characterize various CSCs specific in each type of cancer. Therefore, extracellular matrix scaffolds made of biomaterial will modulate the interactions and facilitate the investigation of CSC associated with biological phenomena simplifying the complexity of the microenvironment. In this review, we summarize latest advances in biomaterial scaffolds, which are exploited to mimic CSC microenvironment, and their chemical and biological requirements with discussion. The discussion includes the possible effects on both cells in tumors and microenvironment to propose what the critical factors are in controlling the CSC microenvironment focusing the future investigation. Our insights on their availability in drug screening will also follow the discussion.

scholarly journals Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bei Liu ◽  
Shi Chen ◽  
Yaxing Xu ◽  
Yulin Lyu ◽  
Jinlin Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Culture Condition ◽  
Human Fibroblast ◽  
Culture System ◽  
Disease Modeling ◽  
Directed Differentiation

AbstractExtended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.

Isolation, Culture and Identification of Bovine Skeletal Stem Cells

2021 ◽  
Vol 11 (12) ◽  
pp. 2337-2345
Author(s):  
Junhui Lai ◽  
Qin Yang ◽  
Ruining Liang ◽  
Weijun Guan ◽  
Xiuxia Li
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Bone Formation ◽  
Growth Plate ◽  
Bone Development ◽  
Long Bone ◽  
Biological Characterization ◽  
Self Renewal ◽  
And Cartilage

The growth plate is essential in long bone formation and contains a wealth of skeletal stem cells (SSCs). Though the origin and the mechanism for SSCs generation remain uncertain, recent studies demonstrate the transition from cartilage to bone that in the lineage for bone development. SSCs possesses the ability to differentiate into bone and cartilage in vitro. In this research, we aimed to isolate and culture the skeletal stem cells from bovine cattle and then studied its biological characterization. The results showed that these bovine SSCs are positive for PDPN+CD73+CD164+CD90+CD44+ cell surface bio-markers, they are capable of self-renewal and differentiation. Our dates proved that SSCs exists in bovine’s long bone.

scholarly journals Adipose-derived stromal/stem cells improve epidermal homeostasis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mariko Moriyama ◽  
Shunya Sahara ◽  
Kaori Zaiki ◽  
Ayumi Ueno ◽  
Koichi Nakaoji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Culture System ◽  
Wound Repair ◽  
Cell Types ◽  
Skin Wound ◽  
Epidermal Keratinocytes ◽  
Stromal Stem Cells ◽  
Collagen Vitrigel

AbstractWound healing is regulated by complex interactions between the keratinocytes and other cell types including fibroblasts. Recently, adipose-derived mesenchymal stromal/stem cells (ASCs) have been reported to influence wound healing positively via paracrine involvement. However, their roles in keratinocytes are still obscure. Therefore, investigation of the precise effects of ASCs on keratinocytes in an in vitro culture system is required. Our recent data indicate that the epidermal equivalents became thicker on a collagen vitrigel membrane co-cultured with human ASCs (hASCs). Co-culturing the human primary epidermal keratinocytes (HPEK) with hASCs on a collagen vitrigel membrane enhanced their abilities for cell proliferation and adhesion to the membrane but suppressed their differentiation suggesting that hASCs could maintain the undifferentiated status of HPEK. Contrarily, the effects of co-culture using polyethylene terephthalate or polycarbonate membranes for HPEK were completely opposite. These differences may depend on the protein permeability and/or structure of the membrane. Taken together, our data demonstrate that hASCs could be used as a substitute for fibroblasts in skin wound repair, aesthetic medicine, or tissue engineering. It is also important to note that a co-culture system using the collagen vitrigel membrane allows better understanding of the interactions between the keratinocytes and ASCs.

scholarly journals YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/β-catenin signaling pathway

2021 ◽  
Author(s):  
Hong-Chen Yan ◽  
Yu Sun ◽  
Ming-Yu Zhang ◽  
Shu-Er Zhang ◽  
Jia-Dong Sun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Signaling Pathway ◽  
Adult Stem Cells ◽  
Self Renewal ◽  
Human Ascs ◽  
Regulation Mechanisms ◽  
Interfering Rna ◽  
Pluripotency Genes

