Isolation and long-term culture of human spermatogonia stem cell

We have recently demonstrated that 50% of primitive human long-term culture-initiating cells (LTC-IC) are maintained for up to 8 weeks in stroma-dependent cultures in which progenitor-stroma contact is prevented (stroma noncontact), or when progenitors are cultured in medium conditioned by stromal feeders. This indicates that factors responsible for LTC-IC maintenance are present in soluble form in stromal supernatant (SN). Although the picogram concentrations of cytokines present in stromal SN can induce the differentiation of CD34+/HLA-DR- (DR-) cells to clonogenic cells (colony forming cells; CFC), they maintain only 10% of LTC-IC for 5 weeks, suggesting that factors other than these cytokines are required for LTC-IC maintenance. To characterize the factor(s) in stromal SN responsible for LTC-IC maintenance, we purified glycoproteins and proteoglycans (PG) from the SN of the LTC-IC supportive murine marrow stromal fibroblast cell line M2–10B4 by ion exchange high performance liquid chromatography (HPLC). Culture of DR- cells in a combination of M2–10B4-derived PG, but not glycoproteins and picogram concentrations of recombinant human interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), granulocyte- macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1alpha (MIP-1alpha) resulted in the recovery of 96% +/- 8% of LTC-IC maintained in cultures supplemented with unfractionated stromal SN. LTC-IC maintenance was largely retained after digestion of the PG-rich fraction with proteinase K and after dissociative gel filtration chromatography, but was completely abolished following treatment with nitrous acid, which digests heparan sulfate glycosaminoglycans (HS GAG). As for M2–10B4-derived HS GAG, high concentrations of bovine kidney HS GAG, but not bovine tracheal chondroitin sulfate, significantly improved cytokine-mediated LTC-IC maintenance. Maintenance of LTC-IC by these nonmarrow-derived HS GAG was, however, significantly lower than that seen with M2–10B4-derived HS. These studies demonstrate a role for marrow stroma-derived HS GAG in the long-term in vitro maintenance of human LTC-IC. Further structure-function analysis of these HS GAG may have important implications for ex vivo stem cell expansion and gene transfer into hematopoietic progenitors.

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Long-term culture of human mesenchymal stem cell-seeded constructs under ultrasound stimulation: evaluation of chondrogenesis

Biomedical Physics & Engineering Express ◽

10.1088/2057-1976/2/5/055016 ◽

2016 ◽

Vol 2 (5) ◽

pp. 055016 ◽

Cited By ~ 2

Author(s):

Sanjukta Guha Thakurta ◽

Neety Sahu ◽

April Miller ◽

Gaurav Budhiraja ◽

Leonard Akert ◽

...

Keyword(s):

Stem Cell ◽

Mesenchymal Stem Cell ◽

Human Mesenchymal Stem Cell ◽

Long Term Culture ◽

Ultrasound Stimulation

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Rapid decline of chronic myeloid leukemic cells in long-term culture due to a defect at the leukemic stem cell level.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.13.6192 ◽

1992 ◽

Vol 89 (13) ◽

pp. 6192-6196 ◽

Cited By ~ 102

Author(s):

C. Udomsakdi ◽

C. J. Eaves ◽

B. Swolin ◽

D. S. Reid ◽

M. J. Barnett ◽

...

Keyword(s):

Stem Cell ◽

Leukemic Cells ◽

Rapid Decline ◽

Leukemic Stem Cell ◽

Cell Level ◽

Long Term Culture

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Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture

Scientific Reports ◽

10.1038/srep26181 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 88

Author(s):

A. Odawara ◽

H. Katoh ◽

N. Matsuda ◽

I. Suzuki

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Neuronal Networks ◽

Induced Pluripotent Stem Cell ◽

Drug Responses ◽

Long Term Culture ◽

Induced Pluripotent

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Adipose derived stem cell properties in long-term culture- an implications for regenerative medicine

European Urology Supplements ◽

10.1016/s1569-9056(17)31372-6 ◽

2017 ◽

Vol 16 (5) ◽

pp. e2231

Author(s):

M. Rasmus ◽

M. Maj ◽

D. Balcerczyk ◽

M. Buhl ◽

K. Warda ◽

...

