Human Umbilical Mesenchymal Stem Research Articles

Abstract 1055: Screens of targeted agents combined with the ubiquitin activating enzyme inhibitor TAK-243 or the pan-Akt inhibitor ipatasertib identified combinations that are effective in patient-derived complex spheroids

Abstract Combinations of targeted chemotherapeutics can provide new options to treat cancer; however, the preclinical discovery of potentially beneficial therapeutic combinations is challenging given the heterogeneity of cancer. Complex tumor spheroids, consisting of combined patient-derived tumor cells and stromal cells (human umbilical vein endothelial cells and mesenchymal stem cells) serve as a physiologically relevant in vitro model of solid tumors that is amenable to high-throughput screening. In this study, ten anticancer agents with a range of targets including MEK, mTOR, RAF, PI3K, and the proteasome, were tested both alone and in combination with the ubiquitin activating enzyme inhibitor, TAK-243, or the pan-Akt inhibitor, ipatasertib. All anticancer agents were tested over a 3 log range of concentrations up to their clinical Cmax value, if known. The activities of single agents and combinations were evaluated in complex spheroids grown from patient-derived tumor cells from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov), including ten colon and six pancreatic adenocarcinoma and four melanoma cell lines. The spheroids were allowed to grow for three days prior to the addition of anticancer agents and cell viability was measured seven days later using CellTiter-Glo 3D. TAK-243 was cytotoxic against all six pancreatic adenocarcinoma spheroids and all four melanoma spheroids, as well as several colon adenocarcinoma spheroids. The single agent BRAF V600E-selective inhibitor, vemurafenib, showed selective cytotoxicity against melanoma spheroids with the BRAF V600E/K variant. TAK-243 in combination with vemurafenib produced greater than additive cytotoxicity in most complex spheroid models. The overall cytotoxicity of the TAK-243 and vemurafenib combination was similar in melanoma spheroids containing WT BRAF and the BRAF V600E/K variant. The activity of vemurafenib and the BRAF V600E-selective inhibitor dabrafenib were not equivalent in this combination screen and greater than additive cytotoxicity was observed more frequently when TAK-243 was combined with vemurafenib than with dabrafenib. The combination of ipatasertib and vemurafenib had additive and greater than additive cytotoxicity in more than half of the complex spheroid models tested. Other notable agents combined with ipatasertib included the MEK inhibitors trametinib and selumetinib, both of which produced greater than additive cytotoxicity in spheroids derived from pancreatic cancer metastases. These results might inform the prioritization of drug combinations for further investigation by in vivo studies. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Nathan P. Coussens, John J. Wright, Ralph E. Parchment, René Delosh, Julie Laudeman, Russell Reinhart, Chad Ogle, Thomas Silvers, Thomas S. Dexheimer, Joel Morris, James H. Doroshow, Beverly A. Teicher. Screens of targeted agents combined with the ubiquitin activating enzyme inhibitor TAK-243 or the pan-Akt inhibitor ipatasertib identified combinations that are effective in patient-derived complex spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1055.

Optimization of human umbilical cord blood-derived mesenchymal stem cell isolation and culture methods in serum- and xeno-free conditions

BackgroundAlthough umbilical cord blood (UCB) is identified as a source of mesenchymal stem cells (MSCs) with various advantages, the success in cell isolation is volatile. Therefore, it is necessary to optimize methods of cord blood-derived MSC (UCB-MSC) isolation and culture. In this study, we evaluated the efficiency of UCB-MSC isolation and expansion using different commercially available serum- and xeno-free media and investigated the capacity of autologous serum and plasma as a supplement to support cell proliferation. Additionally, we defined the presence of multilineage-differentiating stress-enduring (Muse) cells in the UCB-MSC population. Functions of UCB-MSC in in vitro angiogenesis processes and anti-cancer were also verified.MethodsMononuclear cells were isolated using density gradient separation and cultured in four commercial media kits, as well as four surface coating solutions. UCB-MSCs were characterized and tested on tube formation assay, and co-cultured with SK-MEL cells in a transwell system.ResultsThe results showed that only StemMACS™ MSC Expansion Media is more appropriate to isolate and culture UCB-MSCs. The cells exhibited a high cell proliferation rate, CFU forming capability, MSC surface marker expression, trilineage differentiate potential, and chromosome stability. In addition, the culture conditions with autologous serum coating and autologous plasma supplement enhanced cell growth and colony forming. This cell population contained Muse cells at rate of 0.3%. Moreover, UCB-MSCs could induce the tube formation of human umbilical vein endothelial cells and inhibit more than 50% of SK-MEL cell growth.ConclusionsUCB-MSCs could be high-yield isolated and expanded under serum- and xeno-free conditions by using the StemMACS™ MSC Expansion Media kit. Autologous serum coating and plasma supplement enhanced cell proliferation. These UCB-MSCs had effected the tube formation process and an anti-cancer impact.

