Storage conditions affect the composition of the lyophilized secretome of multipotent mesenchymal stromal cells.

The widespread use of multipotent mesenchymal stromal cell-derived secretome (MSC-sec) requires optimal preservation methods. Lyophilization offers benefits like concentrating the secretome, reducing the storage volume, and making storage conditions more flexible. This study evaluated the influence of storage duration and temperature on lyophilized MSC-sec. The conditioned medium from Wharton's jelly MSCs was stored at - 80 °C or lyophilized with or without trehalose. Lyophilized formulations were kept at - 80 °C, - 20 °C, 4 °C, or room temperature (RT) for 3 and 30 months. After storage and reconstitution, the levels of growth factors and cytokines were assessed using multiplex assay. The storage of lyophilized MSC-sec at - 80 °C ensured biomolecule preservation for 3 and 30 months. Following 3 month storage at 4 °C and RT, a notable decrease occurred in BDNF, bNGF, and sVCAM-1 levels. Prolonged 30 month storage at the same temperatures significantly reduced BDNF, bNGF, VEGF-A, IL-6, and sVCAM-1, while storage at - 20 °C decreased BDNF, bNGF, and VEGF- A levels. Trehalose supplementation of MSC-sec improved the outcome during storage at 4 °C and RT. Proper storage conditions were crucial for the preservation of lyophilized MSC-sec composition. Short-term storage at various temperatures maintained over 60% of the studied growth factors and cytokines; long-term preservation was only adequate at -80 °C.

4-Methylumbeliferone Treatment at a Dose of 1.2 g/kg/Day Is Safe for Long-Term Usage in Rats.

4-methylumbelliferone (4MU) has been suggested as a potential therapeutic agent for a wide range of neurological diseases. The current study aimed to evaluate the physiological changes and potential side effects after 10 weeks of 4MU treatment at a dose of 1.2 g/kg/day in healthy rats, and after 2 months of a wash-out period. Our findings revealed downregulation of hyaluronan (HA) and chondroitin sulphate proteoglycans throughout the body, significantly increased bile acids in blood samples in weeks 4 and 7 of the 4MU treatment, as well as increased blood sugars and proteins a few weeks after 4MU administration, and significantly increased interleukins IL10, IL12p70 and IFN gamma after 10 weeks of 4MU treatment. These effects, however, were reversed and no significant difference was observed between control treated and 4MU-treated animals after a 9-week wash-out period.

Hypothermic Storage of 3D Cultured Multipotent Mesenchymal Stromal Cells for Regenerative Medicine Applications.

The regulatory requirements in cell processing, in the choice of a biomaterial scaffold and in quality control analysis, have to be followed in the clinical application of tissue-engineered grafts. Confirmation of sterility during quality control studies requires prolonged storage of the cell-based construct. After storage, preservation of the functional properties of the cells is an important prerequisite if the cells are to be used for cell-based tissue therapies. The study presented here shows the generation of 3D constructs based on Wharton's jelly multipotent mesenchymal stromal cells (WJ-MSCs) and the clinically-acceptable HyaloFast® scaffold, and the effect of two- and six-day hypothermic storage of 3D cell-based constructs on the functional properties of populated cells. To study the viability, growth, gene expression, and paracrine secretion of WJ-MSCs within the scaffolds before and after storage, xeno-free culture conditions, metabolic, qPCR, and multiplex assays were applied. The WJ-MSCs adhered and proliferated within the 3D HyaloFast®. Our results show different viability of the cells after the 3D constructs have been stored under mild (25 °C) or strong (4 °C) hypothermia. At 4 °C, the significant decrease of metabolic activity of WJ-MSCs was detected after 2 days of storage, with almost complete cell loss after 6 days. In mild hypothermia (25 °C) the decrease in metabolic activity was less remarkable, confirming the suitability of these conditions for cell preservation in 3D environment. The significant changes were detected in gene expression and in the paracrine secretion profile after 2 and 6 days of storage at 25 °C. The results presented in this study are important for the rapid transfer of tissue engineering approaches into clinical applications.

