In Utero Transplantation of Placenta-Derived Mesenchymal Stromal Cells for Potential Fetal Treatment of Hemophilia A.

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the factor VIII ( FVIII) gene leading to deficient blood coagulation. The current standard of care is frequent infusions of plasma-derived FVIII or recombinant B-domain-deleted FVIII (BDD-FVIII). While this treatment is effective, many patients eventually develop FVIII inhibitors that limit the effectiveness of the infused FVIII. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Several studies have investigated allogeneic stem cell therapy targeting in utero or postnatal treatment of HA but have not been successful in completely correcting HA. Autologous in utero transplantation of mesenchymal stem cells is promising for treatment of HA due to the naive immune status of the fetal environment as well as its potential to prevent transplant rejection and long-term FVIII inhibitor formation. HA can be diagnosed by chorionic villus sampling performed during the first trimester (10 to 13 wk) of gestation. In this study, we used an established protocol and isolated placenta-derived mesenchymal stromal cells (PMSCs) from first trimester chorionic villus tissue and transduced them with lentiviral vector encoding the BDD-FVIII gene. We show that gene-modified PMSCs maintain their immunophenotype and multipotency, express, and secrete high levels of active FVIII. PMSCs were then transplanted at embryonic day 14.5 (E14.5) into wild-type fetuses from time-mated pregnant mice. Four days after birth, pups were checked for engraftment, and varying levels of expression of human green fluorescent protein were found in the organs tested. This study shows feasibility of the approach to obtain PMSCs from first trimester chorionic villus tissue, genetically modify them with the FVIII gene, and transplant them in utero for cell-mediated gene therapy of HA. Future studies will involve evaluation of long-term engraftment, phenotypic correction in HA mice, and prevention of FVIII inhibitor development by this approach

Placental Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Myelin Regeneration in an Animal Model of Multiple Sclerosis.

Mesenchymal stem/stromal cells (MSCs) display potent immunomodulatory and regenerative capabilities through the secretion of bioactive factors, such as proteins, cytokines, chemokines as well as the release of extracellular vesicles (EVs). These functional properties of MSCs make them ideal candidates for the treatment of degenerative and inflammatory diseases, including multiple sclerosis (MS). MS is a heterogenous disease that is typically characterized by inflammation, demyelination, gliosis and axonal loss. In the current study, an induced experimental autoimmune encephalomyelitis (EAE) murine model of MS was utilized. At peak disease onset, animals were treated with saline, placenta-derived MSCs (PMSCs), as well as low and high doses of PMSC-EVs. Animals treated with PMSCs and high-dose PMSC-EVs displayed improved motor function outcomes as compared to animals treated with saline. Symptom improvement by PMSCs and PMSC-EVs led to reduced DNA damage in oligodendroglia populations and increased myelination within the spinal cord of treated mice. In vitro data demonstrate that PMSC-EVs promote myelin regeneration by inducing endogenous oligodendrocyte precursor cells to differentiate into mature myelinating oligodendrocytes. These findings support that PMSCs' mechanism of action is mediated by the secretion of EVs. Therefore, PMSC-derived EVs are a feasible alternative to cellular based therapies for MS, as demonstrated in an animal model of the disease

In utero repair of myelomeningocele with autologous amniotic membrane in the fetal lamb model

BackgroundDespite advances in prenatal repair, myelomeningocele (MMC) still produces devastating neurologic deficits. The amniotic membranes (AM) are a biologically active tissue that has been used anecdotally for human fetal MMC repair. This study evaluated the use of autologous AM compared to skin closure in an established fetal MMC model.MethodsSeven fetal lambs underwent surgical creation of MMC at gestational age of 75days followed by in utero repair at gestational age of 100days. Lambs were repaired with an autologous AM patch followed by skin closure (n=4) or skin closure alone (n=3). Gross necropsy and histopathology of the spinal cords were performed at term to assess neuronal preservation at the lesion.ResultsAn increase in preserved motor neurons and a larger area of spinal cord tissue were seen in AM-repaired lambs, as was decreased wound healing of the overlying skin. Loss of nearly all spinal cord tissue with limited motor neuron preservation was seen in skin only-repaired lambs.ConclusionsAM-repaired lambs showed increased protection of spinal cord tissue compared to skin only-repaired lambs, but the overlying skin failed to close in AM-repaired lambs. These results suggest a potential role for AM in fetal MMC repair that warrants further study

In Utero Transplantation of Placenta-Derived Mesenchymal Stromal Cells for Potential Fetal Treatment of Hemophilia A.