Treatment of Radiation Bone Injury with Transplanted hUCB-MSCs via Wnt/β-Catenin.

Radiation-induced bone injury (RIBI) is one of the complications after radiotherapy for malignant tumors. However, there are no effective measures for the treatment of RIBI in clinical practice, and the mechanism of RIBI is unclear. We use a single high-dose ionizing radiation (6Gy) to analyze the effect of radiotherapy on osteoblast function. Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) were cocultured with irradiated osteoblasts to examine their therapeutic effects and mechanisms on osteoblast injury. The hUCB-MSC transplantation mouse model is used to confirm the in vivo role of hUCB-MSC treatment in radiation bone injury. Western blot analysis, qRT-PCR, immunohistochemistry, and immunofluorescence staining were used to analyze gene expression and angiogenesis. The apoptosis and migration of osteoblasts were measured by Hoechst staining, scratch test, and transwell. The differentiation of osteoblasts was measured by ALP and Alizarin red staining and transmission electron microscopy. The bone-related parameters of mice were evaluated by micro-CT analysis. We found that radiation can damage the DNA of osteoblasts; induce apoptosis; reduce the differentiation, migration, and adhesion of osteoblasts, leading to lipogenesis of bone marrow mesenchymal stem cells (BMSCs) and reducing the source of osteoblasts; and increase the number of osteoclasts in bone tissue, while MSC treatment prevents these changes. Our results reveal the inhibitory effect of radiation on osteoblast function. hUCB-MSCs can be used as a therapeutic target for the development of new therapeutic strategies for radiotherapy of bone injury diseases.

Outcomes of human umbilical cord blood-derived mesenchymal stem cells in enhancing tendon-graft healing in anterior cruciate ligament reconstruction: an exploratory study

BackgroundThe study investigated whether allogeneic human umbilical cord blood-derived MSCs (hUCB-MSCs) could be safely used without treatment-related adverse events, reducing tunnel enlargement, and improve clinical results in human anterior cruciate ligament (ACL) reconstruction.MethodsThirty patients were enrolled consecutively. They were divided into three groups by randomization. In the negative control group, ACL reconstruction surgery without additional treatment was performed. In the experimental group, a hUCB-MSC and hyaluronic acid mixture was applied to the tendon-bone interface of the femoral tunnels during ACL reconstruction surgery. In the positive control group, only hyaluronic acid was applied. Finally, 27 patients were analyzed after the exclusion of three patients. The incidence of treatment-related adverse events, clinical outcomes, including second-look arthroscopic findings, and the amount of tunnel enlargement, were evaluated.ResultsThere were no treatment-related adverse events in the treatment groups. Tunnel enlargement in the experimental group (579.74 ± 389.85 mm3) was not significantly different from those in the negative (641.97 ± 455.84 mm3) and positive control (421.96 ± 274.83 mm3) groups (p = 0.6468). There were no significant differences between the groups in clinical outcomes such as KT-2000 measurement (p = 0.793), pivot shift test (p = 0.9245), International Knee Documentation Committee subjective score (p = 0.9195), Tegner activity level (p = 0.9927), and second-look arthroscopic findings (synovial coverage of the graft, p = 0.7984; condition of the graft, p = 0.8402).ConclusionsAllogeneic hUCB-MSCs were used safely for ACL reconstruction without treatment-related adverse event in a 2-year follow-up. However, our study did not suggest any evidence to show clinical advantage such as the prevention of tunnel enlargement postoperatively and a decrease in knee laxity or improvement of clinical outcomes.Trial registrationCRIS, Registration Number: KCT0000917. Registered on 12 November 2013; https://cris.nih.go.kr/cris/index.jsp

Effects of single and multiple transplantations of human umbilical cord mesenchymal stem cells on the recovery of ovarian function in the treatment of premature ovarian failure in mice