A Comparative Analysis of Multipotent Mesenchymal Stromal Cells derived from Different Sources, with a Focus on Neuroregenerative Potential.

Multipotent mesenchymal stromal cells (MSCs) can be considered an accessible therapeutic tool for regenerative medicine. Here, we compared the growth kinetics, immunophenotypic and immunomodulatory properties, gene expression and secretome profile of MSCs derived from human adult bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and Wharton's jelly (WJ-MSCs) cultured in clinically-relevant conditions, with the focus on the neuroregenerative potential. All the cell types were positive for CD10/CD29/CD44/CD73/CD90/CD105/HLA-ABC and negative for CD14/CD45/CD235a/CD271/HLA-DR/VEGFR2 markers, but they differed in the expression of CD34/CD133/CD146/SSEA-4/MSCA-1/CD271/HLA-DR markers. BM-MSCs displayed the highest immunomodulatory activity compared to AT- and WJ-MSCs. On the other hand, BM-MSCs secreted the lower content and had the lower gene expression of neurotrophic growth factors compared to other cell lines, which may be caused by the higher sensitivity of BM-MSCs to nutrient limitations. Despite the differences in growth factor secretion, the MSC secretome derived from all cell sources had a pronounced neurotrophic potential to stimulate the neurite outgrowth of DRG-neurons and reduce the cell death of neural stem/progenitor cells after H2O2 treatment. Overall, our study provides important information for the transfer of basic MSC research towards clinical-grade manufacturing and therapeutic applications.

The Effect of Wharton Jelly-Derived Mesenchymal Stromal Cells and Their Conditioned Media in the Treatment of a Rat Spinal Cord Injury.

The transplantation of Wharton's jelly derived mesenchymal stromal cells (WJ-MSCs) possesses therapeutic potential for the treatment of a spinal cord injury (SCI). Generally, the main effect of MSCs is mediated by their paracrine potential. Therefore, application of WJ-MSC derived conditioned media (CM) is an acknowledged approach for how to bypass the limited survival of transplanted cells. In this study, we compared the effect of human WJ-MSCs and their CM in the treatment of SCI in rats. WJ-MSCs and their CM were intrathecally transplanted in the three consecutive weeks following the induction of a balloon compression lesion. Behavioral analyses were carried out up to 9 weeks after the SCI and revealed significant improvement after the treatment with WJ-MSCs and CM, compared to the saline control. Both WJ-MSCs and CM treatment resulted in a higher amount of spared gray and white matter and enhanced expression of genes related to axonal growth. However, only the CM treatment further improved axonal sprouting and reduced the number of reactive astrocytes in the lesion area. On the other hand, WJ-MSCs enhanced the expression of inflammatory and chemotactic markers in plasma, which indicates a systemic immunological response to xenogeneic cell transplantation. Our results confirmed that WJ-MSC derived CM offer an alternative to direct stem cell transplantation for the treatment of SCI.

A Combination of Intrathecal and Intramuscular Application of Human Mesenchymal Stem Cells Partly Reduces the Activation of Necroptosis in the Spinal Cord of SOD1G93A Rats.

