An Efficient Protocol for Deriving Liver Stem Cells from Neonatal Mice: Validating Its Differentiation Potential

The success of liver regeneration depends on the availability of suitable cell types and their potential to differentiate into functional hepatocytes. To identify the stem cells which have the ability to differentiate into hepatocytes, we used neonatal liver as source. However, the current protocol for isolating stem cells from liver involves enzymes like collagenase, hyaluronidase exposed for longer duration which limits the success. This results in the keen interest to develop an easy single step enzyme digestion protocol for isolating stem cells from liver for tissue engineering approaches. Thus, the unlimited availability of cell type favors setting up the functional recovery of the damaged liver, ensuring ahead success towards treating liver diseases. We attempted to isolate liver stem derived cells (LDSCs) from mouse neonatal liver using single step minimal exposure to enzyme followed byin vitroculturing. The cells isolated were characterized for stem cell markers and subjected to lineage differentiation. Further, LDSCs were induced to hepatocyte differentiation and validated with hepatocyte markers. Finally, we developed a reproducible, efficient protocol for isolation of LDSCs with functional hepatocytes differentiation potential, which further can be used asin vitromodel system for assessing drug toxicity assays in various preclinical trials.

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022. Isolation of stem cells from embryos and adult bovine tissues

Reproduction Fertility and Development ◽

10.1071/srb05abs022 ◽

2005 ◽

Vol 17 (9) ◽

pp. 67

Author(s):

P. J. Verma ◽

K. Upton ◽

H. Mc Connell ◽

I. Vassiliev

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Es Cells ◽

Cell Types ◽

Invasive Technique ◽

Stem Cell Markers ◽

Rt Pcr ◽

Differentiation Potential ◽

Cell Therapies

The isolation of stem cells has become an area of increasing interest due to their potential uses in animal reproduction, somatic cell nuclear transfer and cell therapies. The most attractive options are the isolation of stem cells from individual embryos or adult somatic tissues. In addition, for cell therapy, the use of autologous stem cells is considered to have an advantage over heterologous cell based therapies in that immune rejection issues would be circumvented. Here we report on our attempts to isolate stem cells from both these sources in a bovine model. Bovine ES-like (bES) cells were successfully isolated from embryos and maintained in vitro for up to six passages. These cells retained the morphology characteristic of bES cells: small cytoplasmic/nuclear ratio, nuclei with multiple nucleoli, and multiple lipid inclusions in cytoplasm. bES cell colonies grew as monolayers, as islands of ES cells surrounded by trophectoderm (TE) cells. Immunohistochemical detection of SSEA-1 and SSEA-4 demonstrated expression of these markers in bES cells but not in TE cells. Further, the expression of the pluripotent markers Oct-4, Rex-1 and SSEA-1 by RT-PCR was also detected in bES cells but not in TE cells. On spontaneous differentiation, these cells were able to form a variety of cell types including beating muscle with the cells displaying a propensity to differentiate in a manner reminiscent of human ES cells. (2) We also report the isolation of putative stem cells from adult bovine skin biopsies, which express the stem cell markers Oct-4 and SSEA-1 analysed by RT-PCR and are capable of forming 3-dimensional colonies. These cells are obtained from a skin biopsy, a relatively non-invasive technique that makes them useful as donors for therapeutic applications. In summary, we have identified populations of stem cells from embryonic and adult bovine tissues, which are readily isolated. Further characterization of the differentiation potential of these cells is needed to identify the suitability of this population for use in autologous stem cell therapies.

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3D printable Sodium alginate-Matrigel (SA-MA) hydrogel facilitated ectomesenchymal stem cells (EMSCs) neuron differentiation

Journal of Biomaterials Applications ◽

10.1177/0885328220961261 ◽

2020 ◽

Vol 35 (6) ◽

pp. 709-719 ◽

Cited By ~ 1

Author(s):

Yang Li ◽

Xia Cao ◽

Wenwen Deng ◽

Qingtong Yu ◽

Congyong Sun ◽

...