BackgroundCurrently, there is no effective treatment for premature ovarian failure (POF), and stem cell therapy is considered the most promising treatment. Human umbilical cord blood mesenchymal stem cells (hUC-MSCs) have shown good regenerative ability in various diseases, including POF; however, their underlying mechanism and dosage for POF treatment remain unclear. This study aimed to compare the effect of single and multiple injections of hUC-MSCs on ovarian function repair in chemotherapy-induced POF.MethodsFemale mice were intraperitoneally injected with 30 mg/kg busulfan and 120 mg/kg cyclophosphamide (CTX) to induce POF. In the single hUC-MSC injection group, hUC-MSCs were transplanted into mice D7 after CTX and busulfan administration, while in the multiple injection group, hUC-MSCs were transplanted on D7, D14, and D21 after CTX and busulfan administration. We evaluated the ovarian morphology, fertility, follicle-stimulating hormone and estradiol concentrations, follicle count, POF model, and cell transplantation results. In addition, real-time polymerase chain reaction, immunohistochemistry, and miRNA and mRNA chips were used to evaluate the effect of the cell therapy.ResultsOvary size, number of follicle at all developmental stages, and fertility were significantly reduced in the POF group compared with the control. Under hUC-MSC treatment, the ovarian morphology and follicle count were significantly restored, and fertility was significantly increased. By comparing the single and multiple hUC-MSC injection groups, we found that the anti-Müllerian hormone and Ki-67 levels were significantly increased in the multiple hUC-MSC group on D60 after chemotherapy. The expression of stimulating hormone receptors, inhibin α, and inhibin β was significantly restored, and the therapeutic effect was superior to that of the single hUC-MSC injection group.ConclusionThese results indicate that hUC-MSCs can restore the structure of injured ovarian tissue and its function in chemotherapy-induced POF mice and ameliorate fertility. Multiple hUC-MSC transplantations have a better effect on the recovery of ovarian function than single hUC-MSC transplantation in POF.

Preparation of Dopamine Nanoparticles and Its Application in the Treatment of Neonatal Scleredema

The polydopamine (PDA) nanoparticles were prepared by the aqueous phase oxidation, and doxorubicin (DOX) was applied on surface of PDA under appropriate temperature and Pondus Hydrogenii (pH) conditions. The human umbilical vein-derived mesenchymal stem cells (hUV-MSCs) were sub-cultured, and then destroyed by lysing and freeze-thaw to obtain the stem cell membrane (SSM) fragments, which were squeezed using the polycarbonate porous membrane and then wrapped in the surfaces of the PDA nanoparticles by means of water bath ultrasound. In this way, the biomimetic nano medicine was synthesized. In the experiment, the DOX release amount of the nanomedicine under different conditions was analyzed. Under the precondition that the concentration of the drug was changed, and it was used to explore proliferation activity of human esophageal cancer cell (Eca-109). Mice with sclerosed skin caused by scleredema were selected as the test subjects to observe the inhibitory effect of the prepared drugs on its fibrosis. In addition, 90 cases of neonatal scleredema were selected and grouped to verify the improvement effect of nano-medicine injection on their symptoms. The results relaved that average particle size (APS) of synthesized drug was 77.6±8.5 nm, and the drug loading of DOX loaded on the surface of PDA was 18.4%. The transmission electron microscopy (TEM) displayed that surface of prepared drug was structured with cell membrane, and the release of DOX was correlated with the pH of the drug solution. Under the condition of a certain DOX concentration, the prepared nano-drug could kill the Eca-109 cells strongly (P < 0.05). In the sclerosing phase, the skin thickness and collagen index of the mice could be reduced by injecting different doses of the drug solutions (P < 0.05), while the content of transforming growth factor β (TGF-β)/platelet-derived growth factor A-chain (PDGFA) was decreased (P < 0.05). The severity score of newborn children was greatly reduced after drug injection treatment; and the temperature recovery time, sclerosis disappearance time, and hospitalization time were shortened to a great extent (P < 0.05).

Transplantation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Improves Cartilage Repair in a Rabbit Model.

The aim of this study was to investigate the therapeutic efficacy and safety of transplanting human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in the treatment of cartilage injury. First, the articular cartilage defect model in rabbits was constructed. Then, the identified hUCB-MSCs and rabbit bone marrow stem cells (rBM-MSCs) were transplanted into the bone defect, respectively, and the cartilage repair effect was observed by hematoxylin-eosin (HE) staining and immunohistochemistry. Besides, the glycosaminoglycan (GAG) content and biomechanics of the restoration area were also evaluated. In our study, hUCB-MSCs and rBM-MSCs exhibited typical MSC characteristics, with positive expressions of CD73, CD105, and CD90 and negative for CD45, CD34, CD14, and HLA-DR. After the transplantation of hUCB-MSCs and rBM-MSCs, the overall quality of cartilage tissue was significantly improved, and the recipients did not show significant side effects in general. However, the expression of matrix metalloproteinase-13 (MMP-13) in the de novo tissues of the hUCB-MSCs and rBM-MSCs groups was both increased, indicating that the novel tissues may have some potential osteoarthritic changes. In conclusion, our results suggest the therapeutic effect of hUCB-MSCs transplantation in cartilage regeneration, providing a promising future in the clinical treatment of cartilage injury.