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the factor VIII ( FVIII) gene leading to deficient blood coagulation. The current standard of care is frequent infusions of plasma-derived FVIII or recombinant B-domain-deleted FVIII (BDD-FVIII). While this treatment is effective, many patients eventually develop FVIII inhibitors that limit the effectiveness of the infused FVIII. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Several studies have investigated allogeneic stem cell therapy targeting in utero or postnatal treatment of HA but have not been successful in completely correcting HA. Autologous in utero transplantation of mesenchymal stem cells is promising for treatment of HA due to the naive immune status of the fetal environment as well as its potential to prevent transplant rejection and long-term FVIII inhibitor formation. HA can be diagnosed by chorionic villus sampling performed during the first trimester (10 to 13 wk) of gestation. In this study, we used an established protocol and isolated placenta-derived mesenchymal stromal cells (PMSCs) from first trimester chorionic villus tissue and transduced them with lentiviral vector encoding the BDD-FVIII gene. We show that gene-modified PMSCs maintain their immunophenotype and multipotency, express, and secrete high levels of active FVIII. PMSCs were then transplanted at embryonic day 14.5 (E14.5) into wild-type fetuses from time-mated pregnant mice. Four days after birth, pups were checked for engraftment, and varying levels of expression of human green fluorescent protein were found in the organs tested. This study shows feasibility of the approach to obtain PMSCs from first trimester chorionic villus tissue, genetically modify them with the FVIII gene, and transplant them in utero for cell-mediated gene therapy of HA. Future studies will involve evaluation of long-term engraftment, phenotypic correction in HA mice, and prevention of FVIII inhibitor development by this approach

Myosin Light Chain Kinase Signaling in Endothelial Barrier Dysfunction

Microvascular barrier dysfunction is a serious problem that occurs in many inflammatory conditions, including sepsis, trauma, ischemia–reperfusion injury, cardiovascular disease, and diabetes. Barrier dysfunction permits extravasation of serum components into the surrounding tissue, leading to edema formation and organ failure. The basis for microvascular barrier dysfunction is hyperpermeability at endothelial cell–cell junctions. Endothelial hyperpermeability is increased by actomyosin contractile activity in response to phosphorylation of myosin light chain by myosin light chain kinase (MLCK). MLCK-dependent endothelial hyperpermeability occurs in response to inflammatory mediators (e.g., activated neutrophils, thrombin, histamine, tumor necrosis factor alpha, etc.), through multiple cell signaling pathways and signaling molecules (e.g., Ca++, protein kinase C, Src kinase, nitric oxide synthase, etc.). Other signaling molecules protect against MLCK-dependent hyperpermeability (e.g., sphingosine-1-phosphate or cAMP). In addition, individual MLCK isoforms play specific roles in endothelial barrier dysfunction, suggesting that isoform-specific inhibitors could be useful for treating inflammatory disorders and preventing multiple organ failure. Because endothelial barrier dysfunction depends upon signaling through MLCK in many instances, MLCK-dependent signaling comprises multiple potential therapeutic targets for preventing edema formation and multiple organ failure. The following review is a discussion of MLCK-dependent mechanisms and cell signaling events that mediate endothelial hyperpermeability. © 2012 Wiley Periodicals, Inc. Med. Res. Rev., 00, No. 00, 1-23, 201

Age Does Matter: A Pilot Comparison of Placenta-Derived Stromal Cells for in utero Repair of Myelomeningocele Using a Lamb Model.

