scholarly journals Long-term expansion and differentiation of adult murine epidermal stem cells in 3D organoid cultures

2019 ◽  
Vol 116 (29) ◽  
pp. 14630-14638 ◽  
Author(s):  
Kim E. Boonekamp ◽  
Kai Kretzschmar ◽  
Dominique J. Wiener ◽  
Priyanca Asra ◽  
Sepideh Derakhshan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture System ◽  
Adult Life ◽  
Mouse Skin ◽  
Bmp Signaling ◽  
Epidermal Stem Cells ◽  
High Calcium ◽  
Expansion Media

Mammalian epidermal stem cells maintain homeostasis of the skin epidermis and contribute to its regeneration throughout adult life. While 2D mouse epidermal stem cell cultures have been established decades ago, a long-term, feeder cell- and serum-free culture system recapitulating murine epidermal architecture has not been available. Here we describe an epidermal organoid culture system that allows long-term, genetically stable expansion of adult epidermal stem cells. Our epidermal expansion media combines atypically high calcium concentrations, activation of cAMP, FGF, and R-spondin signaling with inhibition of bone morphogenetic protein (BMP) signaling. Organoids are established robustly from adult mouse skin and expand over at least 6 mo, while maintaining the basal-apical organization of the mouse interfollicular epidermis. The system represents a powerful tool to study epidermal homeostasis and disease in vitro.

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.

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.

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1623-1636 ◽  
Author(s):  
Chu-Chih Shih ◽  
Mickey C.-T. Hu ◽  
Jun Hu ◽  
Jeffrey Medeiros ◽  
Stephen J. Forman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
In Vitro Culture System

Abstract We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

Leukemia Bone Marrow Primary Cells Long-Term Culture in a Biomimetic Hematopoietic Niche.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4114-4114
Author(s):  
Li Hou ◽  
Ting Liu ◽  
Jing Tan ◽  
Wentong Meng ◽  
Li Deng
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Culture System ◽  
3D Culture ◽  
Primary Cells ◽  
Marrow Mesenchymal Stem Cells ◽  
3D Culture System ◽  
2D System

Abstract We have constructed a biomimetic hematopoietic niche (3D culture system) with bio-derived bone as framework, composited with human marrow mesenchymal stem cells, and induced the cells into osteoblasts. Our primary results showed that the biomimetic 3D culture system is capable to allow maintenance and expansion of primitive hematopoietic progenitor cells in vitro. But so far, leukemia primary cells long-term culture from patients marrow are still difficult because it is not clear how does the regulation of leukemic cells grow ex vivo, and lack of adequate investigation between leukemic stem cells with stromal cells. Based on our previous research, we cultured bone marrow mesenchymal stem cells from chronic myelogenous leukemia (CML) patients, and conceived a “pathologic biomimetic osteoblast niche”, to explore the growth of leukemia bone marrow primary cells from CML patients. Bio-derived bone was composited with marrow mesenchymal stem cells from CML patients and constructed a 3D biomimetic osteoblast niche. The mononuclear cells (MNCs) were collected with standard Ficoll-Paque separation from newly diagnosed CML patients. The MNCs were cultured for 2∼5 weeks in the 3D culture system and compared with 2D culture system. The results showed that the proportion of CD34+ cells are increased either in 3D or 2D culture systems. Compared to input, the proportion of CD34+ cells were increased 6.52(1.87∼9)vs. 3.18(1.07∼6.8)times at 2 weeks culture, and 13.6(3.59∼26.31)vs. 7.86(0.78∼18.0)times at 5 weeks culture. The proportion of CD34+/CD38- was higher in 3D culture system than 2D system. It was 5.55(2.1∼11.7)% vs. 2.4(0.9∼3.4)%, and 13.5(3.4∼34.2)% vs. 4.83(2.1∼8.9)% at 2 weeks and 5 weeks respectively. The function of cultured cells was evaluated in colony forming unit (CFU) assay and long term culture initial cell (LTC-IC) assay. 3D system produced more colonies than 2D system {103.33(82∼144)vs. 79(53∼122)} at 2 week culture and 47(33∼66)vs. 21.67(16∼27)at 5 week culture. LTC-IC are widely used as a surrogate in vitro culture for pluripotent stem cells, and those primitive progenitor cells responsible for leukemia in mice are named SL-IC or leukemia stem cells (LSCs). 3D system showed higher frequency of LTC-IC than that of 2D system after 2-week culture(2.23E-05(1.73∼2.56)vs.1.40E-05(1.21∼1.73)). FISH showed the proportion of Ph+ cells declined in both system during the culture, but not as rapidly as it did in 2D system{65%(3D)vs.63%(2D)at 2 week, 55%(3D)vs.35%(2D)at 5 week}, and the Ph+ cells were predominant derived from 3D culture. Our 3D culture system constructed with induced osteoblasts from mesnchymal stem cells in CML patients might provide a more suitable microenvironment for leukemic cells growing in vitro. The leukemic stem cells seemed to be regulated by the molecular signals mediated by osteoblast, and the biological characteristics of leukemia stem cells at least partially is maintained. It may be become a new method for studying leukemic HSCs/HPCs behavior in vitro.