Human umbilical cord-derived mesenchymal stem cells conditioned medium ameliorates CCl4-induced liver fibrosis through regulation of expression and activity of liver lysyl oxidase

The effects of human umbilical cord blood mesenchymal stem cells conditioned medium (hMSC-CM) on Lysyl oxidase (LOX) family gene expression and proteins were evaluated. Fifty-four male Wistar rats were divided into nine groups. The control groups received olive oil and phosphate-buffered saline (PBS) while untreated groups received CCl4. The treated groups received hMSC-CM after the 4th week of CCl4 injection. LOX family expression and proteins were evaluated by RT-PCR and western blot. CCl4 enhanced significantly the expression of LOX genes and proteins. hMSC-CM significantly decreased the expression of the LOX genes and proteins. hMSC-CM decreased cross-linked collagen in the treated groups. hMSC-CM improved liver fibrosis through decreased LOX family gene expression.

Effects of shear stress on morphology, adhesion and proliferation of human umbilical cord blood mesenchymal stem cells

Objective To explore the effects of shear stress on morphology, adhesion and proliferation of human umbilical cord blood mesenchymal stem cells(hUC-MSCs). Methods The hUC-MSCs were cultured in vitro until confluence and then placed in a flow system.The cells were subjected to different shear stress (1, 2, 3, 4 dye/cm2) for 2 hours and 6 hours, and no shear stress treatment cells served as a static control.The morphological changes of hUC-MSCs in different groups were analyzed by phase contrast microscopy and immunofluorescence.The mRNA expression levels of intercellular adhesion molecule-1 (ICAM-1) and Ki67 were detected by real-time PCR. Results Compared with the static control group, the hUC-MSCs cells were arranged in the direction of fluid after treated with shear stress.The immunofluorescence results showed that the cytoskeletal protein F-actin filaments was prolonged after shear stress.The cytoskeleton was further elongated in the 2 dye/cm2 for 6 hours group when compared with 2 dye/cm2 for 2 hours group, and the cytoskeleton was loosened when time extended to 6 hours.Real-time PCR results showed that the relative expressions of ICAM-1 mRNA and Ki67 mRNA in the static control group and different gradient shear stress groups were significantly different, with significant differences among them (F=17.141, P=0.000; F=11.336, P=0.001). The relative expression of ICAM-1 mRNA in the 1, 2, 3, 4 dye/cm2 shear stress group was 2.74±0.32, 9.77±1.19, 6.70±0.92 and 5.69±0.72, respectively, which was significantly higher than 1.00±0.28 in the static control group, with significant differences between them (all at P<0.05). The relative expression of Ki67 mRNA in the 3 dye/cm2 and 4 dye/cm2 shear stress group was 0.39±0.09 and 0.04±0.02, respectively, which was significantly lower than 1.00±0.24 in the static control group, with statistically significant differences between them (both at P<0.05). Conclusions After treated with fluid shear stress, hUC-MSCs are arranged in the direction of fluid.Shear stress can promote the adhesion of hUC-MSCs.As the increase of shear stress intensity, cell proliferation is inhibited. Key words: Fluid shear stress; Human umbilical cord blood mesenchymal stem cells; Corneal endothelial cells; Intercellular adhesion molecule-1

The protective effects and mechanisms of human umbilical cord blood mesenchymal stem cells-derived microvesicles on the injury of rat retinal ganglion cells induced by high glucose condition

