Potential surrogate quantitative immunomodulatory potency assay for monitoring human umbilical cord-derived mesenchymal stem cells production.

Mesenchymal stem cells (MSCs) play an important role as immune modulator through interaction with several immune cells, including macrophages. In this study, the immunomodulatory potency of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) was demonstrated in the in vivo middle cerebral artery occlusion (MCAo)-induced brain injury rat model and in vitro THP-1-derived macrophages model. At 24 h after induction of MCAo, hUC-MSCs was administered via tail vein as a single dose. Remarkably, hUC-MSCs could inhibit M1 polarization and promote M2 polarization of microglia in vivo after 14 days induction of MCAo. Compared with THP-1-derived macrophages which had been stimulated by lipopolysaccharide, the secretion of proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interferon-γ inducible protein (IP-10), were significantly reduced in the presence of hUC-MSCs. Moreover, the secretion of anti-inflammatory cytokine, interleukin-10 (IL-10), was significantly increased after cocultured with hUC-MSCs. Prostaglandins E2 (PGE2), secreted by hUC-MSCs, is one of the crucial immunomodulatory factors and could be inhibited in the presence of COX2 inhibitor, NS-398. PGE2 inhibition suppressed hUC-MSCs immunomodulatory capability, which was restored after addition of synthetic PGE2, establishing the minimum amount of PGE2 required for immunomodulation. In conclusion, our data suggested that PGE2 is a crucial potency marker involved in the therapeutic activity of hUC-MSCs through macrophages immune response modulation and cytokines regulation. This study provides the model for the development of a surrogate quantitative potency assay of immunomodulation in stem cells production.

Transcriptomic and Proteomic Profiling of Human Mesenchymal Stem Cell Derived from Umbilical Cord in the Study of Preterm Birth.

OBJECTIVE: Mesenchymal stem cells (MSCs) hold great therapeutic potential in morbidities associated with preterm birth. However, the molecular expressions of MSCs in preterm birth infants are not systematically evaluated. In this study, the dual-omics analyses of umbilical-cord (UC)-derived MSCs to identify the dysregulated cellular functions are presented. MATERIALS AND METHODS: The UC-MSCs are collected from ten full-term and eight preterm birth infants for microarray and iTRAQ-based proteome profiling. RESULTS: The integrative analysis of dual-omics data discovered 5615 commonly identified genes/proteins of which 29 genes/proteins show consistent up- or downregulation in preterm birth. The Gene Ontology analysis reveals that dysregulation of mitochondrial translation and cellular response to oxidative stress are mainly enriched in 290 differential expression proteins (DEPs) while the 412 differential expression genes (DEGs) are majorly involved in single-organism biosynthetic process, cellular response to stress, and mitotic cell cycle in preterm birth. Besides, a 13-protein module involving CUL2 and CUL3 is identified, which plays an important role in cullin-RING-based ubiquitin ligase complex, as potential mechanism for preterm birth. CONCLUSION: The dual-omics data not only provide new insights to the molecular mechanism but also identify panel of candidate markers associated with preterm birth.

Modulation of parietal cytokine and chemokine gene profiles by mesenchymal stem cell as a basis for neurotrauma recovery.

BACKGROUND & PURPOSE: Following traumatic brain injury (TBI), primary mechanical injury to the brain may cause blood-brain-barrier damage followed by secondary injury, ultimately culminating in cell death. We aimed to test whether one injection of mesenchymal stem cells (MSC) derived from the human umbilical cord can modulate brain cytokine and chemokine gene profiles and attenuate neurological injury in rats with TBI. METHODS: One-day post-TBI, the injured rats were treated with one injection of MSC (4 × 106/rat, i.v.). Three days later, immediately after assessment of neurobehavioral function, animals were sacrificed for analysis of neurological injury (evidenced by both brain contusion volume and neurological deficits) and parietal genes encoding 84 cytokines and chemokines in the injured brain by qPCR methods. RESULTS: Three days post-TBI, rats displayed both neurological injury and upgrade of 11 parietal genes in the ipsilateral brain. One set of 8 parietal genes (e.g., chemokine [C-X-C motif] ligand 12, platelet factor 4, interleukin-7, chemokine [C-C motif] ligand (CCL)19, CCL 22, secreted phosphoprotein 1, pro-platelet basic protein 1, and CCL 2) differentially upgraded by TBI was related to pro-inflammatory and/or neurodegenerative processes. Another set of 3 parietal genes up-graded by TBI (e.g., glucose-6-phosphate isomerase, bone morphogenetic protein (BMP) 2, and BMP 4) was related to anti-inflammatory/neuroregenerative events. Administration of MSC attenuated neurological injury, down-regulated these 8 parietal pro-inflammatory genes, and up-regulated these 3 parietal anti-inflammatory genes in the rats with TBI. CONCLUSION: Our data suggest that modulation of parietal cytokines and chemokines gene profiles by MSC as a basis for neurotrauma recovery.