An increasing number of studies have demonstrated the beneficial effects of human mesenchymal stem cells (hMSC) in the treatment of amyotrophic lateral sclerosis (ALS). We compared the effect of repeated intrathecal applications of hMSC or their conditioned medium (CondM) using lumbar puncture or injection into the muscle (quadriceps femoris), or a combination of both applications in symptomatic SOD1G93A rats. We further assessed the effect of the treatment on three major cell death pathways (necroptosis, apoptosis, and autophagy) in the spinal cord tissue. All the animals were behaviorally tested (grip strength test, Basso Beattie Bresnahan (BBB) test, and rotarod), and the tissue was analyzed immunohistochemically, by qPCR and Western blot. All symptomatic SOD1 rats treated with hMSC had a significantly increased lifespan, improved motor activity and reduced number of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells. Moreover, a combined hMSC delivery increased motor neuron survival, maintained neuromuscular junctions in quadriceps femoris and substantially reduced the levels of proteins involved in necroptosis (Rip1, mixed lineage kinase-like protein, cl-casp8), apoptosis (cl-casp 9) and autophagy (beclin 1). Furthermore, astrogliosis and elevated levels of Connexin 43 were decreased after combined hMSC treatment. The repeated application of CondM, or intramuscular injections alone, improved motor activity; however, this improvement was not supported by changes at the molecular level. Our results provide new evidence that a combination of repeated intrathecal and intramuscular hMSC applications protects motor neurons and neuromuscular junctions, not only through a reduction of apoptosis and autophagy but also through the necroptosis pathway, which is significantly involved in cell death in rodent SOD1G93A model of ALS. Stem Cells Translational Medicine 2019;8:535-547.

Clinically Relevant Solution for the Hypothermic Storage and Transportation of Human Multipotent Mesenchymal Stromal Cells.

The wide use of human multipotent mesenchymal stromal cells (MSCs) in clinical trials requires a full-scale safety and identity evaluation of the cellular product and subsequent transportation between research/medical centres. This necessitates the prolonged hypothermic storage of cells prior to application. The development of new, nontoxic, and efficient media, providing high viability and well-preserved therapeutic properties of MSCs during hypothermic storage, is highly relevant for a successful clinical outcome. In this study, a simple and effective trehalose-based solution was developed for the hypothermic storage of human bone marrow MSC suspensions for further clinical applications. Human bone marrow MSCs were stored at 4°C for 24, 48, and 72 hrs in the developed buffered trehalose solution and compared to several research and clinical grade media: Plasma-Lyte® 148, HypoThermosol® FRS, and Ringer's solution. After the storage, the preservation of viability, identity, and therapeutically associated properties of MSCs were assessed. The hypothermic storage of MSCs in the new buffered trehalose solution provided significantly higher MSC recovery rates and ability of cells for attachment and further proliferation, compared to Plasma-Lyte® 148 and Ringer's solution, and was comparable to research-grade HypoThermosol® FRS. There were no differences in the immunophenotype, osteogenic, and adipogenic differentiation and the immunomodulatory properties of MSCs after 72 hrs of cold storage in these solutions. The obtained results together with the confirmed therapeutic properties of trehalose previously described provide sufficient evidence that the developed trehalose medium can be applied as a low-cost and efficient solution for the hypothermic storage of MSC suspensions, with a high potential for translation into clinical practice.

The Effect of Human Mesenchymal Stem Cells Derived from Wharton's Jelly in Spinal Cord Injury Treatment Is Dose-Dependent and Can Be Facilitated by Repeated Application.

Human mesenchymal stem cells derived from Wharton's jelly (WJ-MSCs) were used for the treatment of the ischemic-compression model of spinal cord injury in rats. To assess the effectivity of the treatment, different dosages (0.5 or 1.5 million cells) and repeated applications were compared. Cells or saline were applied intrathecally by lumbar puncture for one week only, or in three consecutive weeks after injury. Rats were assessed for locomotor skills (BBB, rotarod, flat beam) for 9 weeks. Spinal cord tissue was morphometrically analyzed for axonal sprouting, sparing of gray and white matter and astrogliosis. Endogenous gene expression (Gfap, Casp3, Irf5, Cd86, Mrc1, Cd163) was studied with quantitative Real-time polymerase chain reaction (qRT PCR). Significant recovery of functional outcome was observed in all of the treated groups except for the single application of the lowest number of cells. Histochemical analyses revealed a gradually increasing effect of grafted cells, resulting in a significant increase in the number of GAP43+ fibers, a higher amount of spared gray matter and reduced astrogliosis. mRNA expression of macrophage markers and apoptosis was downregulated after the repeated application of 1.5 million cells. We conclude that the effect of hWJ-MSCs on spinal cord regeneration is dose-dependent and potentiated by repeated application.