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1623-1636 ◽  
Author(s):  
Chu-Chih Shih ◽  
Mickey C.-T. Hu ◽  
Jun Hu ◽  
Jeffrey Medeiros ◽  
Stephen J. Forman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
In Vitro Culture System

We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

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.

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

Unexpected and Sustained Symmetry in the Repopulation Programs Displayed by Clonally Amplified Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1651-1651
Author(s):  
Brad Dykstra ◽  
David Kent ◽  
Melisa Hamilton ◽  
Merete Kristiansen ◽  
Kristin Lyons ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Adult Life ◽  
Mouse Bone Marrow ◽  
Recipient Mouse ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Over Time

Abstract Heterogeneity in progeny output by individual pluripotent hematopoietic cells is a well documented but poorly understood paradigm. Importantly, the extent to which this functional heterogeneity is pre-determined by intrinsic mechanisms that specify distinct programs, as opposed to conditions that result in a series of stochastic events, is still debated. The prospective isolation of phenotypically defined subpopulations with more restricted behaviors has lent recent support to the concept of predetermined hierarchies with preset, but alternative pathways of lineage restriction and differentiated cell output. Here we have used highly purified starting populations to compare the long-term cell output dynamics of individual multipotent repopulating cells in sublethally irradiated W41/W41 mice transplanted with single Ly-5 congenic CD45midlin−Rho−SP adult mouse bone marrow cells (158 mice) or their clonal progeny generated after 4 days in vitro in 300 ng/ml SF, 20 ng/ml IL-11 and 1 ng/ml Flt3-L (194 mice). WBC samples collected 4, 8, 12, 16, and 24 weeks post-transplant were analyzed for donor contributions to the myeloid (GM) and lymphoid (B and T) lineages. In 49 of the 158 mice (31%) and 44 of the 194 mice (23%), the cells produced in vivo contributed ≥1% of all the WBCs present at ≥16 weeks. The overall and lineage-specific contributions to the WBCs in each recipient mouse varied widely both over time post-transplant and between mice. However, examination of the ratio of the donor contributions to the myeloid and lymphoid lineages (GM:B+T) in each mouse at 16 weeks post-transplant allowed 4 patterns to be readily identified: α and β with GM:B+T ratios of ≥2 and 0.25–2, respectively; γ, with a GM:B+T ratio of <0.25 including a ≥1% contribution to both lymphoid and myeloid lineages at 16 weeks; and δ, also with a GM:B+T ratio of <0.25, but with contribution only to the lymphoid lineages at this time. Secondary transplants performed after 24 weeks showed long-term repopulation (≥16 weeks) of most recipients of type α and β progeny (10/11 and 11/12, respectively) but none of the recipients of type γ and δ progeny were repopulated (0/6 and 0/17, respectively). Interestingly, the variation over time in both the overall and lineage-specific contributions was remarkably similar in pairs of secondary recipients injected with cells from the same primary donor. In addition, the lineage contribution ratios seen in the secondary recipients tended to recapitulate that of the primary donors (i.e., α or β), and these trends remained obvious when tertiary transplants were performed. Preservation of stem cell programming was also evident from sequential analyses of multiple mice injected with aliquots of the same clones generated in vitro after 10 days from single CD45midlin−Rho−SP cells. Very similar patterns of total and lineage-specific contributions were again observed amongst the different recipients of cells from the same clones. Collectively, these findings indicate that by early adult life hematopoietic stem cells have acquired intrinsically fixed patterns of lineage specification that can be stably transmitted through many self-renewal generations.

scholarly journals GABA exists as a negative regulator of cell proliferation in spermaogonial stem cells

2013 ◽  
Vol 18 (2) ◽  
Author(s):  
Yong Du ◽  
Zhao Du ◽  
Hongping Zheng ◽  
Dan Wang ◽  
Shifeng Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture System ◽  
Histone H3 ◽  
Negative Regulator ◽  
Peripheral Tissues ◽  
Inhibitory Neurotransmitter ◽  
Amino Butyric Acid

Abstractγ-amino butyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA is also found in many peripheral tissues, where it has important functions during development. Here, we identified the existence of the GABA system in spermatogonial stem cells (SSCs) and found that GABA negatively regulates SSC proliferation. First, we demonstrated that GABA and its synthesizing enzymes were abundant in the testes 6 days postpartum (dpp), suggesting that GABA signaling regulates SSCs function in vivo. In order to directly examine the effect of GABA on SSC proliferation, we then established an in vitro culture system for long-term expansion of SSCs. We showed that GABAA receptor subunits, including α1, α5, β1, β2, β3 and γ3, the synthesizing enzyme GAD67, and the transporter GAT-1, are expressed in SSCs. Using phosphorylated histone H3 (pH3) staining, we demonstrated that GABA or the GABAAR-specific agonist muscimol reduced the proliferation of SSCs. This GABA regulation of SSC proliferation was shown to be independent of apoptosis using the TUNEL assay. These results suggest that GABA acts as a negative regulator of SSC proliferation to maintain the homeostasis of spermatogenesis in the testes.

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