Keyword(s):

Stem Cells ◽

Sodium Alginate ◽

Neuronal Differentiation ◽

Adult Stem Cells ◽

Three Dimensional ◽

Cell Types ◽

Differentiation Potential ◽

Lineage Differentiation ◽

Cord Injury

Ectomesenchymal stem cells (EMSCs) are typical adult stem cells obtained from the cranial neural crest. They have the potential to differentiate into various cell types, such as osseous cells, neurons and glial cells. Three-dimensional (3 D) printing is a novel method to construct biological structures by rapid prototyping. Previously, our group reported on the stemness and multi-lineage differentiation potential of EMSCs on gels. However, the exploration of EMSCs in 3 D printing and then evaluation of the growth and neuronal differentiation of EMSCs on extruded 3 D printable hybrid hydrogels has not been reported. Therefore, the current study explored the novel hybrid Sodium alginate-Matrigel (SA-MA) hydrogel extruded 3 D printing to design an in vitro scaffold to promote the differentiation and growth of EMSCs. In addition, the physical properties of the hydrogel were characterized and its drug-releasing property determined. Notably, the results showed that the construct exhibited a sustain-released effect of growth factor BDNF in accordance with the Higuchi equation. Moreover, the cell survival rate on the 3 D printed scaffold was 88.22 ± 1.13% with higher neuronal differentiation efficiency compared with 2 D culture. Thus, SA-MA’s ability to enhanced EMSCs neuronal differentiation offers a new biomaterial for neurons regeneration in the treatment of spinal cord injury.

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

10.1101/364059 ◽

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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Mapping SOX9 transcriptional dynamics during multi-lineage differentiation of human mesenchymal stem cells

10.1101/2021.11.29.470107 ◽

2021 ◽

Author(s):

Kannan Govindaraj ◽

Sakshi Khurana ◽

Marcel Karperien ◽

Janine Nicole Post

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Transcription Factor ◽

Single Cell ◽

Adipogenic Differentiation ◽

Human Mesenchymal Stem Cells ◽

Cell Types ◽

Differentiation Potential ◽

Lineage Differentiation ◽

Transcriptional Dynamics

The master transcription factor SOX9 is a key player during chondrocyte differentiation, cartilage development, homeostasis and disease. Modulation of SOX9 and its target gene expression is essential during chondrogenic, osteogenic and adipogenic differentiation of human mesenchymal stem cells (hMSCs). However, lack of sufficient knowledge about the signaling interplay during differentiation remains one of the main reasons preventing successful application of hMSCs in regenerative medicine. We previously showed that Transcription Factor - Fluorescence Recovery After Photobleaching (TF-FRAP) can be used to study SOX9 dynamics at the single cell level. We showed that changes in SOX9 dynamics are linked to its transcriptional activity. Here, we investigated SOX9 dynamics during differentiation of hMSCs into the chondrogenic, osteogenic and adipogenic lineages. We show that there are clusters of cells in hMSCs with distinct SOX9 dynamics, indicating that there are a number of subpopulations present in the heterogeneous hMSCs. SOX9 dynamics data at the single cell resolution revealed novel insights about its activity in these subpopulations (cell types). In addition, the response of SOX9 to differentiation stimuli varied in these subpopulations. Moreover, we identified donor specific differences in the number of cells per cluster in undifferentiated hMSCs, and this correlated to their differentiation potential.

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Adenovirus infection promotes the formation of glioma stem cells by glioblastoma cells through the TLR9/NEAT1/STAT3 pathway

10.21203/rs.3.rs-15785/v1 ◽

2020 ◽

Author(s):

Jian Zang ◽

Min-hua Zheng ◽

Xiu-li Cao ◽

Yi-zhe Zhang ◽

Yu-fei Zhang ◽

...