Injectable hydroxyphenyl derivative of hyaluronic acid hydrogel modified with RGD as scaffold for spinal cord injury repair.

Hydrogel scaffolds which bridge the lesion, together with stem cell therapy represent a promising approach for spinal cord injury (SCI) repair. In this study, a hydroxyphenyl derivative of hyaluronic acid (HA-PH) was modified with the integrin-binding peptide arginine-glycine-aspartic acid (RGD), and enzymatically crosslinked to obtain a soft injectable hydrogel. Moreover, addition of fibrinogen was used to enhance proliferation of human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) on HA-PH-RGD hydrogel. The neuroregenerative potential of HA-PH-RGD hydrogel was evaluated in vivo in acute and subacute models of SCI. Both HA-PH-RGD hydrogel injection and implantation into the acute spinal cord hemisection cavity resulted in the same axonal and blood vessel density in the lesion area after 2 and 8 weeks. HA-PH-RGD hydrogel alone or combined with fibrinogen (HA-PH-RGD/F) and seeded with hWJ-MSCs was then injected into subacute SCI and evaluated after 8 weeks using behavioural, histological and gene expression analysis. A subacute injection of both HA-PH-RGD and HA-PH-RGD/F hydrogels similarly promoted axonal ingrowth into the lesion and this effect was further enhanced when the HA-PH-RGD/F was combined with hWJ-MSCs. On the other hand, no effect was found on locomotor recovery or the blood vessel ingrowth and density of glial scar around the lesion. In conclusion, we have developed and characterized injectable HA-PH-RGD based hydrogel, which represents a suitable material for further combinatorial therapies in neural tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1129-1140, 2018.

Extracellular Matrix Hydrogel Derived from Human Umbilical Cord as a Scaffold for Neural Tissue Repair and Its Comparison with Extracellular Matrix from Porcine Tissues.

Extracellular matrix (ECM) hydrogels prepared by tissue decellularization have been reported as natural injectable materials suitable for neural tissue repair. In this study, we prepared ECM hydrogel derived from human umbilical cord (UC) and evaluated its composition and mechanical and biological properties in comparison with the previously described ECM hydrogels derived from porcine urinary bladder (UB), brain, and spinal cord. The ECM hydrogels did not differ from each other in the concentration of collagen, while the highest content of glycosaminoglycans as well as the shortest gelation time was found for UC-ECM. The elastic modulus was then found to be the highest for UB-ECM. In spite of a different origin, topography, and composition, all ECM hydrogels similarly promoted the migration of human mesenchymal stem cells (MSCs) and differentiation of neural stem cells, as well as axonal outgrowth in vitro. However, only UC-ECM significantly improved proliferation of tissue-specific UC-derived MSCs when compared with the other ECMs. Injection of UC-ECM hydrogels into a photothrombotic cortical ischemic lesion in rats proved its in vivo gelation and infiltration with host macrophages. In summary, this study proposes UC-ECM hydrogel as an easily accessible biomaterial of human origin, which has the potential for neural as well as other soft tissue reconstruction.

Injectable Extracellular Matrix Hydrogels as Scaffolds for Spinal Cord Injury Repair.