EphA2-positive human umbilical cord-derived mesenchymal stem cells exert anti-fibrosis and immunomodulatory activities via secretion of prostaglandin E2.

OBJECTIVE: Previous study has demonstrated that EphA2 is a biomarker of mesenchymal stem cells (MSCs) from human placenta or umbilical cord and is able to distinguish MSCs from fibroblasts. In this study, we further examine the potential efficacy of EphA2+ human umbilical cord-derived MSCs (hUC-MSCs). MATERIALS AND METHODS: MSCs specific markers, EphA2 and CD146 expression on the surface of hUC-MSCs were determined by flow cytometry analysis. Quantitative real time polymerase chain reaction was used to examine pro-fibrotic gene expression of TGF-ß1-stimulated lung fibroblast (MRC-5 cells). On the other hand, ELISA was used to analyze the content of pro-inflammatory cytokines (TNF-ɑ; and IP-10) in the LPS-activated macrophages culture supernatant. RESULTS: The pro-fibrotic gene (TGF-ß1, CTGF, fibronectin, collagen I and TIMP-1) expression in TGF-ß1-activated MRC-5 cells and the pro-inflammatory cytokines (TNF-ɑ and IP-10) in the LPS-activated macrophages culture supernatant were both attenuated when in present of EphA2+ hUC-MSCs. Moreover, once EphA2+ hUC-MSCs treated with prostaglandin E2 specific inhibitor NS-398, both anti-fibrotic and anti-inflammatory effects of EphA2+ hUC-MSCs were abolished. CONCLUSION: EphA2+ hUC-MSCs possess immunomodulatory and anti-fibrotic properties, and PGE2 plays an important role in these activities. This implies that EphA2+ hUC-MSCs have potentially effectiveness for treatment of acute inflammatory and chronic fibrotic lung diseases.

MRI tracking of polyethylene glycol-coated superparamagnetic iron oxide-labelled placenta-derived mesenchymal stem cells toward glioblastoma stem-like cells in a mouse model.

Mesenchymal stem cells (MSCs) that display homing and infiltration properties towards tumor cells are a promising cellular targeting vector for brain tumor therapy but are limited to local-regional delivery in current preclinical models. Here, we investigated whether placenta-derived MSCs (P-MSCs) are a superior cellular vector for systemic targeting of glioblastoma stem-like cells (GSCs), with an imaging modality to real-time monitor the trafficking P-MSCs to glioblastoma sites. Results demonstrated that P-MSCs had greater migratory activity towards GSCs and across blood-brain barrier compared with bone marrow-derived MSCs, and this activity was enhanced by hypoxia precondition. Chemokine ligand 5 was identified as a chemoattractant responsible for the glioblastoma tropism of P-MSCs. Polyethylene glycol-coated superparamagnetic iron oxide (PEG-SPIO) was synthesized for cellular labelling and imaging P-MSCs, displaying high cellular uptake and no cytotoxic effect on P-MSCs cell proliferation or stemness property. The homing effects of intravenously administered PEG-SPIO-labelled P-MSCs towards intracerebral GSCs were able to be detected in mice models through T2-weighted magnetic resonance imaging (MRI). This study suggests the possibility of innovative systemic P-MSC-based cell therapy for aggressive GSCs, developing a state-of-the-art theranostic technique for real-time tracking of therapeutic P-MSCs tumor infiltration through cellular MRI.