Keyword(s):

Stem Cells ◽

Glioma Cells ◽

Tumor Model ◽

Glioma Stem Cells ◽

Stem Cell Markers ◽

Qrt Pcr ◽

Lineage Differentiation ◽

Stat3 Signaling

Abstract BackgroundGlioma stem cells (GSCs) are glioma cells with stemness and are responsible for a variety of malignant behaviors of glioma. Evidence has shown that signals from tumor microenvironment (TME) enhance stemness of glioma cells, but the identity of the signaling molecules and underlying mechanisms have been incompletely elucidated.MethodsHuman samples and glioma cell lines were cultured in vitro to determine the effects of viral infection by sphere formation, qRT-PCR, Western blot, FACS and immunofluorescence; for in vivo analysis, mice subcutaneous tumor model was carried; while bioinformatics analysis and qRT-PCR were applied for further mechanistic studies.ResultsIn this study, we show that infection of patient-derived glioma cells with adenovirus (ADV) increases the formation of tumor spheres. ADV infection upregulated stem cell markers, and the resultant tumor spheres held the capacities of self-renewal and multi-lineage differentiation, and had stronger potential to form xenograft tumors in immune-compromised mice. ADV promoted GSC formation likely via TLR9, because TLR9 was upregulated after ADV infection, and knockdown of TLR9 reduced ADV-induced GSCs. Consistently, MYD88, as well as total STAT3 and phosphorylated (p-)STAT3, were also upregulated in ADV-induced GSCs. Knockdown of MYD88 or pharmaceutical inhibition of STAT3 attenuated stemness of ADV-induced GSCs. Moreover, we found that ADV infection upregulated lncRNA NEAT1, which is downstream to TLRs and play important roles in cancer stem cells via multiple mechanisms including strengthening STAT3 signaling. Indeed, knockdown of NEAT1 impaired stemness of ADV-induced GSCs. Lastly, we show that HMGB1, a damage associated molecular pattern (DAMP) that also triggers TLR signaling, upregulated stemness markers in glioma cells.ConclusionsIn summary, our data indicate that ADV, which has been developed as vectors for gene therapy and oncolytic virus, promotes the formation of GSCs via TLR9/NEAT1/STAT3 signaling.

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Establishment and biological characterization of a dermal mesenchymal stem cells line from bovine

Bioscience Reports ◽

10.1042/bsr20130095 ◽

2014 ◽

Vol 34 (2) ◽

Cited By ~ 7

Author(s):

Tingting Sun ◽

Chao Yu ◽

Yuhua Gao ◽

Chenqiong Zhao ◽

Jinlian Hua ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Types ◽

Neural Cells ◽

Biological Characterization ◽

Multipotent Stem Cells ◽

Reverse Transcription Pcr ◽

Lineage Differentiation

The DMSCs (dermal mesenchymal stem cells) are multipotent stem cells, which can differentiate in vitro into many cell types. Much work has been done on DMSCs from humans, mice, rabbits and other mammals, but the related literature has not been published about these cells in cattle. In this study, we isolated and established the DMSC lines from cattle, thereby initiating further research on these cells, such as growth kinetics, detection of special surface antigen and RT–PCR (reverse transcription–PCR) assays to identify the biological characterization of the cell line. Furthermore, the DMSCs are induced to differentiate into adipocytes, osteoblasts and neural cells in vitro. Our results suggest that DMSCs isolated from cattle possess similar biological characteristics with those from other species. Their multi-lineage differentiation capabilities herald a probable application model in tissue engineering and induced pluripotent stem cells.

