Manufacturing and Banking Canine Adipose-Derived Mesenchymal Stem Cells for Veterinary Clinical Application

Abstract BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. RESULTS: After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. AD-MSCs presented normal karyotype, and did not form in vivo tumors. No adverse events were noted in two cases treated with intravenously AD-MSCs. CONCLUSION: Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

Manufacturing and banking canine adipose-derived mesenchymal stem cells for veterinary clinical application

BMC Veterinary Research ◽

10.1186/s12917-021-02791-3 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Huina Luo ◽

Dongsheng Li ◽

Zhisheng Chen ◽

Bingyun Wang ◽

Shengfeng Chen

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Normal Karyotype ◽

Clinical Applications ◽

Therapeutic Application ◽

Abstract Background Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. Results After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. AD-MSCs presented normal karyotype, and did not form in vivo tumors. No adverse events were noted in the case treated with intravenously AD-MSCs. Conclusions Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

Manufacturing and Banking Canine Adipose-Derived Mesenchymal Stem Cells for Veterinary Clinical Application

10.21203/rs.3.rs-64097/v2 ◽

2020 ◽

Author(s):

Huina Luo ◽

Dongsheng Li ◽

Zhisheng Chen ◽

Bingyun Wang ◽

Shengfeng Chen

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Clinical Applications ◽

Surface Markers ◽

Clinical Use ◽

Therapeutic Application ◽

Abstract BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. RESULTS: After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. No adverse events were noted in two cases treated with intravenously AD-MSCs. CONCLUSION: Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

Manufacturing and Banking Canine Adipose-Derived Mesenchymal Stem Cells for Veterinary Clinical Application

10.21203/rs.3.rs-64097/v1 ◽

2020 ◽

Author(s):

Huina Luo ◽

Dongsheng Li ◽

Zhisheng Chen ◽

Bingyun Wang ◽

Shengfeng Chen

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Clinical Applications ◽

Surface Markers ◽

Clinical Use ◽

Therapeutic Application ◽

Abstract BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. RESULTS: After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. No adverse events were noted in two cases treated with intravenously AD-MSCs. CONCLUSION: Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

191 Canine Mesenchymal Stem Cells as Therapeutic Agents in Spinal Cord Injured Dog

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab191 ◽

2018 ◽

Vol 30 (1) ◽

pp. 236 ◽

Cited By ~ 1

Author(s):

Y.-H. Choe ◽

H.-J. Lee ◽

S.-L. Lee ◽

J.-H. Lee ◽

B.-W. Park ◽

...

Keyword(s):

Spinal Cord ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Therapeutic Agents ◽