CD34- human placenta-derived mesenchymal stem cells protect against heat stroke mortality in rats.

CD34 is a transmembrane phosphoglycoprotein used to selectively enrich bone marrow in hematopoietic stem cells for transplantation. Treating rats with CD34+ cells derived from human umbilical cord blood before or after heat stroke has been shown to promote survival. We investigated whether CD34- human placenta-derived stem cells (PDMSCs) could improve survival following heat stroke in rats. Rats were subjected to heat stress (42°C for 98 min) to induce heat stroke. Intravenous administration of PDMSCs 1 day before or immediately after the onset of heat stroke improved survival by 60% and 20%, respectively. Pre-treatment with CD34- PDMSCs protected against heat stroke injury more effectively than that treatment after injury. PDMSCs treatment attenuated cerebrovascular dysfunction, the inflammatory response, and lipid peroxidation. These data suggest human PDMSCs protect against heat stroke injury in rats. Moreover, these effects do not require the presence of CD34+ cells.

A genome-wide comparison of mesenchymal stem cells derived from human placenta and umbilical cord.

OBJECTIVE: The human umbilical cord and placenta have been considered as attractive alternative sources for noninvasive isolation of human mesenchymal stem cells (hMSCs). Different sources of MSC may have individual differentiation potential and phenotype. In this study, we compared the genome-wide expression data of umbilical cord and placenta derived hMSCs to identify specific differential expression genes (DEGs) and corresponding functions. MATERIALS AND METHODS: We collected human placental tissues and umbilical cord from healthy full-term placenta (n = 17). The genome-wide gene expression data of hMSCs were used to analyze and compare with that of fibroblasts. We identified the differential expression genes (DEGs) based on the Student's t-test and one-way ANOVA. RESULTS: According to the DEGs of umbilical cord and placenta, we used the Venn diagram to evaluate the consistence and specific genes. There are 390 umbilical cord specific DEGs which functions are related to movement of sub-cellular component. Then, the DEGs derived from placenta have two major clusters (i.e., placenta-specific (AM-CM-specific) and UC-like (UC-CD-specific)). 247 placenta-specific DEGs are down-regulated and involved in cell communication. 278 UC-like genes are up-regulated and are involved in the cell cycle, cell division, and DNA repair process. Finally, we also identified 239 umbilical cord-placenta consistence DEGs. According to the umbilical cord-placenta consistence DEGs, 175 genes are down-regulated and involved in cell death, cell growth, cell developmental processes. CONCLUSION: We identified the consistence and specific DEGs of human placenta and umbilical cord based on the genome-wide comparison. Our results indicated that hMSCs derived from umbilical cord and placenta have different gene expression patterns, and most of specific genes are involved in the cell cycle, cell division, cell death, and cell developmental processes.

Human Umbilical Cord Mesenchymal Stem Cells Preserve Adult Newborn Neurons and Reduce Neurological Injury after Cerebral Ischemia by Reducing the Number of Hypertrophic Microglia/Macrophages.

Microglia are the first source of a neuroinflammatory cascade, which seems to be involved in every phase of stroke-related neuronal damage. Two weeks after transient middle cerebral artery occlusion (MCAO), vehicle-treated rats displayed higher numbers of total ionized calcium-binding adaptor molecule 1 (Iba-1)-positive cells, greater cell body areas of Iba-1-positive cells, and higher numbers of hypertrophic Iba-1-positive cells (with a cell body area over 80 µm2) in the ipsilateral ischemic brain regions including the frontal cortex, striatum, and parietal cortex. In addition, MCAO decreased the number of migrating neuroblasts (or DCX- and 5-ethynyl-2'-deoxyuridine-positive cells) in the cortex, subventricular zone, and hippocampus of the ischemic brain, followed by neurological injury (including brain infarct and neurological deficits). Intravenous administration of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs; 1 × 106 or 4 × 106) at 24 h after MCAO reduced neurological injury, decreased the number of hypertrophic microglia/macrophages, and increased the number of newborn neurons in rat brains. Thus, the accumulation of hypertrophic microglia/macrophages seems to be detrimental to neurogenesis after stroke. Treatment with hUC-MSCs preserved adult newborn neurons and reduced functional impairment after transient cerebral ischemia by reducing the number of hypertrophic microglia/macrophages.

