scholarly journals Propagation of Adult SSCs: From Mouse to Human

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Laura A. Martin ◽  
Marco Seandel
Keyword(s):  
Stem Cells ◽  
Genetic Material ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Inherited Disorders ◽  
The Past ◽  
Unique Cell

Adult spermatogonial stem cells (SSCs) represent a distinctive source of stem cells in mammals for several reasons. First, by giving rise to spermatogenesis, SSCs are responsible for the propagation of a father’s genetic material. As such, autologous SSCs have been considered for treatment of infertility and other purposes, including correction of inherited disorders. Second, adult spermatogonia can spontaneously produce embryonic-like stem cellsin vitro, which could be used as an alternative for therapeutic, diagnostic, or drug discovery strategies for humans. Therefore, an increasing urgency is driving efforts to understand the biology of SSCs and improve techniques to manipulate themin vitroas a prerequisite to achieve the aforementioned goals. The characterization of adult SSCs also requires reproducible methods to isolate and maintain them in long-term culture. Herein, we describe recent major advances and challenges in propagation of adult SSCs from mice and humans during the past few years, including the use of unique cell surface markers and defined cultured conditions.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

scholarly journals Phenotype Characterization of Human Adipose-Derived Stem Cell by Flow Cytometry

2021 ◽  
Author(s):  
Celeste Limoli ◽  
Paolo Giuseppe Limoli ◽  
Marcella Nebbioso
Keyword(s):  
Adipose Tissue ◽  
Cell Surface ◽  
Stromal Vascular Fraction ◽  
Surface Markers ◽  
Adipose Derived Stem Cells ◽  
Cell Surface Markers ◽  
Cell Surface Antigens ◽  
Adipose Derived Stem Cell

Background: Developing an efficient and standardized method to isolate and characterize adipose-derived stem cells (ASCs) from the stromal vascular fraction (SVF) of the adipose tissue for clinical application represents one of the major challenges in cell therapy and tissue engineering. Methods: In this study, we proposed an innovative, non-enzymatic protocol to collect clinically useful ASCs within freshly isolated SVF from adipose tissue by centrifugation of the infranatant portion of lipoaspirate and to determine the characteristic cytofluorimetric pattern, prior to in vitro culture. Results: The SVF yielded a mean of 73,32 \pm\ 10,89% cell viability evaluated with CALCEINA-FITC, i.e. cell-permeant dye. The ASCs were positive for PC7-labeled mAb anti-CD34 and negative for both PE-labeled mAb anti-CD31 and APC-labeled mAb anti-CD45. The frequency of ASCs estimated according to the panel of cell surface markers used was 51,06%\ \pm 5,26% versus the unstained ASCs subpopulation that was 0,74%\pm0,84% (P<0.0001). The ASCs events/\muL were 1602,13/\muL \pm 731,87/\muL. Conclusion: Our findings suggested that ASCs found in freshly isolated adipose SVF obtained by centrifugation of lipoaspirate can be immunophenotypically identified with a basic panel of cell surface markers. These findings aimed to provide standardization and contribute to reducing the inconsistency on reported cell surface antigens of ASC derived from the existing literature.

scholarly journals In vitro Comparison of Adipogenic Differentiation in Human Adipose-Derived Stem Cells Cultured with Collagen Gel and Platelet-Rich Fibrin

2021 ◽  
Vol 54 (03) ◽  
pp. 278-283
Author(s):  
Pallavi Priyadarshini ◽  
Soumi Samuel ◽  
Basan Gowda Kurkalli ◽  
Chethan Kumar ◽  
Basavarajappa Mohana Kumar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Soft Tissue ◽  
Cell Surface ◽  
Adipogenic Differentiation ◽  
Collagen Gel ◽  
Surface Markers ◽  
Adipose Derived Stem Cells ◽  
Cell Surface Markers ◽  
Platelet Rich Fibrin