Clinical Applications ◽

Pain Sensation ◽

Na Currents ◽

Spinal Cord Injured

In the recent era of veterinary research, stem cells have gained special attention due to their efficiency and use in clinical applications. Mesenchymal stem cells (MSC) have been extensively studied over decades, and their prospect for clinical application is recognised in human medicine. Despite numerous reports in veterinary clinical trials of stem cells, few studies have been presented regarding the in vitro characterisation of canine mesenchymal stem cells (cMSC). Therefore, their efficacy as therapeutic agents in vitro has not been much elucidated. Canine adipose-derived mesenchymal stem cells (cAMSC) were characterised as per International Society for Cellular Therapy guidelines. Culturing cells showed spindle-like morphology and high proliferation rate. They displayed positive expression of mesenchymal markers CD44, CD90, and CD105, and lacked expression of CD34 and CD45. They were also positive for expression of pluripotency-related transcription factors (Oct3/4, Nanog, and Sox2) and showed differentiation potential towards mesodermal lineages. The cAMSC were further analysed for the neuronal trans-differentiation potential. Under appropriate differentiation conditions, cAMSC displayed distinctive dendritic morphology along with axon projections. Neuronal specific genes including Nestin, β-tubulin, neurofilament protein (NF-M, NF-H), and nerve growth factor (NGF) were also positively expressed. Nevertheless, functional analysis of neuronal differentiated cAMSC displayed voltage dependence and kinetics for transient K+ and Na+ currents (Ito). Both K+ and Na+ currents were recorded in differentiated MSC by voltage steps (between −120 and +60 mV for K+ currents, −40 and +50 mV for Na+ currents), whereas control undifferentiated MSC lacked the currents. Taken together, we concluded that the cAMSC have potential to differentiate into neuron-like cells. Based on these findings, we transplanted cAMSC into the spinal cord injured dogs to evaluate their clinical efficiency under approved medical guidelines set by Gyeongsang National University Animal Medical Center (Korea). Neurological examination showed that the injured dog had undergone hind limb paralysis and lost deep pain sensation due to an L2 spinal cord lesion, as detected by CT and MRI. The dog was diagnosed with traumatic L2 intradural spinal cord contusion, and decompression surgery was performed, but deep pain sensation did not recover. Therefore, each cAMSC (diluted in 0.5 mL of saline) was transplanted into spinal cord segment (L2~L3) 5 times at 1-week intervals. The dog showed mild recovery of deep pain sensation by neurological examinations and exhibited gradual improvement in hind limb function. Finally, we concluded that transplantation of cAMSC has a beneficial therapeutic effect on spinal cord injury. This study also provides a significant advantage in understanding the potential of MSC-based products in veterinary clinical applications.

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 ◽

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.

Comparison of in Vitro Vasculogenesis Potential between ASCs with BM-MSCs

International Journal of Health Sciences and Research ◽

10.52403/ijhsr.20210557 ◽

2021 ◽

Vol 11 (5) ◽

pp. 362-378

Author(s):

Ani Oranda Panjaitan ◽

Dewi Sukmawati ◽

Ria Anggraeni

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Tube Formation ◽

Tube Length ◽

Branch Points ◽

Marrow Mesenchymal Stem Cells ◽

Cd34 Expression

Tube formation assay is the most widely used method as a vasculogenesis/ angiogenesis test in vitro. Mesenchymal stem cells (MSCs) are multipotent adult cells. The paracrine effect of MSCs on neovascularization is well known. In general, MSCs do not express CD34 hematopoietic surface marker, but according to some experts, bone marrow mesenchymal stem cells (BM-MSCs) express CD34 in vivo and lose their expression when they are cultured in vitro, while adipose-derived stem cells (ASCs) still have CD34 expression in the early passages when cultured in vitro. BM-MSCs are the most widely used MSC, but ASCs are also used in stem cell therapy and tissue engineering for angiogenesis purposes. Until now, the potential of vasculogenesis between ASCs and BM-MSCs is still unclear. Expression of CD34 is also unknown whether affecting the quality of tube formation. This study wanted to compare the potential of vasculogenesis between ASC and BM-MSCs through tube formation test and CD34 expression.Measurements of vasculogenesis quality showed higher tube length, number of loopsand mean number of branch points on BM-MSC. Both BM-MSCs and ASCs showed low CD34 levels.BM-MSCs showed better tube formation ability compared with ASCs. No association was found between CD34 levels and MSC vasculogenesis capability. Key words: ASCs, BM-MSCs, CD34, matrigel, tube formation.

Identification of WNT16 as a Predictable Biomarker for Accelerated Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells In Vitro

Stem Cells International ◽

10.1155/2019/8503148 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Yu-Hee Kim ◽

Kyung-Ah Cho ◽

Hyun-Ji Lee ◽

Minhwa Park ◽

Han Su Kim ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Selection Criterion ◽

