Marianne Joerger-Messerli

PreImplantation Factor bolsters neuroprotection via modulating Protein Kinase A and Protein Kinase C signaling

A synthetic peptide (sPIF) analogous to the mammalian embryo-derived PreImplantation Factor (PIF) enables neuroprotection in rodent models of experimental autoimmune encephalomyelitis and perinatal brain injury. The protective effects have been attributed, in part, to sPIF’s ability to inhibit the biogenesis of microRNA let-7, which is released from injured cells during central nervous system (CNS) damage and induces neuronal death. Here, we uncover another novel mechanism of sPIF-mediated neuroprotection. Using a clinically relevant rat newborn brain injury model, we demonstrate that sPIF, when subcutaneously administrated, is able to reduce cell death, reverse neuronal loss and restore proper cortical architecture. We show, both in vivo and in vitro, that sPIF activates cyclic AMP dependent protein kinase (PKA) and calcium-dependent protein kinase (PKC) signaling, leading to increased phosphorylation of major neuroprotective substrates GAP-43, BAD and CREB. Phosphorylated CREB in turn facilitates expression of Gap43, Bdnf and Bcl2 known to have important roles in regulating neuronal growth, survival and remodeling. As is the case in sPIF-mediated let-7 repression, we provide evidence that sPIF-mediated PKA/PKC activation is dependent on TLR4 expression. Thus, we propose that sPIF imparts neuroprotection via multiple mechanisms at multiple levels downstream of TLR4. Given the recent FDA fast-track approval of sPIF for clinical trials, its potential clinical application for treating other CNS diseases can be envisioned.

Intranasal Delivery of Umbilical Cord-Derived Mesenchymal Stem Cells Preserves Myelination in Perinatal Brain Damage

Preterm white matter injury (WMI) is an important cause for long-term disability. Stem cell transplantation has been proposed as a novel therapeutic approach. However, intracerebral transplantation is not feasible for clinical purpose in newborns. Intranasal delivery of cells to the brain might be a promising, noninvasive therapeutic approach to restore the damaged brain. Therefore, our goal is to study the remyelinating potential of human Whartons jelly mesenchymal stem cells (hWJ-MSCs) after intranasal delivery. Wistar rat pups, previously brain-damaged by a combined hypoxic-ischemic and inflammatory insult, received hWJ-MSC (150,000 cells in 3u2009μL) that were intranasally delivered twice to each nostril (600,000 cells total). WMI was assessed by immunohistochemistry and western blot for myelination, astrogliosis, and microgliosis. The expression of preoligodendrocyte markers, and neurotrophic factors, was analyzed by real-time polymerase chain reaction. Animals treated with intranasally delivered hWJ-MSC showed increased myelination and decreased gliosis compared to untreated animals. hWJ-MSC may, therefore, modulate the activation of microglia and astrocytes, resulting in a change of the brain microenvironment, which facilitates the maturation of oligodendrocyte lineage cells. This is the first study to show that intranasal delivery of hWJ-MSC in rats prevented hypomyelination and microgliosis in a model of WMI in the premature rat brain. Further studies should address the dose and frequency of administration.

Mesenchymal Stem Cells from Wharton's Jelly and Amniotic Fluid

The discovery of mesenchymal stem cells (MSCs) in perinatal sources, such as the amniotic fluid (AF) and the umbilical connective tissue, the so-called Whartons jelly (WJ), has transformed them into promising stem cell grafts for the application in regenerative medicine. The advantages of AF-MSCs and WJ-MSCs over adult MSCs, such as bone marrow-derived mesenchymal stem cells (BM-MSCs), include their minimally invasive isolation procedure, their more primitive cell character without being tumourigenic, their low immunogenicity and their potential autologous application in congenital disorders and when cryopreserved in adulthood. This chapter gives an overview of the biology of AF-MSCs and WJ-MSCs, and their regenerative potential based on the results of recent preclinical and clinical studies. In the end, open questions concerning the use of WJ-MSCs and AF-MSCs in regenerative medicine will be emphasized.

