Improved Method forEx Ovo-Cultivation of Developing Chicken Embryos for Human Stem Cell Xenografts

The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potentialin vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary,ex ovo-culture systems avoid such negative side effects. Here, we present an improvedex ovo-cultivation method enabling the embryos to survive 13 daysin vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Ourex ovo-approach closely resembles the development of chicken embryosin ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26–HH28/E5—E6) ofex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, thisex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages.

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Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells

Blood ◽

10.1182/blood.v97.9.2618 ◽

2001 ◽

Vol 97 (9) ◽

pp. 2618-2624 ◽

Cited By ~ 52

Author(s):

Fumihito Tajima ◽

Takao Deguchi ◽

Joseph H. Laver ◽

Haiqun Zeng ◽

Makio Ogawa

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Steady State ◽

Adult Stem Cells ◽

Human Stem Cells ◽

Hematopoietic Stem ◽

Reciprocal Expression

Abstract The effects of activation of adult murine stem cells on their expression of CD38 were studied using a murine transplantation model. First, the published finding that the majority of long-term engrafting cells from normal adult steady-state marrow are CD38+ was confirmed. Next, it was determined that the majority of stem cells activated in vivo by injection of 5-fluorouracil (5-FU) or mobilized by granulocyte colony-stimulating factor are CD38−. Stem cells that were activated in culture with interleukin-11 and steel factor were also CD38−. Previous studies have shown that expression of CD34 by adult stem cells is also modulated by in vivo or in vitro activation. To determine whether there is reciprocal expression of CD38 and CD34, 4 populations of post–5-FU marrow cells were analyzed. The majority of the stem cells were in the CD38−CD34+ fraction. However, secondary transplantation experiments indicated that when the bone marrow reaches steady state, the majority of the stem cells become CD38+CD34−. In addition, the minority populations of CD34+ stem cells that occur in steady-state bone marrow are CD38−. This reversible and reciprocal expression of CD38 and CD34 by murine stem cells may have implications for the phenotypes of human stem cells.

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Effect of Muscle Extract and Graphene Oxide on Muscle Structure of Chicken Embryos

Animals ◽

10.3390/ani11123467 ◽

2021 ◽

Vol 11 (12) ◽

pp. 3467

Author(s):

Jaśmina Bałaban ◽

Marlena Zielińska ◽

Mateusz Wierzbicki ◽

Teresa Ostaszewska ◽

Magdalena Fajkowska ◽

...

Keyword(s):

Cell Proliferation ◽

Chicken Embryo ◽

Muscle Development ◽

Histological Structure ◽

Morphological Development ◽

In Ovo ◽

Chicken Embryos ◽

Muscle Structure

The effects of CEME and it complex with GO injected in ovo on the growth and development of chicken embryo hindlimb muscle were investigated. First, the preliminary in vitro study on primary muscle precursor cell culture obtained from a nine-day-old chicken embryo was performed to assess toxicity (MTT assay) of CEME, GO (100 ppm) and it complex with different concentrations (1, 2, 5, and 10 wt.%). The effect on cell proliferation was investigated by BrdU assay. CEME at concentrations 1–5% increased cell proliferation, but not the complex with GO. In vitro cytotoxicity was highest in 10% and GO groups. Next, the main experiment with chicken embryos was performed with CEME, GO and it complex injected in ovo on day one of embryogenesis. On day 20 of embryogenesis survival, morphological development, histological structure of the muscle, and biochemical parameters of blood serum of the embryos were measured. No negative effect on mortality, body weight, or biochemistry of blood after use of CEME or GO-CEME complexes was observed. Interestingly, the slight toxicity of GO, observed in in vitro studies, was not observed in vivo. The use of CEME at the levels of 2% and 5% improved the structure of the lower limb muscle by increasing the number of cells, and the administration of 2% CEME increased the number of nuclei visible in the stained cross-section of the muscle. The complex GO-CEME did not further improve the muscle structure. The results indicate that CEME can be applied as an in ovo enhancer of muscle development in broilers.

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Quantum Dots Do Not Alter the Differentiation Potential of Pancreatic Stem Cells and Are Distributed Randomly among Daughter Cells

International Journal of Cell Biology ◽

10.1155/2013/918242 ◽

2013 ◽

Vol 2013 ◽

pp. 1-12 ◽

Cited By ~ 9

Author(s):

S. Danner ◽

H. Benzin ◽

T. Vollbrandt ◽

J. Oder ◽

A. Richter ◽

...