Osteogenic Potential ◽

Real Time Quantitative Pcr ◽

Single Cell Cloning

The application of mesenchymal stem cells (MSCs) for treating bone-related diseases shows promising outcomes in preclinical studies. However, cells that are isolated and defined as MSCs comprise a heterogeneous population of progenitors. This heterogeneity can produce variations in the performance of MSCs, especially in applications that require differentiation potential in vivo, such as the treatment of osteoporosis. Here, we aimed to identify genetic markers in tonsil-derived MSCs (T-MSCs) that can predict osteogenic potential. Using a single-cell cloning method, we isolated and established several lines of nondifferentiating (ND) or osteoblast-prone (OP) clones. Next, we performed transcriptome sequencing of three ND and three OP clones that maintained the characteristics of MSCs and determined the top six genes that were upregulated in OP clones. Upregulation of WNT16 and DCLK1 expression was confirmed by real-time quantitative PCR, but only WNT16 expression was correlated with the osteogenic differentiation of T-MSCs from 10 different donors. Collectively, our findings suggest that WNT16 is a putative genetic marker that predicts the osteogenic potential of T-MSCs. Thus, examination of WNT16 expression as a selection criterion prior to the clinical application of MSCs may enhance the therapeutic efficacy of stem cell therapy for bone-related complications, including osteoporosis.

The Hypoxic Preconditioning Effect On Senescence Cell Process In Cultured Adipose-Derived Mesenchymal Stem Cells (AMSCs)

Indonesian Journal of Cardiology ◽

10.30701/ijc.v39i3.802 ◽

2019 ◽

Vol 39 (3) ◽

Author(s):

Nadiar Dwi Nuarisa ◽

I Gde Rurus Suryawan ◽

Andrianto Andrianto

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Life Span ◽

Hypoxic Preconditioning ◽

Hypoxic Precondition ◽

Significant Difference

Introduction : Stem cell therapy for myocardial regeneration is expected to increase cardiomyocyte proliferation and trigger neovascularization to improve cardiomyocytes. Mesenchymal Stem Cells (MSCs) are ideal candidates for regenerative medicine and immunotherapy. But low viability of MSCs is a major challenge in this alternative therapy. Therefore, a cytoprotective strategy is needed, one of them is hypoxic preconditioning which can significantly increase survival stem cells after being transplanted. MSCs are known to have a limited life span, after experiencing several splits MSC will enter the senescence process. It is known that hypoxia can also increase cell proliferation and differentiation potential in vitro and in vivo through the role of Octamer-4 (Oct-4) as a regulator of the pluripotency gene. Methods : Experimental laboratory studies (in vitro studies) using human-AMSCs which were given hypoxic preconditioning, observed as a immunocytochemistry. Results : The results showed that hypoxic precondition (1% O2) inhibited the senescence process. It can be seen in the lower expression of senescence in hypoxic conditions at P6, P7, P8, P9, P10 compared to normoxic ((p=0,004, p=0,001, p=0,009, p=0,013, p=0,024. There is a significant difference in the senescence expression of each passage in hypoxic and normoxic conditions with the highest expression at P10. In addition, we also observed AMSCs differentiation through the Oct-4 expression. It is showed that Oct-4 expression were higher in hypoxia compared to normoxia on P7, P8, P9, P10 (p=0,009, p=0,009, p=0,030, p=0,0001). Conclusions : Hypoxic preconditioning have the effect of inhibiting the senescence process on Adipose-derived MSCs (AMSCs) or prolonging their life span. The longer life span of AMSCs is also seen by higher cell differentiation potential from increased expression of Oct-4. However, the mechanism of inhibiting the senescence process in hypoxia in stem cells is still remain unknown. Keywords: human-Adipose derived Mesenchymal Stem Cell Cultures (h-AMSCs), Hypoxic Preconditioning, Senescence cell, Oct-4.