Preeclampsia enhances neuroglial marker expression in umbilical cord Wharton's jelly-derived mesenchymal stem cells

Abstract Objective: The aim of the study was to compare the neuroglial phenotype of Whartons jelly-derived mesenchymal stem cells (WJ-MSC) from pregnancies complicated with preeclampsia and gestational age (GA)-matched controls. Methods: WJ-MSC were isolated from umbilical cords from both groups and analyzed for the cell surface expression of MSC markers and the gene and protein expression of neuroglial markers. Results: All WJ cells were highly positive for the MSC markers CD105, CD90 and CD73, but negative for markers specific for hematopoietic (CD34) and immunological cells (CD45, CD14, CD19 and HLA-DR). WJ-MSC from both groups expressed neuroglial markers (MAP-2, GFAP, MBP, Musashi-1 and Nestin) at the mRNA and protein level. The protein expressions of neuronal (MAP-2) and oligodendrocytic (MBP) markers were significantly increased in WJ-MSC from preeclampsia versus GA-matched controls. Conclusions: WJ-MSC from preeclamptic patients are possibly more committed to neuroglial differentiation through the activation of pathways involved both in the pathophysiology of the disease and in neurogenesis.

Wharton's Jelly Mesenchymal Stem Cells Protect the Immature Brain in Rats and Modulate Cell Fate.

The development of a mammalian brain is a complex and long-lasting process. Not surprisingly, preterm birth is the leading cause of death in newborns and children. Advances in perinatal care reduced mortality, but morbidity still represents a major burden. New therapeutic approaches are thus desperately needed. Given that mesenchymal stem/stromal cells (MSCs) emerged as a promising candidate for cell therapy, we transplanted MSCs derived from the Whartons Jelly (WJ-MSCs) to reduce the burden of immature brain injury in a murine animal model. WJ-MSCs transplantation resulted in protective activity characterized by reduced myelin loss and astroglial activation. WJ-MSCs improved locomotor behavior as well. To address the underlying mechanisms, we tested the key regulators of responses to DNA-damaging agents, such as cyclic AMP-dependent protein kinase/calcium-dependent protein kinase (PKA/PKC), cyclin-dependent kinase (CDK), ataxia-telangiectasia-mutated/ATM- and Rad3-related (ATM/ATR) substrates, protein kinase B (Akt), and 14-3-3 binding protein partners. We characterized WJ-MSCs using a specific profiler polymerase chain reaction array. We provide evidence that WJ-MSCs target pivotal regulators of the cell fate such as CDK/14-3-3/Akt signaling. We identified leukemia inhibitory factor as a potential candidate of WJ-MSCs induced modifications as well. We hypothesize that WJ-MSCs may exert adaptive responses depending on the type of injury they are facing, making them prominent candidates for cell therapy in perinatal injuries.

Extracellular vesicles derived from Wharton’s jelly mesenchymal stem cells prevent and resolve programmed cell death mediated by perinatal hypoxia-ischemia in neuronal cells

Hypoxic-ischemic (HI) insult in the perinatal phase harbors a high risk of encephalopathy in the neonate. Brain cells undergo apoptosis, initiating neurodegeneration. So far, therapeutic approaches such as cooling remain limited. Transplantation of mesenchymal stem cells (MSCs) exhibits therapeutic success despite the short-time survival in the host brain, providing strong evidence that their beneficial effects are largely based on secreted factors, including extracellular vesicles (EVs). The aim of this study was to investigate the effects of human Wharton’s jelly MSC (hWJ-MSC)-derived EVs on neuroprotection and neuroregeneration, using an in vitro model of oxygen–glucose deprivation/reoxygenation (OGD/R) mimicking HI injury in the mouse neuroblastoma cell line neuro2a (N2a). hWJ-MSC-derived EVs were isolated from cell culture supernatants by multistep centrifugation and identified by endosomal marker expression and electron microscopy. OGD/R significantly increased DNA fragmentation and caspase 3 (Casp3) transcription in N2a cells relative to undamaged cells. OGD/R-mediated DNA fragmentation and Casp3 expression could be prevented as well as resolved by the addition of hWJ-MSC-derived EV before and after OGD, respectively. hWJ-MSC-derived EV also tended to increase the phosphorylation of the B cell lymphoma 2 (Bcl2) family member Bcl-2-antagonist of cell death (BAD) in N2a cells, when added prior or post OGD, thereby inactivating the proapoptotic function of BAD. Fluorescence confocal microscopy revealed the close localization of hWJ-MSC-derived EVs to the nuclei of N2a cells. Furthermore, EVs released their RNA content into the cells. The expression levels of the microRNAs (miRs) let-7a and let-7e, known regulators of Casp3, were inversely correlated to Casp3. Our data suggest that hWJ-MSC-derived EVs have the potential to prevent and resolve HI-induced apoptosis in neuronal cells in the immature neonatal brain. Their antiapoptotic effect seems to be mediated by the transfer of EV-derived let-7-5p miR.