Keyword(s):

Stem Cells ◽

Quantum Dots ◽

Animal Models ◽

Adult Stem Cells ◽

Cell Labeling ◽

Differentiation Potential ◽

Daughter Cells ◽

Pancreatic Stem Cells

With the increasing relevance of cell-based therapies, there is a demand for cell-labeling techniques forin vitroandin vivostudies. For the reasonable tracking of transplanted stem cells in animal models, the usage of quantum dots (QDs) for sensitive cellular imaging has major advances. QDs could be delivered to the cytoplasm of the cells providing intense and stable fluorescence. Although QDs are emerging as favourable nanoparticles for bioimaging, substantial investigations are still required to consider their application for adult stem cells. Therefore, rat pancreatic stem cells (PSCs) were labeled with different concentrations of CdSe quantum dots (Qtracker 605 nanocrystals). The QD labeled PSCs showed normal proliferation and their usual spontaneous differentiation potentialin vitro. The labeling of the cell population was concentration dependent, with increasing cell load from 5 nM QDs to 20 nM QDs. With time-lapse microscopy, we observed that the transmission of the QD particles during cell divisions was random, appearing as equal or unequal transmission to daughter cells. We report here that QDs offered an efficient and nontoxic way to label pancreatic stem cells without genetic modifications. In summary, QD nanocrystals are a promising tool for stem cell labeling and facilitate tracking of transplanted cells in animal models.

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Human muscle stem/progenitor cells are bi-potential adult stem cells

10.21203/rs.3.rs-594603/v1 ◽

2021 ◽

Author(s):

Xiexiang Shao ◽

Xin Fu ◽

Lili Han ◽

Sheng Li ◽

Hongye Wang ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Adult Stem Cells ◽

Tendon Injury ◽

Human Muscle ◽

Human Stem Cells ◽

Differentiation Potential ◽

Mouse Muscle ◽

Muscle Stem Cells

Abstract Tendon injury occurs at high frequency and is difficult to repair. Identification human stem cells being able to regenerate tendon will greatly facilitate the development of regenerative medicine for tendon injury. We identified human muscle stem/progenitor cells having tendon differentiation potential both in vitro and in vivo. Interestingly, the tendon differentiation potential is not present in mouse muscle stem cells. These findings reveal that human muscle stem cells are bi-potential adult stem cells and can serve as a new source for tendon regeneration.

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High glucose induces early senescence in adipose-derived stem cells by accelerating p16 and mTOR

Biomedical Research and Therapy ◽

10.15419/bmrat.v6i6.548 ◽

2019 ◽

Vol 6 (6) ◽

pp. 3213-3221

Author(s):

Hieu Liem Pham ◽

Phuc Van Pham

Keyword(s):

Stem Cells ◽

High Glucose ◽

Glucose Concentration ◽

Culture Medium ◽

Diabetic Patients ◽

Adipose Derived Stem Cells ◽

Differentiation Potential ◽

Normal Glucose

Introduction: The senescence of stem cells is the primary reason that causes aging of stem cell-containing tissues. Some hypotheses have suggested that high glucose concentration in diabetic patients is the main factor that causes senescence of cells in those patients. This study aimed to evaluate the effects of high glucose concentrations on the senescence of adipose-derived stem cells (ADSCs). Methods: ADSCs were isolated and expanded from human adipose tissues. They were characterized and confirmed as mesenchymal stem cells (MSCs) by expression of surface markers, their shape, and in vitro differentiation potential. They were then cultured in 3 different media- that contained 17.5 mM, 35 mM, or 55 mM of D-glucose. The senescent status of ADSCs was recorded by the expression of the enzyme beta-galactosidase, cell proliferation, and doubling time. Real-time RT-PCR was used to evaluate the expression of p16, p21, p53 and mTOR. Results: The results showed that high glucose concentrations (35 mM and 55 mM) in the culture medium induced senescence of human ADSCs. The ADSCs could progress to the senescent status quicker than those cultured in the lower glucose-containing medium (17.5 mM). The senescent state was related to the up-regulation of p16 and mTOR genes. Conclusion: These results suggest that high glucose in culture medium can trigger the expression of p16 and mTOR genes which cause early senescence in ADSCs. Therefore, ADSCs should be cultured in low glucose culture medium, or normal glucose concentration, to extend their life in vitro as well as in vivo.