Keyword(s):

Stem Cell ◽

Regenerative Medicine ◽

Long Term Culture ◽

Adipose Derived Stem Cell

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Biophysical study of human induced Pluripotent Stem Cell-Derived cardiomyocyte structural maturation during long-term culture

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2018.03.198 ◽

2018 ◽

Vol 499 (3) ◽

pp. 611-617 ◽

Cited By ~ 18

Author(s):

Tiago P. Dias ◽

Sandra N. Pinto ◽

Juliana I. Santos ◽

Tiago G. Fernandes ◽

Fábio Fernandes ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Long Term Culture ◽

Biophysical Study ◽

Induced Pluripotent

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Isolation and Expansion of Mesenchymal Stem/Stromal Cells, Functional Assays and Long-Term Culture Associated Alterations of Cellular Properties

10.5772/intechopen.100286 ◽

2021 ◽

Author(s):

Chenghai Li

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mhc Class Ii ◽

Stromal Cells ◽

Replicative Senescence ◽

Cell Types ◽

Long Term Culture ◽

Medicine Research

Mesenchymal stem cell/stromal cells (MSCs) can differentiate into a variety of cell types, including osteocytes, adipocytes and chondrocytes. MSCs are present in the multiple types of adult tissue, such as bone marrow, adipose tissue, and various neonatal birth-associated tissues. Given their self-renewal and differentiation potential, immunomodulatory and paracrine properties, and lacking major histocompatibility complex (MHC) class II molecules, MSCs have attracted much attention for stem cell-based translational medicine research. Due to a very low frequency in different types of tissue, MSCs can be isolated and expanded in vitro to derive sufficient cell numbers prior to the clinical applications. In this chapter, the methodology to obtain primary bone marrow-derived MSCs as well as their in vitro culture expansion will be described. To assess the functional properties, differentiation assays, including osteogenesis, chondrogenesis and adipogenesis, 3-D culture of MSCs and co-culture of MSCs and tumor cells are also provided. Finally, the long-term culture associated alterations of MSCs, such as replicative senescence and spontaneous transformation, will be discussed for better understanding of the use of MSCs at the early stages for safe and effective cell-based therapy.

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Distinct Requirements for Optimal Growth and In Vitro Expansion of Human CD34+CD38− Bone Marrow Long-Term Culture-Initiating Cells (LTC-IC), Extended LTC-IC, and Murine In Vivo Long-Term Reconstituting Stem Cells

Blood ◽

10.1182/blood.v94.12.4093 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4093-4102 ◽

Cited By ~ 60

Author(s):

Veslemøy Ramsfjell ◽

David Bryder ◽

Helga Björgvinsdóttir ◽

Sten Kornfält ◽

Lars Nilsson ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Activity ◽

Optimal Growth ◽

Cell Manipulation ◽

Long Term Culture ◽

Human Cd34

Abstract Recently, primitive human bone marrow (BM) progenitors supporting hematopoiesis in extended (>60 days) long-term BM cultures were identified. Such extended long-term culture-initiating cells (ELTC-IC) are of the CD34+CD38− phenotype, are quiescent, and are difficult to recruit into proliferation, implicating ELTC-IC as the most primitive human progenitor cells detectable in vitro. However, it remains to be established whether ELTC-IC can proliferate and potentially expand in response to early acting cytokines. Here, CD34+CD38− BM ELTC-IC (12-week) were efficiently recruited into proliferation and expanded in vitro in response to early acting cytokines, but conditions for expansion of ELTC-IC activity were distinct from those of traditional (5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and maintenance or expansion of murine long-term reconstituting activity and human LTC-IC, they dramatically depleted ELTC-IC activity. In contrast, KL, flt3 ligand (FL), and megakaryocyte growth and development factor (MGDF) (and KL + FL + IL-3) expanded murine long-term reconstituting activity as well as human LTC-IC and ELTC-IC. Expansion of LTC-IC was most optimal after 7 days of culture, whereas optimal expansion of ELTC-IC activity required 12 days, most likely reflecting the delayed recruitment of quiescent CD34+CD38− progenitors. The need for high concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free conditions was more critical for expansion of ELTC-IC than of LTC-IC. The distinct requirements for expansion of ELTC-IC activity when compared with traditional LTC-IC suggest that the ELTC-IC could prove more reliable as a predictor for true human stem cell activity after in vitro stem cell manipulation.

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