Regionally-derived cell populations and skeletal stem cells from human foetal femora exhibit specific osteochondral and multi-lineage differentiation capacity in vitro and ex vivo

Mesenchymal stem cells (MSCs) showed in vitro mesoderm-lineage differentiation and self-renew capacity. However, no comparative study was reported on the biological characteristics of stem cells derived from skeletal muscle (SM-MSCs), dermal skin (DS-MSCs), and adipose tissues (A-MSCs) from a single donor in camels. The present study aimed to evaluate the influence of MSCs source on stem cell characteristics. We evaluated proliferation capacity and mesoderm-lineage differentiation potential from SM-MSCs, DS-MSCs, and A-MSCs. They showed spindle-like morphology after homogenization. The proliferation ability was no significant difference in all groups. Furthermore, the portion of the cell cycle and expression of pluripotent markers (Oct4, Sox2, and Nanog) were similar in all cell lines at passage 3. The differentiation capacity of A-MSCs into adipocytes was significantly higher than that of SM-MSCs and DS-MSCs. However, the osteoblast differentiation capacity of A-MSCs was significantly lower than that of SM-MSCs and DS-MSCs. Additionally, after osteoblast differentiation, the ALP activity and calcium content was significantly decreased in A-MSCs as compared to SM-MSCs and DS-MSCs. To the best of our knowledge, we primally established MSCs from the single camel and demonstrated their comparative characteristics including expression of pluripotent factors and proliferation, and in vitro differentiation capacity into adipocytes and osteoblasts.

Download Full-text

Selective Activation of STAT5 Unveils Its Role in the Maintenance of Hematopoietic Stem Cells and the Development of Myeloproliferative Disorder.

Blood ◽

10.1182/blood.v104.11.3549.3549 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3549-3549

Author(s):

Yuko Kato ◽

Atsushi Iwama ◽

Hiromitsu Nakauchi

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Janus Kinase ◽

Selective Activation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Lineage Differentiation

Abstract Recent studies have implicated the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the maintenance of stem cells, such as mouse embryonic stem cells and Drosophila germ cells. We have previously reported that thrombopoietin (TPO) can support in vitro self-renewal division of murine hematopoietic stem cells (HSCs) (CD34−/lowc-Kit+Sca-1+lineage marker-negative; CD34−KSL cells). Signal transducers and activators of transcription 5 (STAT5) is one of the major signaling molecules that mediate TPO signals. All these findings suggest that STAT5 could be an attractive candidate for therapeutic manipulation of HSCs. Cytokines activate JAK/STAT5 pathway along with other signaling pathways, causing difficulty to dissect STAT5-specific functions in hematopoietic stem cells (HSCs). Here we took advantage of constitutively active STAT5 mutants to selectively activate STAT5 signaling pathway in HSCs. The mutants used are STAT5A 1*6 that harbors two amino acid mutations S710F and H298R in the effecter domain, and STAT5A #2 that harbors a point mutation N642H in the SH2 domain. Retroviral transduction of either STAT5 1*6 or STAT5#2 mutant into purified CD34−KSL HSCs caused a drastic expansion of multipotential progenitors in vitro and promoted multi-lineage differentiation in vitro. During 7 days of culture supplemented with SCF and TPO, the number of high proliferative potential colonies (HPPC) increased ten-fold compared with the GFP control and half of them were derived from multipotential progenitor cells. Notably, even in the culture supplemented with SCF only, expression of STAT5 mutants in HSCs supported a similar mode of expansion of progenitors cells and multi-lineage differentiation, indicating that activation of STAT5 can substitute major biological effects of TPO in HSCs. In all in vitro experiments, STAT5 1*6 showed stronger effects than STAT5#2. To evaluate the effect of STAT5A mutants in the maintenance of long-term bone marrow repopulating HSC ex vivo, cultured transduced cells corresponding to 30 initial CD34−KSL HSCs were transplanted into lethally irradiated mice 7 to 10 days after transduction. Although rapid hamatopoietic repopulation was observed with HSCs expressing STAT5A 1*6, mice developed myeloproliferative disease (MPD) and succumbed to death within two months. In contrast, HSCs expressing STAT5A #2 presented significantly higher long-term repopulating capacity than the GFP control. These data indicate that selective activation of STAT5 maintains long-term repopulating ability of HSCs ex vivo. Oncostatin M, a well known STAT5 target gene, has been postulated to be involved in the development of MPD and was actually induced STAT5A 1*6-expressing cells. However, transplantation of OSM−/− HSCs expressing STAT5A 1*6 similarly caused a lethal MPD in wild-type mice, indicating that Oncostatin is not the main target for STAT5 in MPD development. Taken together, our findings establish a role for STAT5 in the self-renewal of HSCs and provide STAT5 as novel target for therapeutic manipulation of HSCs ex vivo.