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Different Concentrations of C5a Affect Human Dental Pulp Mesenchymal Stem Cells Differentiation

10.21203/rs.3.rs-156495/v1 ◽

2021 ◽

Author(s):

Jie Liu ◽

Xiaoling Wei ◽

Junlong Hu ◽

Xiaohan Tan ◽

Xiaocui Kang ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Dental Pulp ◽

Inhibitory Effect ◽

Stem Cell Markers ◽

High Concentration ◽

Differentiation Potential ◽

Human Dental Pulp ◽

Dentin Sialoprotein

Abstract Background During the process of deep decay, when decay approaches the pulp, an immune response is triggered inside the pulp, which activates the complement cascade. The effect of complement component 5a (C5a) on the differentiation of dental pulp mesenchymal stem cells (DPSCs) is related to dentin reparation. The aim of the present study was to stimulate DPSCs with different concentrations of C5a and evaluate the differentiation of odontoblasts using dentin sialoprotein (DSP). Methods DPSCs were divided into the following six groups: i) Control; ii) DPSCs treated with 50 ng/ml C5a; iii) DPSCs treated with 100 ng/ml C5a; iv) DPSCs treated with 200 ng/ml C5a; v) DPSCs treated with 300 ng/ml C5a; and vi) DPSCs treated with 400 ng/ml C5a. Flow cytometry and multilineage differentiation potential were used to identify DPSCs. Mineralization induction, Real-time PCR and Western blot were conducted to evaluate the differentiation of odontoblast in the 6 groups.Result DPSCs can express mesenchymal stem cell markers, including CD105, CD90, CD73 and, a less common marker, mesenchymal stromal cell antigen-1. In addition, DPSCs can differentiate into adipocytes, neurocytes and osteoblasts. All six groups formed mineralized nodules after 28 days of culture. Reverse transcription-quantitative PCR and western blotting indicated that the high concentration C5a groups expressed higher DSP levels and promoted DPSC differentiation, whereas the low concentration C5a groups displayed an inhibitory effect.Conclusion In this study, the increasing concentration of C5a, which accompanies the immune process in the dental pulp, has demonstrated an enhancing effect on odontoblast differentiation at higher C5a concentrations in vitro.

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Progress and Future Challenges of Human Induced Pluripotents Stem Cell in Regenerative Medicine

The Indonesian Biomedical Journal ◽

10.18585/inabj.v3i2.138 ◽

2011 ◽

Vol 3 (2) ◽

pp. 76

Author(s):

Anna Meiliana ◽

Andi Wijaya

Keyword(s):

Stem Cells ◽

Somatic Cell ◽

Expression Profiles ◽

Human Fibroblasts ◽

Embryonic Stem ◽

Cell Types ◽

Differentiation Potential ◽

Human Ipscs

BACKGROUND: Less than a decade ago the prospect for reprogramming the human somatic cell looked bleak at best. It seemed that the only methods at our disposal for the generation of human isogenic pluripotent cells would have to involve somatic cell nuclear transfer (SCNT). Shinya Yamanaka in August 2006 in his publication (Cell) promised to change everything by showing that it was apparently very simple to revert the phenotype of a differentiated cell to a pluripotent one by overexpressing four transcription factors in murine fibroblasts.CONTENT: Mouse and human somatic cells can be genetically reprogrammed into induced pluripotent stem cells (iPSCs) by the expression of a defined set of factors (Oct4, Sox2, c-Myc, and Klf4, as well as Nanog and LIN28). iPSCs could be generated from mouse and human fibroblasts as well as from mouse liver, stomach, pancreatic, neural stem cells, and keratinocytes. Similarity of iPSCs and embryonic stem cells (ESCs) has been demonstrated in their morphology, global expression profiles, epigenetic status, as well as in vitro and in vivo differentiation potential for both mouse and human cells. Many techniques for human iPSCs (hiPSCs) derivation have been developed in recent years, utilizing different starting cell types, vector delivery systems, and culture conditions. A refined or perfected combination of these techniques might prove to be the key to generating clinically applicable hiPSCs.SUMMARY: iPSCs are a revolutionary tool for generating in vitro models of human diseases and may help us to understand the molecular basis of epigenetic reprogramming. Progress of the last four years has been truly amazing, almost verging on science fiction, but if we can learn to produce such cells cheaply and easily, and control their differentiation, our efforts to understand and fight disease will become more accessible, controllable and tailored. Ability to safely and efficiently derive hiPSCs may be of decisive importance to the future of regenerative medicine.KEYWORDS: iPSCs, ESC, reprogramming factor, reprogramming efficiency, somatic cell