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Organoid based personalized medicine: from bench to bedside

Cell Regeneration ◽

10.1186/s13619-020-00059-z ◽

2020 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Yaqi Li ◽

Peiyuan Tang ◽

Sanjun Cai ◽

Junjie Peng ◽

Guoqiang Hua

Keyword(s):

Stem Cells ◽

Personalized Medicine ◽

Adult Stem Cells ◽

Three Dimensional ◽

Basic Research ◽

Normal Tissues ◽

Technology Applications ◽

Genetically Manipulated

AbstractThree-dimensional cultured organoids have become a powerful in vitro research tool that preserves genetic, phenotypic and behavioral trait of in vivo organs, which can be established from both pluripotent stem cells and adult stem cells. Organoids derived from adult stem cells can be established directly from diseased epithelium and matched normal tissues, and organoids can also be genetically manipulated by CRISPR-Cas9 technology. Applications of organoids in basic research involve the modeling of human development and diseases, including genetic, infectious and malignant diseases. Importantly, accumulating evidence suggests that biobanks of patient-derived organoids for many cancers and cystic fibrosis have great value for drug development and personalized medicine. In addition, organoids hold promise for regenerative medicine. In the present review, we discuss the applications of organoids in the basic and translational research.

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Gene transfer into normal human hematopoietic cells using in vitro and in vivo assays

Blood ◽

10.1182/blood.v78.3.624.624 ◽

1991 ◽

Vol 78 (3) ◽

pp. 624-634 ◽

Cited By ~ 57

Author(s):

JE Dick ◽

S Kamel-Reid ◽

B Murdoch ◽

M Doedens

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Gene Transfer ◽

Hematopoietic Cells ◽

Human Stem Cells ◽

In Vivo Assays ◽

Genetically Manipulated ◽

Deficient Mice

Abstract The ability to transfer new genetic material into human hematopoietic cells provides the foundation for characterizing the organization and developmental program of human hematopoietic stem cells. It also provides a valuable model in which to test gene transfer and long-term expression in human hematopoietic cells as a prelude to human gene therapy. At the present time such studies are limited by the absence of in vivo assays for human stem cells, although recent descriptions of the engraftment of human hematopoietic cells in immune-deficient mice may provide the basis for such an assay. This study focuses on the establishment of conditions required for high efficiency retrovirus- mediated gene transfer into human hematopoietic progenitors that can be assayed in vitro in short-term colony assays and in vivo in immune- deficient mice. Here we report that a 24-hour preincubation of human bone marrow in 5637-conditioned medium, before infection, increases gene transfer efficiency into in vitro colony-forming cells by sixfold; interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) provide the same magnitude increase as 5637-conditioned medium. In contrast, incubation in recombinant growth factors IL-1, IL-3, and granulocyte- macrophage colony-stimulating factor increases gene transfer efficiency by 1.5- to 3-fold. Furthermore, preselection in high concentrations of G418 results in a population of cells significantly enriched for G418- resistant progenitors (up to 100%). These results, obtained using detailed survival curves based on colony formation in G418, have been substantiated by directly detecting the neo gene in individual colonies using the polymerase chain reaction. Using these optimized protocols, human bone marrow cells were genetically manipulated with a neo retrovirus vector and transplanted into immune-deficient bg/nu/xid mice. At 1 month and 4 months after the transplant, the hematopoietic tissues of these animals remained engrafted with genetically manipulated human cells. More importantly, G418-resistant progenitors that contained the neo gene were recovered from the bone marrow and spleen of engrafted animals after 4 months. These experiments establish the feasibility of characterizing human stem cells using the unique retrovirus integration site as a clonal marker, similar to techniques developed to elucidate the murine stem cell hierarchy.

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Implications and Current Limitations of Oogenesis from Female Germline or Oogonial Stem Cells in Adult Mammalian Ovaries

Cells ◽

10.3390/cells8020093 ◽

2019 ◽

Vol 8 (2) ◽

pp. 93 ◽

Cited By ~ 16

Author(s):

Jessica Martin ◽

Dori Woods ◽

Jonathan Tilly

Keyword(s):

Stem Cells ◽

Adult Stem Cells ◽

Ovarian Function ◽

Mammalian Species ◽

Large Body ◽

The Body ◽

Human Female ◽

Female Germline

A now large body of evidence supports the existence of mitotically active germ cells in postnatal ovaries of diverse mammalian species, including humans. This opens the possibility that adult stem cells naturally committed to a germline fate could be leveraged for the production of female gametes outside of the body. The functional properties of these cells, referred to as female germline or oogonial stem cells (OSCs), in ovaries of women have recently been tested in various ways, including a very recent investigation of the differentiation capacity of human OSCs at a single cell level. The exciting insights gained from these experiments, coupled with other data derived from intraovarian transplantation and genetic tracing analyses in animal models that have established the capacity of OSCs to generate healthy eggs, embryos and offspring, should drive constructive discussions in this relatively new field to further exploring the value of these cells to the study, and potential management, of human female fertility. Here, we provide a brief history of the discovery and characterization of OSCs in mammals, as well as of the in-vivo significance of postnatal oogenesis to adult ovarian function. We then highlight several key observations made recently on the biology of OSCs, and integrate this information into a broader discussion of the potential value and limitations of these adult stem cells to achieving a greater understanding of human female gametogenesis in vivo and in vitro.