Download Full-text

Comparative Study of Biological Characteristics, and Osteoblast Differentiation of Mesenchymal Stem Cell Established from Camelus dromedarius Skeletal Muscle, Dermal Skin, and Adipose Tissues

Animals ◽

10.3390/ani11041017 ◽

2021 ◽

Vol 11 (4) ◽

pp. 1017

Author(s):

Young-Bum Son ◽

Yeon Ik Jeong ◽

Yeon Woo Jeong ◽

Mohammad Shamim Hossein ◽

Alex Tinson ◽

...

Keyword(s):

Skeletal Muscle ◽

Stem Cells ◽

Stem Cell ◽

Comparative Study ◽

Osteoblast Differentiation ◽

Biological Characteristics ◽

Adipose Tissues ◽

Differentiation Capacity ◽

Lineage Differentiation

Mesenchymal stem cells (MSCs) showed in vitro mesoderm-lineage differentiation and self-renewal capacity. However, no comparative study was reported on the biological characteristics of stem cells derived from skeletal muscle (SM-MSCs), dermal skin (DS-MSCs), and adipose tissues (A-MSCs) from a single donor in camels. The present study aimed to evaluate the influence of MSCs source on stem cell characteristics. We evaluated proliferation capacity and mesoderm-lineage differentiation potential from SM-MSCs, DS-MSCs, and A-MSCs. They showed spindle-like morphology after homogenization. The proliferation ability was not significantly difference in any of the groups. Furthermore, the portion of the cell cycle and expression of pluripotent markers (Oct4, Sox2, and Nanog) were similar in all cell lines at passage 3. The differentiation capacity of A-MSCs into adipocytes was significantly higher than that of SM-MSCs and DS-MSCs. However, the osteoblast differentiation capacity of A-MSCs was significantly lower than that of SM-MSCs and DS-MSCs. Additionally, after osteoblast differentiation, the alkaline phosphatase (ALP) activity and calcium content significantly decreased in A-MSCs compared to SM-MSCs and DS-MSCs. To the best of our knowledge, we primarily established MSCs from the single camel and demonstrated their comparative characteristics, including expression of pluripotent factors and proliferation, and in vitro differentiation capacity into adipocytes and osteoblasts.

Download Full-text

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200225091339 ◽

2020 ◽

Vol 15 ◽

Author(s):

Fatima Aerts-Kaya

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Stress Conditions ◽

Stress Factors ◽

Hematopoietic Stem ◽

Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

Download Full-text

Potential Effects of Corneal Cross-Linking upon the Limbus

BioMed Research International ◽

10.1155/2016/5062064 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Johnny E. Moore ◽

Davide Schiroli ◽

C. B. Tara Moore

Keyword(s):

Stem Cells ◽

Potential Risk ◽

Ocular Surface ◽

Ex Vivo ◽

Uv Exposure ◽

Cross Linking ◽

Clinical Complications ◽

Cellular Dna ◽

Pathological Consequence

Corneal cross-linking is nowadays the most used strategy for the treatment of keratoconus and recently it has been exploited for an increasing number of different corneal pathologies, from other ectatic disorders to keratitis. The safety of this technique has been widely assessed, but clinical complications still occur. The potential effects of cross-linking treatment upon the limbus are incompletely understood; it is important therefore to investigate the effect of UV exposure upon the limbal niche, particularly as UV is known to be mutagenic to cellular DNA and the limbus is where ocular surface tumors can develop. The risk of early induction of ocular surface cancer is undoubtedly rare and has to date not been published other than in one case after cross-linking. Nevertheless it is important to further assess, understand, and reduce where possible any potential risk. The aim of this review is to summarize all the reported cases of a pathological consequence for the limbal cells, possibly induced by cross-linking UV exposure, the studies donein vitroorex vivo, the theoretical bases for the risks due to UV exposure, and which aspects of the clinical treatment may produce higher risk, along with what possible mechanisms could be utilized to protect the limbus and the delicate stem cells present within it.

Download Full-text

Comparison of in vitro Neuronal Differentiation Capacity Between Mouse Epiblast Stem Cells Derived From Nuclear Transfer and Naturally Fertilized Embryos

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2018.00392 ◽

2018 ◽

Vol 11 ◽

Author(s):

Tong Li ◽

Yi Zheng ◽

Yan Li ◽

Danna Ye

Keyword(s):

Stem Cells ◽

Neuronal Differentiation ◽

Nuclear Transfer ◽

Differentiation Capacity ◽

Epiblast Stem Cells

Download Full-text

Exploring the Role of Mesenchymal Stem Cells During Normothermic Organ Perfusion: A New Paradigm to Enhance Outcome Following Allograft Transplantation

The Open Stem Cell Journal ◽

10.2174/1876893801805010047 ◽

2018 ◽

Vol 5 (1) ◽

pp. 47-52

Author(s):

Mohamed Morsy ◽

Mohammad Ayaz Hossain ◽

Atul Bagul

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Organ Transplantation ◽

Ex Vivo ◽

Solid Organ Transplantation ◽

Short Review ◽

Solid Organ ◽

New Paradigm ◽

Liver And Kidney

Background: Normothermic Machine Perfusion (NMP) has been established in the field of solid organ transplantation for both liver and kidney allografts. The ability to perfuse organs at body temperature enables viability assessment as well as optimisation prior to implantation. Discussion: A recent in vitro report of the use of Mesenchymal Stem Cells (MSCs) in the use of a normothermic lung perfusion circuit has raised the possibility of their use in solid organ transplantation. The aim of this short review is to outline the potential uses of bone marrow derived MSCs for their use in renal allograft ex vivo NMP. An overview is provided of current literature of NMP as well as theorised uses for MSCs.