Mesenchymal stem cell-based treatments for stroke, neural trauma, and heat stroke.

BACKGROUND: Mesenchymal stem cell (MSC) transplantation has been reported to improve neurological function following neural injury. Many physiological and molecular mechanisms involving MSC therapy-related neuroprotection have been identified. METHODS: A review is presented of articles that pertain to MSC therapy and diverse brain injuries including stroke, neural trauma, and heat stroke, which were identified using an electronic search (e.g., PubMed), emphasize mechanisms of MSC therapy-related neuroprotection. We aim to discuss neuroprotective mechanisms that underlie the beneficial effects of MSCs in treating stroke, neural trauma, and heatstroke. RESULTS: MSC therapy is promising as a means of augmenting brain repair. Cell incorporation into the injured tissue is not a prerequisite for the beneficial effects exerted by MSCs. Paracrine signaling is believed to be the most important mediator of MSC therapy in brain injury. The multiple mechanisms of action of MSCs include enhanced angiogenesis and neurogenesis, immunomodulation, and anti-inflammatory effects. Microglia are the first source of the inflammatory cascade during brain injury. Cytokines, including tumor necrosis factor-α, interleukin-1ß, and interleukin-6, are significantly produced by microglia in the brain after experimental brain injury. The proinflammatory M1 phenotype of microglia is associated with tissue destruction, whereas the anti-inflammatory M2 phenotype of microglia facilitates repair and regeneration. MSC therapy may improve outcomes of ischemic stroke, neural trauma, and heatstroke by inhibiting the activity of M1 phenotype of microglia but augmenting the activity of M2 phenotype of microglia. CONCLUSION: This review offers a testable platform for targeting microglial-mediated cytokines in clinical trials based upon the rational design of MSC therapy in the future. MSCs that are derived from the placenta provide a great choice for stem cell therapy. Although targeting the microglial activation is an important approach to reduce the burden of the injury, it is not the only one. This review focuses on this specific aspect.

EphA2 is a biomarker of hMSCs derived from human placenta and umbilical cord.

OBJECTIVE: The heterogeneous nature of mesenchymal stem cells (MSCs) and the absence of known MSC-specific biomarkers make it challenging to define MSC phenotypes and characteristics. In this study, we compared the phenotypic and functional features of human placenta-derived MSCs with those of human dermal fibroblasts in vitro in order to identify a biomarker that can be used to increase the purity of MSCs in a primary culture of placenta-derived cells. MATERIALS AND METHODS: Liquid chromatography-tandem mass spectrometry analysis was used to analyze and compare the proteome of human placenta-derived MSCs with that of fibroblasts. Quantitative real-time polymerase chain reaction, immunofluorescence, and flow cytometry were used to determine expression levels of EphA2 in placenta-derived MSCs. EphA2-positive cells were enriched by magnetic-activated cell sorting or with a cell sorter. An shRNA-mediated EphA2 knockdown was used to assess the role of EphA2 in MSC response to Tumor necrosis factor (TNF)-α stimulation. RESULTS: Analysis of proteomics data from MSCs and fibroblasts resulted in the identification of the EphA2 surface protein biomarker, which could reliably distinguish MSCs from fibroblasts. EphA2 was significantly upregulated in placenta-derived MSCs when compared to fibroblasts. EphA2 played an important role in MSC migration in response to inflammatory stimuli, such as TNF-α. EphA2-enriched MSCs were also more responsive to inflammatory stimuli in vitro when compared to unsorted MSCs, indicating a role for EphA2 in the immunomodulatory functionality of MSCs. CONCLUSION: EphA2 can be used to distinguish and isolate MSCs from a primary culture of placenta-derived cells. EphA2-sorted MSCs exhibited superior responsiveness to TNF-α signaling in an inflammatory environment compared with unsorted MSCs or MSC-like cells.