Cell surface receptor expression profile of human synovial mesenchymal stem cells in-vivo predicts their differentiation potential in-vitro

Osteoarthritis and Cartilage ◽

10.1016/j.joca.2016.01.840 ◽

2016 ◽

Vol 24 ◽

pp. S460-S461 ◽

Cited By ~ 1

Author(s):

N.U. Aljezani ◽

A. Affan ◽

P. Railton ◽

J. Powell ◽

R. Krawetz

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Surface ◽

Expression Profile ◽

Receptor Expression ◽

Cell Surface Receptor ◽

Surface Receptor

CD10/Neprilysin Enrichment in Infrapatellar Fat Pad–Derived Mesenchymal Stem Cells Under Regulatory-Compliant Conditions: Implications for Efficient Synovitis and Fat Pad Fibrosis Reversal

The American Journal of Sports Medicine ◽

10.1177/0363546520917699 ◽

2020 ◽

Vol 48 (8) ◽

pp. 2013-2027 ◽

Cited By ~ 4

Author(s):

Dimitrios Kouroupis ◽

Annie C. Bowles ◽

Thomas M. Best ◽

Lee D. Kaplan ◽

Diego Correa

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Ex Vivo ◽

Infrapatellar Fat Pad ◽

Therapeutic Effects ◽

Fat Pad ◽

In Vitro Proliferation ◽

Differentiation Potential

Background: Synovitis and infrapatellar fat pad (IFP) fibrosis participate in various conditions of the knee. Substance P (SP), a neurotransmitter secreted within those structures and historically associated with nociception, also modulates local neurogenic inflammatory and fibrotic responses. Exposure of IFP mesenchymal stem cells (IFP-MSCs) to a proinflammatory/profibrotic environment (ex vivo priming with TNFα, IFNγ, and CTGF) induces their expression of CD10/neprilysin, effectively degrading SP in vitro and in vivo. Purpose/Hypothesis: The purpose was to test the therapeutic effects of IFP-MSCs processed under regulatory-compliant protocols, comparing them side-by-side with standard fetal bovine serum (FBS)–grown cells. The hypothesis was that when processed under such protocols, IFP-MSCs do not require ex vivo priming to acquire a CD10-rich phenotype efficiently degrading SP and reversing synovitis and IFP fibrosis. Study Design: Controlled laboratory study. Methods: Human IFP-MSCs were processed in FBS or either of 2 alternative conditions—regulatory-compliant pooled human platelet lysate (hPL) and chemically reinforced medium (Ch-R)—and then subjected to proinflammatory/profibrotic priming with TNFα, IFNγ, and CTGF. Cells were assessed for in vitro proliferation, stemness, immunophenotype, differentiation potential, transcriptional and secretory profiles, and SP degradation. Based on a rat model of acute synovitis and IFP fibrosis, the in vivo efficacy of cells degrading SP plus reversing structural signs of inflammation and fibrosis was assessed. Results: When compared with FBS, IFP-MSCs processed with either hPL or Ch-R exhibited a CD10High phenotype and showed enhanced proliferation, differentiation, and immunomodulatory transcriptional and secretory profiles (amplified by priming). Both methods recapitulated and augmented the secretion of growth factors seen with FBS plus priming, with some differences between them. Functionally, in vitro SP degradation was more efficient in hPL and Ch-R, confirmed upon intra-articular injection in vivo where CD10-rich IFP-MSCs also dramatically reversed signs of synovitis and IFP fibrosis even without priming or at significantly lower cell doses. Conclusion: hPL and Ch-R formulations can effectively replace FBS plus priming to induce specific therapeutic attributes in IFP-MSCs. The resulting fine-tuned, regulatory-compliant, cell-based product has potential future utilization as a novel minimally invasive cell therapy for the treatment of synovitis and IFP fibrosis. Clinical Relevance: The therapeutic enhancement of IFP-MSCs manufactured under regulatory-compliant conditions suggests that such a strategy could accelerate the time from preclinical to clinical phases. The therapeutic efficacy obtained at lower MSC numbers than currently needed and the avoidance of cell priming for efficient results could have a significant effect on the design of clinical protocols to potentially treat conditions involving synovitis and IFP fibrosis.