Placental glucose transporter (GLUT)-1 is down-regulated in preeclampsia

INTRODUCTIONnTransplacental fetal glucose supply is predominantly regulated by glucose transporter-1 (GLUT1). Altered expression and/or function of GLUT1 may affect the intrauterine environment, which could compromise fetal development and may contribute to fetal programming. To date it is unknown whether placental GLUT1 is affected by preeclampsia, which is often associated with intrauterine growth restriction (IUGR). We addressed the hypothesis that preeclampsia leads to decreased expression and function of placental GLUT1.nnnMETHODSnPlacentae were obtained following normal pregnancy and from pregnancies affected by preeclampsia. Washed villous tissue fragments were used to prepare syncytial microvillous (MVM) and basal plasma membranes (BM) microvesicles. GLUT1 protein and mRNA expression was assessed by western blot analysis and qPCR using Fast SYBR Green. A radio-labeled glucose up-take assay using placenta-derived syncytial microvesicles was used to analyze GLUT1 function.nnnRESULTSnGLUT1 protein expression was significantly down-regulated in (apical) MVM of the syncytiotrophoblast in preeclampsia (nxa0=xa06) compared to controls (nxa0=xa06) (0.40xa0±xa00.04 versus 1.00xa0±xa00.06, arbitrary units, Pxa0<xa00.001, Students t-test), while GLUT1 mRNA expression did not show a significant difference. In addition, the functional assay in syncytial microvesicles showed a significantly decreased glucose transport activity in preeclampsia (61.78xa0±xa06.48%, Pxa0<xa00.05) compared to controls. BM GLUT1 protein expression was unchanged and glucose up-take into BM microvesicles showed no differences between the preeclampsia and control groups.nnnDISCUSSIONnOur study shows for the first time that in preeclampsia placental GLUT1 expression and function are down-regulated at the apical plasma membrane of the syncytiotrophoblast. Further studies are needed to assess whether these changes occur also inxa0vivo and contribute to the development of IUGR in preeclampsia.

Mesenchymal stromal cells from umbilical cord Wharton's jelly trigger oligodendroglial differentiation in neural progenitor cells through cell-to-cell contact

BACKGROUND AIMSnWhartons jelly mesenchymal stromal cells (WJ-MSCs) might be ideal candidates to treat perinatal brain damage. Their secretome has been shown to have beneficial effects on neuroregeneration, in part through interaction with neural progenitor cells (NPCs). However, it remains unclear whether cell-to-cell contact decisively contributes to this positive effect. The objective of this study was to elucidate the mechanism through which differentiation in NPCs is triggered after exposure to WJ-MSCs. Furthermore, given that WJ-MSCs can be derived from term (tWJ-MSCs) or preterm (ptWJ-MSCs) deliveries and that WJ-MSCs might be used for transplantations independent of gestational age, the influence of tWJ-MSCs versus ptWJ-MSCs on the differentiation capacities of NPCs was studied.nnnMETHODSnThe effect of tWJ-MSCs and ptWJ-MSCs on the expression of neuroglial markers in NPCs was assessed in co-culture (CC), conditioned medium (CM) or transwell CC experiments by immunocytochemistry, real-time polymerase chain reaction and Western blot. Additionally, mass spectrometry was used to study their secretomes.nnnRESULTSnNPCs showed an increased expression of glial markers after CC with WJ-MSCs or exposure to WJ-MSC-CMs. CC had a more prominent effect on the expression of glial markers compared with CM or transwell CCs. tWJ-MSCs more strongly induced the expression of mature oligodendroglial markers compared with ptWJ-MSCs. A possible role in enhancing this maturation could be attributed to the laminin α2-subunit.nnnCONCLUSIONSnCell-to-cell contact between WJ-MSCs and NPCs induces oligodendrogenesis on NPCs, whereas trophic factor secretion is sufficient to promote astrogenesis. Thus, transplanting WJ-MSCs may promote endogenous neuroregeneration in perinatal brain damage.