Abstract Background: Adipose-derived stem cells (ADSCs) are the most preferred cell type, based on their phenotypic characteristics, plasticity, and favorable immunological properties for applications in soft-tissue augmentation. Hence, the present in vitro study was aimed to evaluate the adipogenic differentiation potential of human ADSCs upon culturing individually with collagen gel and platelet-rich fibrin (PRF). Materials and methods: The collected lipoaspirate was used for establishing ADSCs using enzymatic digestion method. Then, the cells were analyzed for their morphology, viability, proliferation rate, population doubling time (PDT), colony-forming ability, cell surface markers expression, and osteogenic differentiation as biological properties. Further, ADSCs were evaluated for their adipogenicity using induction media alone, and by culturing with collagen gel and PRF individually for prospective tissue augmentation. Results: ADSCs were successfully established in vitro and exhibited a fibroblast-like morphology throughout the culture period. Cells had higher viability, proliferation potential and showed their ability to form colonies. The positive expression of cell surface markers and osteogenic ability confirmed the potency of ADSCs. The ADSCs cultured on collagen gel and PRF, individually, showed higher number of differentiated adipocytes than ADSCs grown with adipogenic induction medium alone. Conclusion: The extent of lipid accumulation by ADSCs was slightly higher when cultured on collagen gel than on PRF. Additional experiments are required to confirm better suitability of scaffold materials for soft-tissue regeneration.

scholarly journals CD150− side population cells represent a functionally distinct population of long-term hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2444-2451 ◽  
Author(s):  
David C. Weksberg ◽  
Stuart M. Chambers ◽  
Nathan C. Boles ◽  
Margaret A. Goodell
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Side Population ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Slam Family

Hematopoietic stem cells (HSCs) are a self-renewing population of bone marrow cells that replenish the cellular elements of blood throughout life. HSCs represent a paradigm for the study of stem-cell biology, because robust methods for prospective isolation of HSCs have facilitated rigorous characterization of these cells. Recently, a new isolation method was reported, using the SLAM family of cell-surface markers, including CD150 (SlamF1), to offer potential advantages over established protocols. We examined the overlap between SLAM family member expression with an established isolation scheme based on Hoechst dye efflux (side population; SP) in conjunction with canonical HSC cell-surface markers (Sca-1, c-Kit, and lineage markers). Importantly, we find that stringent gating of SLAM markers is essential to achieving purity in HSC isolation and that the inclusion of canonical HSC markers in the SLAM scheme can greatly augment HSC purity. Furthermore, we observe that both CD150+ and CD150− cells can be found within the SP population and that both populations can contribute to long-term multilineage reconstitution. Thus, using SLAM family markers to isolate HSCs excludes a substantial fraction of the marrow HSC compartment. Interestingly, these 2 subpopulations are functionally distinct, with respect to lineage output as well as proliferative status.

scholarly journals Expansion and characterization of epithelial stem cells with potential for cyclical hair regeneration

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Makoto Takeo ◽  
Kyosuke Asakawa ◽  
Koh-ei Toyoshima ◽  
Miho Ogawa ◽  
JingJing Tong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hair Follicles ◽  
Regenerative Therapy ◽  
Functional Heterogeneity ◽  
Epithelial Stem Cells ◽  
Hair Regeneration ◽  
Bulge Region

AbstractIn mammals, organ induction occurs only during embryonic development except for hair follicles (HFs). However, HF-resident epithelial stem cells (HFSCs), which are responsible for repetitive HF regeneration, are not fully characterized. Here, we establish in vitro culture systems that are capable of controlling the ability of HFSCs to regenerate HFs. Based on a method that precisely controlled the number of HFs for regeneration, functional analysis revealed that CD34/CD49f/integrin β5 (Itgβ5)-triple-positive (CD34+/CD49f+/Itgβ5+) cells have multipotency and functional significance for continual hair regeneration. In native HFs, these cells reside in the uppermost area of the bulge region, which is surrounded by tenascin in mice and humans. This study unveils the subpopulation of HFSCs responsible for long-term hair cycling of HFs regenerated from bioengineered HF germ, suggesting the presence of functional heterogeneity among bulge HFSCs and the utility of our culture system to achieve HF regenerative therapy.

scholarly journals A new protocol for long‐term culture of a specific subpopulation of liver cancer stem cells enriched by cell surface markers

FEBS Open Bio ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1737-1747 ◽  
Author(s):  
Biao Zhang ◽  
Hai‐Yang Wang ◽  
Dong‐Xing Wang ◽  
Quan Zeng ◽  
Zeng Fan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Liver Cancer ◽  
Cell Surface ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Long Term Culture ◽  
Liver Cancer Stem Cells

scholarly journals CD66e Enrichment Enhances Repopulation of Human Long-Term Hematopoietic Stem Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2156-2156
Author(s):  
Kuiying Ma ◽  
Riguo Fang ◽  
Lingling Yu ◽  
Yongjian Zhang ◽  
Chao Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Surface Markers ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Differentiation Pattern ◽  
Human Hscs