IntroductionFetal amniotic membranes (FM) have been shown to preserve spinal cord histology in the fetal sheep model of myelomeningocele (MMC). This study compares the effectiveness of placenta-derived mesenchymal stromal cells (PMSCs) from early-gestation versus term-gestation placenta to augment FM repair to improve distal motor function in a sheep model.MethodsFetal lambs (n = 4) underwent surgical MMC creation followed by repair with FM patch with term-gestation PMSCs (n = 1), FM with early-gestation PMSCs (n = 1), FM only (n = 1), and skin closure only (n = 1). Histopathology and motor assessment was performed.ResultsHistopathologic analysis demonstrated increased preservation of spinal cord architecture and large neurons in the lamb repaired with early-gestation cells compared to all others. Lambs repaired with skin closure only, FM alone, and term-gestation PMSCs exhibited extremely limited distal motor function; the lamb repaired with early-gestation PMSCs was capable of normal ambulation.DiscussionThis pilot study is the first in vivo comparison of different gestational-age placenta-derived stromal cells for repair in the fetal sheep MMC model. The preservation of large neurons and markedly improved motor function in the lamb repaired with early-gestation cells suggest that early-gestation placental stromal cells may exhibit unique properties that augment in utero MMC repair to improve paralysis

Age Does Matter: A Pilot Comparison of Placenta-Derived Stromal Cells for in utero Repair of Myelomeningocele Using a Lamb Model.

IntroductionFetal amniotic membranes (FM) have been shown to preserve spinal cord histology in the fetal sheep model of myelomeningocele (MMC). This study compares the effectiveness of placenta-derived mesenchymal stromal cells (PMSCs) from early-gestation versus term-gestation placenta to augment FM repair to improve distal motor function in a sheep model.MethodsFetal lambs (n = 4) underwent surgical MMC creation followed by repair with FM patch with term-gestation PMSCs (n = 1), FM with early-gestation PMSCs (n = 1), FM only (n = 1), and skin closure only (n = 1). Histopathology and motor assessment was performed.ResultsHistopathologic analysis demonstrated increased preservation of spinal cord architecture and large neurons in the lamb repaired with early-gestation cells compared to all others. Lambs repaired with skin closure only, FM alone, and term-gestation PMSCs exhibited extremely limited distal motor function; the lamb repaired with early-gestation PMSCs was capable of normal ambulation.DiscussionThis pilot study is the first in vivo comparison of different gestational-age placenta-derived stromal cells for repair in the fetal sheep MMC model. The preservation of large neurons and markedly improved motor function in the lamb repaired with early-gestation cells suggest that early-gestation placental stromal cells may exhibit unique properties that augment in utero MMC repair to improve paralysis

Placental Mesenchymal Stem Cells and Extracellular Vesicles on an Extracellular Matrix ImprovedMotor Function Recovery After Acute Spinal Cord Injury

Spinal cord injury (SCI) is a devasting disease with no effective cure. We have shown that placental mesenchymal stromal cells (PMSCs) applied in utero improve ambulatory function in an ovine model of spina bifida and have begun the first-inhuman clinical trial for fetal spina bifida. PMSCs and extracellular vesicles (EVs)have neuroprotective properties and we hypothesized that PMSCs and PMSC-EVs would provide a similar neuroprotective effect in SCI

Recovering fetal signals transabdominally through interferometric near-infrared spectroscopy (iNIRS).

Noninvasive transabdominal fetal pulse oximetry can provide clinicians critical assessment of fetal health and potentially contribute to improved management of childbirth. Conventional pulse oximetry through continuous wave (CW) light has challenges measuring the signals from deep tissue and separating the weak fetal signal from the strong maternal signal. Here, we propose a new approach for transabdominal fetal pulse oximetry through interferometric near-infrared spectroscopy (iNIRS). This approach provides pathlengths of photons traversing the tissue, which facilitates the extraction of fetal signals by rejecting the very strong maternal signal from superficial layers. We use a multimode fiber combined with a mode-field converter at the detection arm to boost the signal of iNIRS. Together, we can detect signals from deep tissue (>∼1.6 cm in sheep abdomen and in human forearm) at merely 1.1 cm distance from the source. Using a pregnant sheep model, we experimentally measured and extracted the fetal heartbeat signals originating from deep tissue. This validated a key step towards transabdominal fetal pulse oximetry through iNIRS and set a foundation for further development of this method to measure the fetal oxygen saturation