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Osteogenic Potential of Multipotent Adult Progenitor Cells for Calvaria Bone Regeneration

Advances in Medicine ◽

10.1155/2016/2803081 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11

Author(s):

Dong Joon Lee ◽

Yonsil Park ◽

Wei-Shou Hu ◽

Ching-Chang Ko

Keyword(s):

Bone Regeneration ◽

Progenitor Cells ◽

Adult Stem Cells ◽

Single Cells ◽

Osteogenic Potential ◽

Type I ◽

Differentiation Potential ◽

Multipotent Adult Progenitor Cells

Osteogenic cells derived from rat multipotent adult progenitor cells (rMAPCs) were investigated for their potential use in bone regeneration. rMAPCs are adult stem cells derived from bone marrow that have a high proliferation capacity and the differentiation potential to multiple lineages. They may also offer immunomodulatory properties favorable for applications for regenerative medicine. rMAPCs were cultivated as single cells or as 3D aggregates in osteogenic media for up to 38 days, and their differentiation to bone lineage was then assessed by immunostaining of osteocalcin and collagen type I and by mineralization assays. The capability of rMAPCs in facilitating bone regeneration was evaluatedin vivoby the direct implantation of multipotent adult progenitor cell (MAPC) aggregates in rat calvarial defects. Bone regeneration was examined radiographically, histologically, and histomorphometrically. Results showed that rMAPCs successfully differentiated into osteogenic lineage by demonstrating mineralized extracellular matrix formationin vitroand induced new bone formation by the effect of rMAPC aggregatesin vivo. These outcomes confirm that rMAPCs have a good osteogenic potential and provide insights into rMAPCs as a novel adult stem cell source for bone regeneration.

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Treprostinil indirectly regulates endothelial colony forming cell angiogenic properties by increasing VEGF-A produced by mesenchymal stem cells

Thrombosis and Haemostasis ◽

10.1160/th14-11-0907 ◽

2015 ◽

Vol 114 (10) ◽

pp. 735-747 ◽

Cited By ~ 13

Author(s):

Marilyne Levy ◽

Lan Huang ◽

Elisa Rossi ◽

Adeline Blandinières ◽

Dominique Israel-Biet ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cord Blood ◽

Proliferative Potential ◽

Differentiation Potential ◽

Endothelial Colony Forming Cells ◽

Pulmonary Vasodilators ◽

High Level

SummaryPulmonary vasodilators and prostacyclin therapy in particular, have markedly improved the outcome of patients with pulmonary hypertension (PH). Endothelial dysfunction is a key feature of PH, and we previously reported that treprostinil therapy increases number and proliferative potential of endothelial colony forming cells (ECFC) isolated from PH patients’ blood. In the present study, the objective was to determine how treprostinil contributes to the proangiogenic functions of ECFC. We examined the effect of treprostinil on ECFC obtained from cord blood in terms of colony numbers, proliferative and clonogenic properties in vitro, as well as in vivo vasculogenic properties. Surprisingly, treprostinil inhibited viability of cultured ECFC but did not modify their clonogenic properties or the endothelial differentiation potential from cord blood stem cells. Treprostinil treatment significantly increased the vessel-forming ability of ECFC combined with mesenchymal stem cells (MSC) in Matrigel implanted in nude mice. In vitro, ECFC proliferation was stimulated by conditioned media from treprostinil-pretreated MSC, and this effect was inhibited either by the use of VEGF-A blocking antibodies or siRNA VEGF-A in MSC. Silencing VEGF-A gene in MSC also blocked the pro-angiogenic effect of treprostinil in vivo. In conclusion, increased VEGF-A produced by MSC can account for the increased vessel formation observed during treprostinil treatment. The clinical relevance of these data was confirmed by the high level of VEGF-A detected in plasma from patients with paediatric PH who had been treated with treprostinil. Moreover, our results suggest that VEGF-A level in patients could be a surrogate biomarker of treprostinil efficacy.

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