Download Full-text

Low Oxygen Tension Potentiates Proliferation and Stemness but Not Multilineage Differentiation of Caprine Male Germline Stem Cells

10.21203/rs.3.rs-420611/v1 ◽

2021 ◽

Author(s):

Shiva Pratap Singh ◽

Suresh Dinkar Kharche ◽

Manisha Pathak ◽

Ravi Ranjan ◽

Yogesh Kumar Soni ◽

...

Keyword(s):

Stem Cells ◽

Ex Vivo ◽

Growth Characteristics ◽

Population Doubling ◽

Population Doubling Time ◽

Germline Stem Cells ◽

Multilineage Differentiation ◽

Low Oxygen ◽

Differentiation Potential

Abstract The milieu of testicular germline stem cells (mGSCs) is characterized as low oxygen (O2) environment, whereas, there in-vitro expansion is typically performed under normoxia (20-21% O2). Here, we evaluated and compared the culture and multilineage differentiation characteristics of enriched (through differential platting and percoll density centrifugation) caprine mGSCs (cmGSCs) under hypoxic (5% O2) and normoxic (21% O2) culture conditions. For this, in addition to growth characteristics and population-doubling time (PDT); viability, proliferation, senescence, and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated and compared under normoxia and hypoxia. Moreover, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) was assessed. The survival, viability and proliferation were significantly promoted and PDT was reduced (p < 0.05), thus yielding a higher number of viable cells with larger colonies under hypoxia. Furthermore, expression of stemness and adhesion markers was distinctly increased under lowered O2 condition. Conversely, the presence of differentiated regions and expression of differentiation specific key genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxic conditions. These data demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics, and stemness but not the multilineage differentiation potential of cmGSCs as compared with normoxia. These data are important for the development of robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

Download Full-text

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.

Download Full-text

Intramuscular Injection of Botox Induces Tendon Atrophy and Senescence of Tendon-Derived Stem Cells

10.21203/rs.3.rs-70180/v1 ◽

2020 ◽

Author(s):

Peilin Chen ◽

Ziming Chen ◽

Christopher Mitchell ◽

Junjie Gao ◽

Lianzhi Chen ◽

...

Keyword(s):

Stem Cells ◽

Mechanical Loading ◽

Patellar Tendon ◽

Intramuscular Injection ◽

Vastus Lateralis ◽

Collagen Fibre ◽

Differentiation Capacity ◽

Botox Injection

Abstract Background: Botulinum toxin (Botox) injection is in widespread clinical use for the treatment of muscle spasms and tendinopathy but the mechanism of action is poorly understood. Hypothesis: We hypothesised that the reduction of patellar-tendon mechanical-loading following intra-muscular injection of Botox results in tendon atrophy that is at least in part mediated by the induction of senescence of tendon-derived stem cells (TDSCs). Study Design: Controlled laboratory study Methods: A total of 36 mice were randomly divided in 2 groups (18 Botox-injected and 18 vehicle-only control). Mice were injected into to right vastus lateralis of quadriceps muscles either with Botox to induce mechanical stress deprivation of the patellar tendon or with normal saline as control. At 2 weeks post-injection, animals were euthanized prior to tissues harvest for either evaluation of tendon morphology or in vitro studies. TDSCs were isolated by cell-sorting prior to determination of viability, differentiation capacity and senescence markers, as well as assessing their response to mechanical loading in a bioreactor. Finally, to examine the mechanism of tendon atrophy in vitro, key proteins in the PTEN/AKT pathway were evaluated in TDSCs in both groups. Results: Two weeks after Botox injection, patellar tendons displayed atrophic features including tissue volume reduction and collagen fibre misalignment and increased degradation. The colony formation assay revealed the significantly reduced colony units of TDSCs in Botox injected group compared to controls. Multipotent differentiation capacity of TDSCs has also diminished after Botox injection. To examine if mechanical deprived TDSC is capable of forming tendon tissue, we used an isolated bioreactor system to culture 3D TDSCs constructs. The result showed that TDSCs from the Botox-treated group failed to restore tenogenic differentiation after appropriate mechanical loading. Examination of PTEN/AKT signalling pathway revealed that injection of Botox into quadriceps muscle causes PTEN/AKT mediated cell senescence of TDSCs. Conclusion: Intramuscular injection of Botox interferes with tendon homeostasis by inducing tendon atrophy and senescence of TDSCs. Botox injection may have long-term adverse consequences for the treatment of tendinopathy. Clinical relevance: Intramuscular Botox injection for tendinopathy and tendon injury could cause adverse effects in human tendons and re-evaluation of its long-term efficacy is warranted.

Download Full-text