Restoration of lost neuronal function after spinal cord injury (SCI) still remains a big challenge for current medicine. One important repair strategy is bridging the SCI lesion with a supportive and stimulatory milieu that would enable axonal rewiring. Injectable extracellular matrix (ECM)-derived hydrogels have been recently reported to have neurotrophic potential in vitro. In this study, we evaluated the presumed neuroregenerative properties of ECM hydrogels in vivo in the acute model of SCI. ECM hydrogels were prepared by decellularization of porcine spinal cord (SC) or porcine urinary bladder (UB), and injected into a spinal cord hemisection cavity. Histological analysis and real-time qPCR were performed at 2, 4, and 8 weeks postinjection. Both types of hydrogels integrated into the lesion and stimulated neovascularization and axonal ingrowth into the lesion. On the other hand, massive infiltration of macrophages into the lesion and rapid hydrogel degradation did not prevent cyst formation, which progressively developed over 8 weeks. No significant differences were found between SC-ECM and UB-ECM. Gene expression analysis revealed significant downregulation of genes related to immune response and inflammation in both hydrogel types at 2 weeks post SCI. A combination of human mesenchymal stem cells with SC-ECM did not further promote ingrowth of axons and blood vessels into the lesion, when compared with the SC-ECM hydrogel alone. In conclusion, both ECM hydrogels bridged the lesion cavity, modulated the innate immune response, and provided the benefit of a stimulatory substrate for in vivo neural tissue regeneration. However, fast hydrogel degradation might be a limiting factor for the use of native ECM hydrogels in the treatment of acute SCI.

Analysis of marker expression in porcine cell lines derived from blastocysts produced in vitro and in vivo.

The present study was designed to extensively characterize cell lines derived from porcine blastocysts by several methodical approaches, including morphological observation, cytogenetic analysis, estimation of alkaline phosphatase activity and detection of specific marker expression at the mRNA/protein level. A comparison was made between the properties of cell lines isolated from in vivo- and in vitro-obtained blastocysts. Our results showed that 57.1% of the in vivo-obtained blastocysts attached to the feeder layer and that 33.3% of them started to grow in a monolayer. The percentage of attached in vitro-produced blastocysts was lower (24.6%), and only 6.9% of them started to grow. Outgrowths from the in vitro-produced blastocysts formed mainly trophectoderm or epithelial-like monolayer, whereas the in vivo-obtained blastocysts formed heterogeneous outgrowths that also contained cells with embryonic stem (ES)-like morphology. Detailed analyses showed that the primary outgrowths with ES-like morphology expressed the pluripotency markers OCT-4 and NANOG and revealed intensive alkaline phosphatase staining, while they did not express markers of differentiation. The majority of passaged cells, including those with ES-like morphology, lacked OCT-4 protein and revealed expression of specific differentiation markers (cytokeratin 18, lamins A/C, transferrin, α-fetoprotein and GATA-4), although they still expressed NANOG and exhibited weak alkaline phosphatase activity. Moreover, these cells spontaneously differentiated into neural, fibroblast or epithelial-like cells, even in the presence of leukaemia inhibitory factor. Our results show that complex analysis of markers of pluripotency as well as differentiation markers is necessary for proper interpretation of data in porcine embryonic stem cell studies.

Viability, acrosome morphology and fertilizing capacity of boar spermatozoa treated with strontium chloride.

The positive effect of strontium ions (Sr2+) on sperm motility, capacitation and acrosome reaction has been demonstrated in the mouse, human, guinea pig and hamster. In the present study, we have evaluated the effect of Sr2+ on the viability and acrosome morphology of boar spermatozoa, and on the fertilization and development after the microinjection of Sr(2+)-treated spermatozoa into porcine oocytes. Before incubation, 79% of spermatozoa were classified as propidium iodide (PI)-negative (live) using the LIVE/DEAD Sperm Viability Kit. After incubation with strontium chloride (SrCl2), 39% (0 mM; no divalent cations), 25% (1.9 mM) and 24% (7.5 mM) of them were classified as PI-negative. The proportion of spermatozoa that had initiated the acrosome reaction was higher in Sr(2+)-containing medium than in Sr(2+)-free medium, when assessed by electron microscopy. There was no significant difference in percentage of spermatozoa initiating the acrosome reaction between Sr2+-treated groups (1.9 mM: 22%, 7.5 mM: 33%, p>0.05). After the microinjection of spermatozoa incubated with SrCl2, 67% (1.9 mM) and 61% (7.5 mM) of injected oocytes were successfully fertilized, and then 43% (1.9 mM) and 41% (7.5 mM) contained a fully decondensed sperm head. Sham-injected oocytes were significantly activated at a lower rate than Sr(2+)-treated groups (27%, p<0.05). Next, after microinjection of spermatozoa incubated with 1.9 mM SrCl2 (n=51), 45% of injected oocytes cleaved on day 2, and 18% developed to blastocysts on day 7 (sham-injection, n=48: 15% to cleavage and 0% to blastocyst). These results demonstrate that Sr2+ is likely to positively affect the fertilizing capacity of spermatozoa in the pig.