Objective To observe the effect and mechanism of human umbilical cord blood mesenchymal stem cells-derived microvesicles (hUMSCs-MVs) on the injury of the primary rat retinal ganglion cells (RGCs) by high glucose environment. Methods The primary RGCs of Sprague Dawley rats were cultured in vitro, hUMSCs-MVs were isolated and extracted by ultra-centrifugation. hUMSCs-MVs were internalized with RGCs. The RGCs were divided into 4 groups under the conditions below: normal control group (group A), high glucose condition group (group B, RGCs+glucose 33 mmol/L), normal RGCs co-cultured with hUMSCs-MVs group (group C, RGCs+hUMSCs-MVs), and RGCs co-cultured with hUMSCs-MVs in high glucose condition group (group D, RGCs+hUMSCs-MVs+glucose 33 mmol/L). The cell activity was detected by CCK-8 test. Annexin Ⅴ/PI staining detected the cell apoptosis rate by flow cytometry. And the relative expression levels of the genes such as Bcl-2, Bax and Caspase-3 were detected by fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Statistical analysis was performed by using One-way analysis of variance and SNK-q test was used for the comparison between groups. Results The hUMSCs-MVs were extracted by ultra-centrifugation, which were characterized as single or cluster of circular membranous vesicle-like structure with diameter ranging from 100 nm to 1000 nm. The flow cytometry analysis showed that hUMSCS-MVs were highly positived by the surface markers of CD44, CD29, CD73, and CD105 whereas been poorly expressed the integrin (CD49f), HLA class Ⅱ , CD34, CD45. There were significant differences in the cell activity and the apoptosis rate among 4 groups, the cell apoptosis rate of group B was higher significantly than that of group A and group D (F=107.92, P=0.000), the cell activity of group B was lower than that of group A and group D (F=382.11, P=0.000). The results of RT-PCR and Western blot showed that the relative mRNA (F=219.79, 339.198, 1 071.21; P=0.000, 0.000, 0.000) and protein (F=544.28, 295.79, 224.75; P=0.000, 0.000, 0.000) expression of Bcl-2, Bax, Caspase-3 and the protein expression of cleaved Capspase-3 (F=533.18, P=0.000) in group B and D were higher significantly than those in group A and C. The relative expression of Bcl-2 mRNA and protein in group B was significantly lower than that of in group D(P>0.05). The relative expression of Bax, Caspase-3 mRNA and protein in group B was higher than that in group D (P>0.05). The relative expression of cleaved Caspase-3 protein in group B was higher significantly than that in group D (P>0.05). Conclusion The hUMSCs-MVs can protect the cultured rat RGCs from the damage of the high glucose condition through increasing the cell activity and reducing the apoptosis rate of RGCs by promoting the Bcl-2 expression, decreasing the expression of Bax and Caspase-3 and inhibiting the Caspase-3 into the activity form of cleaved Caspase-3. Key words: Mesenchymal stem cells; Microvesicles; Retinal ganglion cells

BICD1 mediates HIF1\u03b1 nuclear translocation in mesenchymal stem cells during hypoxia adaptation

Hypoxia inducible factor 1α (HIF1α) is a master regulator leading to metabolic adaptation, an essential physiological process to maintain the survival of stem cells under hypoxia. However, it is poorly understood how HIF1α translocates into the nucleus in stem cells under hypoxia. Here, we investigated the role of a motor adaptor protein Bicaudal D homolog 1 (BICD1) in dynein-mediated HIF1α nuclear translocation and the effect of BICD1 regulation on hypoxia adaptation and its therapeutic potential on human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). In our results, silencing of BICD1 but not BICD2 abolished HIF1α nuclear translocation and its activity. BICD1 overexpression further enhanced hypoxia-induced HIF1α nuclear translocation. Hypoxia stimulated direct bindings of HIF1α to BICD1 and the intermediate chain of dynein (Dynein IC), which was abolished by BICD1 silencing. Akt inhibition reduced the binding of BICD1 to HIF1α and nuclear translocation of HIF1α. Conversely, Akt activation or GSK3β silencing further enhanced the hypoxia-induced HIF1α nuclear translocation. Furthermore, BICD1 silencing abolished hypoxia-induced glycolytic reprogramming and increased mitochondrial ROS accumulation and apoptosis in UCB-MSCs under hypoxia. In the mouse skin wound healing model, the transplanted cell survival and skin wound healing capacities of hypoxia-pretreated UCB-MSCs were reduced by BICD1 silencing and further increased by GSK3β silencing. In conclusion, we demonstrated that BICD1-induced HIF1α nuclear translocation is critical for hypoxia adaptation, which determines the regenerative potential of UCB-MSCs.

Safety of hopoxia-cultured human umbilical cord blood-derived mesenchymal stem cells: subchronic toxicity, tumorigenicity

Safety of hopoxia-cultured human umbilical cord blood-derived mesenchymal stem cells: subchronic toxicity, tumorigenicity

Preparation of CS/PVA Nanofibrous Membrane with Tunable Mechanical Properties for Tympanic Member Repair

The wound healing for tympanic membrane (TM) perforations is one of the most common clinic treatment. New designs of biomaterials, and lately tissue-engineered composites and grafts, have been applied for the management of TM perforation. In this work, a series of chitosan (CS)/polyvinyl alcohol (PVA) nanofibrous films have been fabricated as a kind of patch for repairing TM perforation. The scaffolds were crosslinked by glutaraldehyde solution to prevent them from being dissolved in the culture medium. The morphology of nanofibers from different precursors has been observed by using scanning electron microscopy (SEM). And the crosslinked films have been characterized by SEM and Fourier transform infrared spectroscopy for confirming its morphology and chemical structure. We found the films with crosslinking showed stronger tensile strength and modulus compared to the uncrosslinked one. And the mechanical properties could be tuned by the crosslinking time. In addition, after crosslinked, the films showed good thermostability and water-resistance, characterized by thermo gravimetric analyzer and contact angle meter. The cell viability studies with human umbilical vein endothelial cells, fibroblasts and mesenchymal stem cells demonstrated that the nanofibrous scaffolds formed by electrospinning process had no side effects for cell proliferation. However, a better way of crosslinking CS/PVA films needs to be further studied.