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Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

BMC Oral Health ◽

10.1186/s12903-021-01833-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jie Liu ◽

Xiaoling Wei ◽

Junlong Hu ◽

Xiaohan Tan ◽

Xiaocui Kang ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Dental Pulp ◽

Inhibitory Effect ◽

Stem Cell Markers ◽

High Concentration ◽

Differentiation Potential ◽

Human Dental Pulp ◽

Dentin Sialoprotein

Abstract Background During the process of deep decay, when decay approaches the pulp, an immune response is triggered inside the pulp, which activates the complement cascade. The effect of complement component 5a (C5a) on the differentiation of dental pulp mesenchymal stem cells (DPSCs) is related to dentin reparation. The aim of the present study was to stimulate DPSCs with different concentrations of C5a and evaluate the differentiation of odontoblasts using dentin sialoprotein (DSP). Methods DPSCs were divided into the following six groups: (i) Control; (ii) DPSCs treated with 50 ng/ml C5a; (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a. Flow cytometry and multilineage differentiation potential were used to identify DPSCs. Mineralization induction, Real-time PCR and Western blot were conducted to evaluate the differentiation of odontoblast in the 6 groups. Result DPSCs can express mesenchymal stem cell markers, including CD105, CD90, CD73 and, a less common marker, mesenchymal stromal cell antigen-1. In addition, DPSCs can differentiate into adipocytes, neurocytes, chondrocytes and odontoblasts. All six groups formed mineralized nodules after 28 days of culture. Reverse transcription-quantitative PCR and western blotting indicated that the high concentration C5a groups expressed higher DSP levels and promoted DPSC differentiation, whereas the low concentration C5a groups displayed an inhibitory effect. Conclusion In this study, the increasing concentration of C5a, which accompanies the immune process in the dental pulp, has demonstrated an enhancing effect on odontoblast differentiation at higher C5a concentrations in vitro.

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Multipotent Human Mesenchymal Stem/Stromal Cells: An Updated Review on Historical Background, Recent Trends and Advances in their Clinical Applications

10.20944/preprints202103.0373.v1 ◽

2021 ◽

Author(s):

Kevin Dzobo

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Clinical Trials ◽

Cell Types ◽

Clinical Applications ◽

Historical Background ◽

Differentiation Potential ◽

Lineage Differentiation ◽

Different Cell Types ◽

Stromal Stem Cells

Early reports demonstrated the presence of cells with stem-like properties in bone marrow, with these cells having both hematopoietic and mesenchymal lineages. Over the years, various investigations have purified and characterized mesenchymal stromal/stem cells (MSCs) from different human tissues as cells with multi-lineage differentiation potential under the appropriate conditions. Due to their appealing characteristics and potential, MSCs are leveraged in many applications including medicine, oncology, bioprinting and as recent as treatment of COVID-19. To date, reports indicate mesenchymal stromal/stem cells have varied differentiation capabilities into different cell types and demonstrate immunomodulating and anti-inflammatory properties. Reports indicate that different MSCs microenvironments or niche and the resulting heterogeneity may influence their behavior and differentiation capacity. The potential clinical applications of mesenchymal stromal/stem cells have led to an avalanche of research reports on their properties and hundreds of clinical trials being undertaken. The future looks bright and promising for mesenchymal stem cell research with many clinical trials under way to prove their utility in many applications and in the clinic. This report provides an update on the potential broader use of mesenchymal stromal/stem cells, review early observations of the presence of these cells in the bone marrow and their magnificent differentiation capabilities and immunomodulation.

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