797: Wharton's jelly stem cell-derived extracellular vesicles drive neural progenitor cells towards oligodendroglial identity

OBJECTIVE: Perinatal brain damage is accompanied by oligodendrocyte progenitor cell loss. The neuroregenerative effects of transplanted mesenchymal stem cells (MSC) in animal models of perinatal brain damage are presumed to rely on secreted factors such nas MSC-derived extracellular vesicles (EV). Thus, the aim of this study is to evaluate the capacity of EV from human Wharton’s jelly-derived MSC (WJ-MSC) to prime neural progenitor cells (NPC) towards oligodendroglial cell fate specification. nSTUDY DESIGN: WJ-MSC-derived EV were isolated from culture supernatants by serial high-speed and ultracentrifugations. EV microRNA (miRNA) content was assessed by real-time PCR. After the culture with WJ-MSC-derived EV, NPC were evaluated for the nexpression of markers involved in oligodendrogenic specification and differentiation by real-time PCR. nRESULTS: WJ-MSC-derived EV contained miRNA that are involved in oligodendrogenic cell fate determination and differentiation (miR-338, miR-9, miR-19b, miR-138). The expression of miR-338-3p and miR-219-5p, known to regulate oligodendrocyte specification and differentiation, were significantly increased in NPC after 72 h of co-culture with EV. Furthermore, the gene expression of the transcription factor neurogenic differentiation 1 (Neurod1), which blocks oligodendrogenic specification, was reduced in NPC after coculture with EV for 72 h. nCONCLUSION: In conclusion, we showed that isolated WJ-MSCderived EV contained miRNA having a role in oligodendrogenesis. Furthermore, EV primed NPC towards oligodendrogenic identity, ascribing a potential neuroregenerative role to WJ-MSC-derived EV.

798: Intranasal administration of extracellular vesicles derived from human umbilical cord mesenchymal stem cells as a potential treatment for perinatal brain damage

OBJECTIVE: Every year, an estimated 15 million babies are born preterm. Survivors of preterm birth are at risk to develop severe necrotizing enterocolitis, bronchopulmonary dysplasia and perinatal brain damage. The latter is caused by cerebral hypoxia-schemia (HI) and systemic maternal-fetal inflammation and leads to severe neurological adverse effects. A promising preclinical approach to treat perinatal brain damage is the administration of extracellular vesicles (EVs) derived from Wharton’s jelly mesenchymal stem cells (WJ-MSCs). The aim of this study is to evaluate the distribution of intranasally administered EVs in a rat model of perinatal brain damage. nSTUDY DESIGN: Consistent with the etiology of perinatal brain damage, 3-day old rat pups were injected with lipopolysaccharides (LPS) and subjected a unilateral carotid artery ligation followed by oxygen deprivation. EVs were isolated from WJ-MSCs culture supernatant by ultracentrifugation. EVs were labeled with an infrared nfluorescent dye and delivered intranasally to the rat pups. The distribution nof the EVs throughout the bodies of the pups was traced 30 min, 3h and 24h after their administration using an infrared imaging system. nRESULTS: EVs were diffusely located within the pup brains already 30 min after their administration, suggesting a rapid translocation of the EVs to the brain. The accumulation of the EVs within the brain reached its peak 3 h after their administration. No more EVs were present in the brain 24 h after administration. A small fraction of the labeled EVs were also detected within the lungs and the gastrointestinal (GI) tract since the rat pups aspirate and swallow some of the EVs during the intranasal administration. No EVs were found within the spleen, indicating that the EVs do not reach the systemic circulation. nCONCLUSION: Intranasal delivery of EVs represents a feasible approach to treat perinatal brain damage by effectively delivering EVs to the brain while bypassing the systemic circulation. Intranasal delivery of EVs could also be suitable to treat other preterm-associated complications such as bronchopulmonary dysplasia and severe nnecrotizing enterocolitis as it was shown that a fraction of EVs also reached the lungs and the GI tract.