Human umbilical cord tissue stem cells and neuronal lineages in an injectable caffeic acid-bioconjugated gelatin hydrogel for transplantation.

Present-day scaffolds are useful in cell therapy to a reasonable extent, but in pursuit of improvising the scaffold to improve the outcome, we tested a new injectable caffeic acid-bioconjugated gelatin hydrogel scaffold (CBGH; with tunable stiffness -10%). Two-dimensional (2D) form of human umbilical cord tissue-derived mesenchymal stem cells (HUCMSCs) culture performed based on our previously reported methods and characterized by using multipotent and pluripotent analysis. In addition, neurogenesis was induced in the presence of retinoic acid or neural growth factor or epidermal growth factor categorized by neuronal markers. The viability, proliferation rate, and vascular endothelial growth factor expression of HUCMSCs increased significantly in the CBGH scaffold. In addition, there was an increase in CD90 and TRA-1-81 phenotypic expressions and SOX-2, MAP-2, TAU, NeuN, and NF, which confirmed the neurogenesis of encapsulated HUCMSCs. Topographical elucidation by scanning electron microscopy data showed that the HUCMSCs proliferated and migrated inside the construct. Hematoxylin and eosin staining demonstrated a more viable structural pattern and cresyl violet staining showed the Nissl synthesis, confirming the presence of functional neurons in the encapsulated form. The molecular-level analysis further substantiated that HUCMSCs cultured in CBGH expressed significantly greater upregulation of stemness, neuronal genes, and protein expression compared with the adherent culture. Correspondingly, this is the first time that we have measured the fluorescence intensity variation of the HUCMSCs-stained cell segmentation process using customized MATLAB code execution to reduce the background noise and autofluorescence. We conclude that this novel CBGH scaffold increases the viability, proliferation, stemness, and also neuronal transdifferentiation of HUCMSCs in a three-dimensional culture than the 2D plastic adherent culture.

Repeated injections of human umbilical cord blood-derived mesenchymal stem cells significantly promotes functional recovery in rabbits with spinal cord injury of two noncontinuous segments

BackgroundSpinal cord injuries (SCIs) are sustained by an increasing number of patients each year worldwide. The treatment of SCIs has long been a hard nut to crack for doctors around the world. Mesenchymal stem cells (MSCs) have shown benefits for the repair of SCI and recovery of function. Our present study aims to investigate the effects of intravenously infused human umbilical cord blood-derived MSCs (hUCB-MSCs) on functional recovery after subacute spinal cord compression injury of two noncontinuous segments. In addition, we compared the effects of single infusion and repeated intravenous (i.v.) injections on the recovery of spinal cord function.MethodsA total of 43 adult rabbits were randomly divided into four groups: control, single injection (SI), repeated injection at a 3-day (3RI) or repeated injection at a 7-day interval (7RI) groups. Non-immunosuppressed rabbits in the transplantation groups were infused with either a single complete dose or three divided doses of 2 × 106 hUCB-MSCs (3-day or 7-day intervals) on the first day post decompression. Behavioural scores and somatosensory evoked potentials (SEPs) were used to evaluate hindlimb functional recovery. The survival and differentiation of the transplanted human cells and the activation of the host glial and inflammatory reaction in the injured spinal cord were studied by immunohistochemical staining.ResultsOur results showed that hUCB-MSCs survived, proliferated, and primarily differentiated into oligodendrocytes in the injured area. Treatment with hUCB-MSCs reduced the extent of astrocytic activation, increased axonal preservation, potentially promoted axonal regeneration, decreased the number of Iba-1+ and TUNEL+ cells, increased the amplitude and decreased the onset latency of SEPs and significantly promoted functional improvement. However, these effects were more pronounced in the 3RI group compared with the SI and 7RI groups.ConclusionsOur results suggest that treatment with i.v. injected hUCB-MSCs after subacute spinal cord compression injury of two noncontinuous segments can promote functional recovery through the differentiation of hUCB-MSCs into specific cell types and the enhancement of anti-inflammatory, anti-astrogliosis, anti-apoptotic and axonal preservation effects. Furthermore, the recovery was more pronounced in the rabbits repeatedly injected with cells at 3-day intervals. The results of this study may provide a novel and useful treatment strategy for the transplantation treatment of SCI.