Abstract Gene-modified hematopoietic stem cells (HSCs) therapy has demonstrated remarkable success for the treatment of inherited blood disorders. As the origin of hematologic hierarchy, HSCs play an essential role in sustaining life-long hematopoiesis. HSCs identification via reliable and robust bio-markers could facilitate the development of HSC gene therapy. Previous studies showed that long-term hematopoietic stem cells (LT-HSCs) were enriched in the Lin -CD34 +CD38 -CD45RA -CD90 +CD49f + population which could support long-term hematopoietic reconstitution. However, several of these surface markers proved to be unreliable when ex vivo culturing, such as CD38 and CD49f. Thus, HSCs characterization is still hindered by lacking bona-fide bio-markers, and consequently identification of long-term HSCs still needs time-consuming in vivo transplantation. To this end, we performed in vitro screening and comprehensive functional evaluation to identify a novel surface marker of human HSCs. During initial screening, a cell surface antigen screen panel (including 242 human cell surface markers) and human CD34 and CD90 antibodies were used to perform flow cytometry analysis on CD34 + HSPCs enriched from umbilical cord blood. Compared with CD34 + cell population, we found that CD66 (a,c,d,e), CD200 and CD48 positive cells were more enriched in CD34 +CD90 + subset. Previous studies indicated that HSCs cannot be maintained during in vitro culturing. By tracking these candidate surface markers based on this principle, CD66e was selected as the potential HSCs bio-marker. Next, we examined the in vivo hematologic repopulating potential of HSCs by limiting dilution assay (LDA) on immune-deficient mouse model. We sorted CD66e + and CD66e - subsets from CD34 +CD90 +CD45RA - subpopulation, and transplanted into irradiated NOD-scid Il2rg −/− (NPG) mice respectively. At week16 post-transplantation, in contrast to the CD66e - group, CD66e + cells exhibited significantly higher reconstitution in peripheral blood (PB), bone marrow (BM) and spleen. Engraftment dynamics revealed that the CD66e - group were only capable of reconstitution 4 weeks post transplantation, even at the highest initial cell dose. Moreover, the CD66e - group displayed impaired multi-lineage differentiation pattern, especially in PB and BM samples, while the CD66e + group presented a robust multi-lineage reconstitution. Notably, LDA results showed that the CD66e + cells within CD34 +CD90 +CD45RA - population contained 1 out of 529 SCID repopulating cells (SRC), almost 60-fold greater than the CD66e - fraction. To further investigate the long-term repopulating potential of the CD66e + cells, we performed the secondary transplantation collected from the BM cells of primary recipients. CD66e + cells presented significant higher repopulating activity than CD66e- subset in the secondary recipients. These findings reveal that the major cells with homing and long-term reconstitution capacity among CD34 +CD90 +CD45RA - cells were CD66e positive. In order to determine the transcriptional profile of CD66e + cells, we performed RNA-sequencing analysis using the population of CD34 + cells, CD34 +CD90 +CD45RA - cells, CD66e + and CD66e - cells within CD34 +CD90 +CD45RA - subset. Remarkably, compared with other groups, the CD66e + cells displayed a bias toward the signature of HSC and early progenitors such as LMPP and CLP. Moreover, gene set enrichment analysis showed that hematopoietic lineage and long-term potentiation-related genes were highly enriched in the CD66e + cells. Further qRT-PCR experiment confirmed that several HSC-related genes were significantly higher expressed in CD34 +CD90 +CD45RA -CD66e + cells, compared to CD66e - population or CD34 + HSPCs, suggesting that the gene expression profile of CD66e + cells is reminiscent of HSC signature. Altogether, we demonstrate that CD66e is a robust functional HSC bio-marker that CD66e-positive population among CD34 +CD90 +CD45RA - cells exhibit typical HSC signature, enhanced in vivo engraftment potential and robust multilineage differentiation pattern, which will provide an invaluable tool to investigate the origin of human HSCs, paving the way for the therapeutic application. Figure 1 Figure 1. Disclosures Fang: EdiGene, Inc.: Current Employment.

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