Putative embryonic stem cell lines from pig embryos.

Embryonic stem cells (ES cells) were first established in the mouse, and they represent a population of pluripotent, undifferentiated cells derived from early embryos that is capable of proliferating without any limitation in an undifferentiated state. These cells retain the ability to differentiate in vitro or in vivo into derivates of all three germ layers, and when injected into blastocysts, they can participate in the formation of all tissues, including gonads (germ-line chimeras). It is possible to transfect them with a gene of interest, and the resulting transgenic cell lines can also be used for production of chimeras. Unfortunately, mammalian germ-line chimeras that can carry an inserted gene into their progeny have only been produced in the mouse. Logically, before application of stem cell therapies into a human medicine, it is necessary to verify the efficiency and safety of these methods with an acceptable animal model. The pig is currently used as a very convenient animal for pre-clinical applications, and therefore establishment of porcine ES cell lines is highly needed; unfortunately, no convincing ES cell lines have been produced in this species (and other domestic animals) to date. In this article, we discuss the recent advances in this field, especially oriented on possible reasons and obstacles why derivation of porcine ES cell lines is still unsuccessful.

The absence of a DNA replication checkpoint in porcine zygotes.

It has been demonstrated that in the zygotes of some mammals a unique checkpoint controls the onset of DNA replication. Thus, DNA replication begins in the maternal pronucleus only after the paternal pronucleus is fully formed. In our experiments we have investigated whether this checkpoint also operates in porcine zygotes produced either by in vitro fertilization (IVF) or by intracytoplasmic sperm injection (ICSI). Our results show that the onset of DNA replication occurs in the maternal pronucleus even in the presence of an intact sperm head in zygotes produced by ICSI, as well as in polyspermic eggs where some sperm heads are intact or male pronuclei are not yet fully developed. We conclude that in porcine zygotes there is an absence of the DNA replication checkpoint that is typical for some other mammals.

Placental abnormalities associated with post-natal mortality in sheep somatic cell clones.

We report on cloning experiments designed to explore the causes of peri- and post-natal mortality of cloned lambs. A total of 93 blastocysts obtained by nuclear transfer of somatic cells (granulosa cells) were transferred into 41 recipient ewes, and pregnancies were monitored by ultrasound scanning. In vitro derived, fertilized embryos (IVF, n=123) were also transferred to assess oocyte competence, and naturally mated ewes (n=120) were analysed as well. Cloned embryos developed to the blastocyst stage and implanted at the same rate as IVF embryos. After day 30 of gestation, however, dramatic losses occurred, and only 12 out of 93 (13%) clones reached full-term development, compared to 51 out of 123 (41.6%) lambs born from the IVF control embryos. Three full-term lamb clones were delivered stillborn, as a result of placental degeneration. A further five clone recipients developed hydroallantois. Their lambs died within 24h following delivery by caesarian section, and displayed degenerative lesions in liver and kidney resulting from the severe hydroallantois. One set of twins was delivered by assisted parturition at day 150, but died 24h later due to respiratory distress syndrome. The remaining two clone recipients underwent caesarian section, and the corresponding two lambs displayed signs of respiratory dysfunction and died at approximately 1 month of age due to a bacterial complication. Blood samples collected from the cloned lambs after birth revealed a wide range of abnormalities indicative of kidney and liver dysfunction. Macroscopical and histopathological examination of the placentae revealed a marked reduction in vascularization, particularly at the apex of the villous processes, as well as a loss of differentiation of the trophoblastic epithelium. Our results strongly suggest that post-mortality in cloned lambs is mainly caused by placental abnormalities.