Abstract TP106: Sex Differences After Mesenchymal Stem Cell Treatment on Post-Stroke Neurological Outcome

Introduction: A major problem exists in stroke management because of the sex differences in frequency and severity of disease and in treatment outcomes. Our recent studies demonstrated the potential of human umbilical cord blood-derived mesenchymal stem cells (HUCB-MSCs) in attenuating the post-stroke brain damage. Methods: Sprague-Dawley rats of both sexes were obtained and randomly assigned to various groups. Rats were subjected to a two-hour middle cerebral artery occlusion (MCAO) procedure by inserting a silicone rubber-coated monofilament suture followed by fourteen days of reperfusion. The body weight of rats on the day of the MCAO procedure and the size of the monofilament for male and female rats were standardized to obtain the same depth of post-stroke infarction. Previously standardized effective dose (0.25 million cells/animal) of HUCB-MSCs were intravenously injected via tail vein 24 hours post-MCAO to the designated groups of rats. Rats were subjected to neurological evaluation at various reperfusion time points by modified neurological severity scores ( mNSS), modified adhesive removal ( sticky-tape) beam walking and accelerating Rotarod performance tests. Results: The fourteen-day post-stroke survival period in males and females is approximately 20% and 70%, respectively. Females are more resistant to stroke-induced mortality. Although the difference in post-stroke percent survival is prominent in both sexes, there was no change in the body weight gain of both sexes. It was evident from the mNSS and sticky-tape tests that the magnitude of natural neurological recovery is the same in both sexes. However, the improvement in coordination and integration of motor function in females is better than males as evidenced by other tests. Stem cell treatment prevented mortality in males without any change in the treatment-induced body weight gain. Further, stem cell treatment improved neurological recovery in males, and the coordination and integration of motor function in females. Conclusions: Sex differences exist in post-stroke mortality and neurological function. Stem cell treatment improved neurological recovery and there was no sex difference in the magnitude of stem cell treatment improved neurological function

Optimization of culture conditions for rapid clinical-scale expansion of human umbilical cord blood-derived mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) have broad-spectrum therapeutic effects in various diseases, and thus have many clinical applications. However, it is difficult to produce sufficient numbers of MSCs for clinical use, and improved culture systems are required. Here, we report the effects of calcium (Ca2+) and hypoxia on the proliferation of human umbilical cord blood-derived MSCs (hUCB-MSCs). In addition, we determined the optimal conditions of these two factors for the large-scale culture of hUCB-MSCs.MethodshUCB-MSCs were maintained under hypoxic conditions (3% O2) with 1.8 mM Ca2+ during long-term culture, and their proliferation was evaluated. To characterize the underlying mechanisms, the effects on hypoxia-inducible factor (HIF)-1α and the extracellular signal-regulated kinase (ERK) signaling pathways were investigated. The therapeutic effects in a mouse emphysema model were analyzed and compared with those of naive MSCs.ResultsThe proliferation of Ca2+/hypoxia-treated hUCB-MSCs was increased compared with that observed using either calcium or hypoxia culture alone, without loss of stem cell marker expression or differentiation ability. The enhancement of the proliferation capacity of hUCB-MSCs by the synergistic effects of Ca2+ and hypoxia was dependent on the expression of HIF-1α and the ERK signaling pathway. The proliferation of Ca2+/hypoxia-treated hUCB-MSCs resulted in a delayed senescence phenotype and increased the expression levels of stemness genes such as Oct4 and Nanog compared to those observed in conventional culture conditions. In addition, Ca2+/hypoxia-treated MSCs transplantation in the mouse emphysema model showed the same therapeutic effects as observed with naive MSCs.ConclusionsThese findings suggest that a Ca2+/hypoxia-based expansion system has applications for the large-scale production of MSCs for therapeutic purposes.

Activated Schwann cells combined with umbilical cord blood mesenchymal stem cell transplantation for repair of spinal cord injury