Abstract Background Skin-derived stem cells (SDSCs) are a class of adult stem cells (ASCs) that have the ability to self-renew and differentiate. The regulation mechanisms involved in the differentiation of ASCs is a hot topic. Porcine models have close similarities to humans and porcine SDSCs (pSDSCs) offer an ideal in vitro model to investigate human ASCs. To date, studies concerning the role of yes-associated protein (YAP) in ASCs are limited, and the mechanism of its influence on self-renewal and differentiation of ASCs remain unclear. In this paper, we explore the link between the transcriptional regulator YAP and the fate of pSDSCs. Results We found that YAP promotes the pluripotent state of pSDSCs by maintaining the high expression of the pluripotency genes Sox2, Oct4. The overexpression of YAP prevented the differentiation of pSDSCs and the depletion of YAP by small interfering RNA (siRNAs) suppressed the self-renewal of pSDSCs. In addition, we found that YAP regulates the fate of pSDSCs through a mechanism related to the Wnt/β-catenin signaling pathway. When an activator of the Wnt/β-catenin signaling pathway, CHIR99021, was added to pSDSCs overexpressing YAP the ability of pSDSCs to differentiate was partially restored. Conversely, when XAV939 an inhibitor of Wnt/β-catenin signaling pathway, was added to YAP knockdown pSDSCs a higher self-renewal ability resulted. Conclusions our results suggested that, YAP and the Wnt/β-catenin signaling pathway interact to regulate the fate of pSDSCs.

scholarly journals Soft Substrate Maintains Proliferative and Adipogenic Differentiation Potential of human Mesenchymal Stem Cells on Long Term Expansion by Delaying Senescence

2018 ◽  
Author(s):  
Sanjay K. Kureel ◽  
Pankaj Mogha ◽  
Akshada Khadpekar ◽  
Vardhman Kumar ◽  
Rohit Joshi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture System ◽  
Human Mesenchymal Stem Cells ◽  
Population Doubling ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Poly Acrylamide

AbstractHuman mesenchymal stem cells (hMSCs), when cultured on tissue culture plate (TCP) for in vitro expansion, they spontaneously lose their proliferative capacity and multi-lineage differentiation potential. They also lose their distinct spindle morphology and become large and flat. After a certain number of population doubling, they enter into permanent cell cycle arrest, called senescence. This is a major roadblock for clinical use of hMSCs which demands large number of cells. A cell culture system is needed which can maintain the stemness of hMSCs over long term passages yet simple to use. In this study, we explore the role of substrate rigidity in maintaining stemness. hMSCs were serially passaged on TCP and 5 kPa poly-acrylamide gel for 20 population doubling. It was found that while on TCP, cell growth reached a plateau at cumulative population doubling (CPD) = 12.5, on 5 kPa gel, they continue to proliferate linearly till we monitored (CPD = 20). We also found that while on TCP, late passage MSCs lost their adipogenic potential, the same was maintained on soft gel. Cell surface markers related to MSCs were also unaltered. We demonstrated that this maintenance of stemness was correlated with delay in onset of senescence, which was confirmed by β-gal assay and by differential expression of vimentin, Lamin A and Lamin B. As preparation of poly-acrylamide gel is a simple, well established, and well standardized protocol, we believe that this system of cell expansion will be useful in therapeutic and research applications of hMSCs.One Sentence SummaryhMSCs retain their stemness when expanded in vitro on soft polyacrylamide gel coated with collagen by delaying senescence.Significance StatementFor clinical applications, mesenchymal stem cells (MSCs) are required in large numbers. As MSCs are available only in scarcity in vivo, to fulfill the need, extensive in vitro expansion is unavoidable. However, on expansion, they lose their replicative and multi-lineage differentiation potential and become senescent. A culture system that can maintain MSC stemness on long-term expansion, without compromising the stemness, is need of the hour. In this paper, we identified polyacrylamide (PAA) hydrogel of optimum stiffness that can be used to maintain stemness of MSCs during in vitro long term culture. Large quantity of MSCs thus grown can be used in regenerative medicine, cell therapy, and in treatment of inflammatory diseases.

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