Objective To observe the variation of neuronal markers after co-transplantation of activated Schwann cells (ASCs) and human umbilical cord blood mesenchymal stem cells (HUCBMSCs)into rat spinal cord injury (SCI). Also, we discuss whether these two types of cells grafted into the injured site of spinal cord have the ability of promoting axonal regeneration and functional recovery after SCI. Methods SCI model in Wistar rat (Female, 8 weeks’ old) was created at T10 segment with NYU Impactor-Ⅱmachine of 10 g×50 mm weight drop.80 SCI rats were classified into 4 groups randomly: blank control group, ASCs transplantation group, HUCBMSCs transplantation group and co-transplantation group of ASCs and HUCBMSCs. 1, 2, 3, 4 weeks after operation, 3 animals of each group were sacrificed, and injured spinal cord tissue were used for Western blotting and Real-time polymerase chain reaction analysis [nuclear factor (NF)-200, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP)]. Basso, Beattie, and Bresnahan (BBB) score was carried out at the 3rd day and at weekly after intervals injury.12 weeks later, 8 rats in each group were selected to carry out 10% BDA anterograde tracing mark.2 weeks later, the animals were sacrificed, the spinal cord tissue with injured lesion was taken out and fast cryostat sections (5 μm) was made followed by BDA developing, hematoxylin and eosin (HE) and immunochemistry (NF-200, MBP, GFAP) staining. Results Western blotting and Real-time PCR analysis revealed that NF-200 and MBP expressed higher with time while the co-graft group is the highest (4 weeks after injury, NF-200: 4.581±0.588, MBP: 3.922±0.259). From the 4th week after cell-transplantation, BBB score in co-graft group was significantly higher than that in other 3 groups (P=0.000). The rats of co-graft group recoverd to the largest extent. BDA anterograde injured site in co-graft group than that in other 3 groups, while little in control group (P=0.000). HE staining showed that the injured cavity was the smallest one in cell co-graft group when compared with other 3 groups. Conclusion The results indicate that the co-transplantation fully takes advantage of ASCs to promote HUCBMSCs differentiate into neurons. This is more effective than single cell transplantation in promoting axonal regeneration and functional recovery after rat’s spinal cord injury. Key words: Umbilical cord blood mesenchymal stem cells; Activated Schwann cells; Spinal cord injury; Neural repair; Differentiation; Co-transplantation

Protein profiling and angiogenic effect of hypoxia-cultured human umbilical cord blood-derived mesenchymal stem cells in hindlimb ischemia.

The aim of the present study was to investigate protein profiles of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) cultured in normoxic (21% O2) and hypoxic (1% O2) conditions, and evaluate oxygenation effects on angiogenesis in an ischemic hindlimb mouse model using a modified ischemic scoring system. Hypoxic conditions did not change the expression of phenotypic markers and increased adipogenesis and chondrogenesis. Epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), TGF-β RII, and vascular endothelial growth factor (VEGF) were upregulated in the conditioned medium of hypoxic hUCB-MSCs, which are commonly related to angiogenesis and proliferation of biological processes by Gene Ontology. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, significant enrichment of the phosphorylation of abelson murine leukemia viral oncogene homolog 1 (ABL1) (Phospho-Tyr204) and B-cell lymphoma-extra large (BCL-XL) (Phospho-Thr47) as anti-apoptotic pathways was observed in hypoxic hUCB-MSCs. Furthermore, hypoxic conditions induced proliferation and migration, and reduced apoptosis of hUCB-MSCs in vitro. Based on the results of protein antibody array, we evaluated the angiogenic effects of injecting normoxic or hypoxic hUCB-MSCs (1×106) into the ischemic hindlimb muscles of mice. Ischemic scores and capillary generation were significantly greater in the hypoxic hUCB-MSC injection group than in the normoxic hUCB-MSC group. Our findings demonstrate that culturing hUCB-MSCs in hypoxic conditions not only significantly enriches phosphorylation in the anti-apoptosis pathway and enhances the secretion of several angiogenic proteins from cells, but also alleviates ischemic injury of hindlimb of mice.

Effect of Electrospun Fibrous Scaffolds with Different Fiber Orientations on the Alignment of Microvessel-Like Structures

Establishing aligned microvessel-like structures is crucial to the engineered construct in vitro for the efficient tissue perfusion. In this study, human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells derived from induced pluripotent stem cells (iPSC-MSCs) were co-cultured on aligned and random scaffolds to verify the hypothesis that contact guidance could direct the formation of aligned microvessel-like structures. As tested, the major discrepancy between aligned and random scaffolds lied in the fiber orientation. Investigated by immunofluorescence staining, co-culturing HUVECs and iPSC-MSCs could form the microvessel-like structures. Applying this co-culturing system to both scaffolds showed that the orientation of microvessel-like structures was closely related to the fiber orientation of scaffold. In comparison with scattered microvessel-like structures on random scaffolds, aligned microvessel-like structures were observed on aligned scaffolds. We firstly indicated that aligned electrospun fibers is sufficient to align microvessel-like structures formed by co-culturing the HUVECs and iPSC-MSCs, which provides a basis for further constructing diverse tissues containing aligned microvessel-like structures in vitro.