Lee Lankford

Placental Mesenchymal Stromal Cells Rescue Ambulation in Ovine Myelomeningocele

Myelomeningocele (MMC)—commonly known as spina bifida—is a congenital birth defect that causes lifelong paralysis, incontinence, musculoskeletal deformities, and severe cognitive disabilities. The recent landmark Management of Myelomeningocele Study (MOMS) demonstrated for the first time in humans that in utero surgical repair of the MMC defect improves lower limb motor function, suggesting a capacity for improved neurologic outcomes in this disorder. However, functional recovery was incomplete, and 58% of the treated children were unable to walk independently at 30 months of age. In the present study, we demonstrate that using early gestation human placenta‐derived mesenchymal stromal cells (PMSCs) to augment in utero repair of MMC results in significant and consistent improvement in neurologic function at birth in the rigorous fetal ovine model of MMC. In vitro, human PMSCs express characteristic MSC markers and trilineage differentiation potential. Protein array assays and enzyme‐linked immunosorbent assay show that PMSCs secrete a variety of immunomodulatory and angiogenic cytokines. Compared with adult bone marrow MSCs, PMSCs secrete significantly higher levels of brain‐derived neurotrophic factor and hepatocyte growth factor, both of which have known neuroprotective capabilities. In vivo, functional and histopathologic analysis demonstrated that human PMSCs mediate a significant, clinically relevant improvement in motor function in MMC lambs and increase the preservation of large neurons within the spinal cord. These preclinical results in the well‐established fetal ovine model of MMC provide promising early support for translating in utero stem cell therapy for MMC into clinical application for patients.

Early gestation chorionic villi-derived stromal cells for fetal tissue engineering

AIM To investigate the potential for early gestation placenta-derived mesenchymal stromal cells (PMSCs) for fetal tissue engineering. METHODS PMSCs were isolated from early gestation chorionic villus tissue by explant culture. Chorionic villus sampling (CVS)-size tissue samples (mean = 35.93 mg) were used to test the feasibility of obtaining large cell numbers from CVS within a clinically relevant timeframe. We characterized PMSCs isolated from 6 donor placentas by flow cytometry immunophenotyping, multipotency assays, and through immunofluorescent staining. Protein secretion from PMSCs was examined using two cytokine array assays capable of probing for over 70 factors in total. Delivery vehicle compatibility of PMSCs was determined using three common scaffold systems: fibrin glue, collagen hydrogel, and biodegradable nanofibrous scaffolds made from a combination of polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA). Viral transduction of PMSCs was performed using a Luciferase-GFP-containing lentiviral vector and efficiency of transduction was tested by fluorescent microscopy and flow cytometry analysis. RESULTS We determined that an average of 2.09 × 10(6) (SD ± 8.59 × 10(5)) PMSCs could be obtained from CVS-size tissue samples within 30 d (mean = 27 d, SD ± 2.28), indicating that therapeutic numbers of cells can be rapidly expanded from very limited masses of tissue. Immunophenotyping by flow cytometry demonstrated that PMSCs were positive for MSC markers CD105, CD90, CD73, CD44, and CD29, and were negative for hematopoietic and endothelial markers CD45, CD34, and CD31. PMSCs displayed trilineage differentiation capability, and were found to express developmental transcription factors Sox10 and Sox17 as well as neural-related structural proteins NFM, Nestin, and S100β. Cytokine arrays revealed a robust and extensive profile of PMSC-secreted cytokines and growth factors, and detected 34 factors with spot density values exceeding 10(3). Detected factors had widely diverse functions that include modulation of angiogenesis and immune response, cell chemotaxis, cell proliferation, blood vessel maturation and homeostasis, modulation of insulin-like growth factor activity, neuroprotection, extracellular matrix degradation and even blood coagulation. Importantly, PMSCs were also determined to be compatible with both biological and synthetic material-based delivery vehicles such as collagen and fibrin hydrogels, and biodegradable nanofiber scaffolds made from a combination of PLA and PLGA. Finally, we demonstrated that PMSCs can be efficiently transduced (> 95%) with a Luciferase-GFP-containing lentiviral vector for future in vivo cell tracking after transplantation. CONCLUSION Our findings indicate that PMSCs represent a unique source of cells that can be effectively utilized for in utero cell therapy and tissue engineering.

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

Introduction: Fetal 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. Methods: Fetal 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. Results: Histopathologic 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. Discussion: This 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.

Manufacture and preparation of human placenta-derived mesenchymal stromal cells for local tissue delivery

BACKGROUND In this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech). METHODS PMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA). RESULTS PMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3-5 × 105 cells/cm2, and seeded cells were >95% viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots. DISCUSSION PMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities.

The key components of Schwann cell-like differentiation medium and their effects on gene expression pattern of adipose-derived stem cells.

BackgroundSchwann cell-like cells differentiated from adipose-derived stem cells may have an important role in peripheral nerve regeneration. Herein, we document the individual effects of growth factors in Schwann cell-like differentiation medium. MethodsThere were 6 groups in the study. In the control group, we supplemented the rat adipose–derived stem cells with normal cell culture medium. In group 1, we fed the cells with Schwann cell-like differentiation medium (normal cell culture medium supplemented with platelet-derived growth factor, basic fibroblast growth factor, forskolin, and glial growth factor). In the other groups, we removed the components of the medium one at a time from the differentiation medium so that group 2 lacked glial growth factor, group 3 lacked forskolin, group 4 lacked basic fibroblast growth factor, and group 5 lacked platelet-derived growth factor. We examined the expression of the Schwann cell–specific genes with quantitative reverse transcription polymerase chain reaction and immunofluorescence staining in each group. ResultsGroups 3 and 4, lacking forskolin and basic fibroblast growth factor, respectively, had the highest expression levels of integrin-&bgr;4, and p75. Group 1 showed a 3.2-fold increase in the expression of S100, but the expressions of integrin-&bgr;4 and p75 were significantly lower compared to groups 3 and 4. Group 2 [glial growth factor (−)] did not express significant levels of Schwann cell–specific genes. The gene expression profile in group 4 most closely resembled Schwann cells. Immunofluorescence staining results were parallel with the quantitative real-time polymerase chain reaction results. ConclusionsGlial growth factor is a key component of Schwann cell-like differentiation medium.

Human amnion extracellular matrix derived bioactive hydrogel for cell delivery and tissue engineering

Author(s): Ryzhuk, Volodymyr; Zeng, Xu-Xin; Wang, Xijun; Melnychuk, Veniamin; Lankford, Lee; Farmer, Diana; Wang, Aijun

Isolation and characterization of canine placenta-derived mesenchymal stromal cells for the treatment of neurological disorders in dogs

Spinal cord injury (SCI) is a devastating disorder that affects humans and dogs. The prognosis of SCI depends on the severity of the injury and can include varying levels of motor and sensory deficits including devastating paraplegia and quadriplegia. Placental mesenchymal stromal cells (PMSCs) have been shown to improve wound healing and possess neuroprotective and immunomodulatory capabilities, but have not yet been clinically tested for the treatment of SCI. This study established a protocol to isolate fetal PMSCs from canine placentas and characterized their paracrine secretion profile and ability to stimulate neurons in vitro to assess their potential as a treatment option for neurological disorders in dogs. Canine PMSCs (cPMSCs) were plastic adherent and capable of trilineage differentiation. cPMSCs expressed typical MSC markers and did not express hematopoietic or endothelial cell markers. Genotyping of cPMSCs revealed fetal rather than maternal origin of the cells. cPMSCs were viable and mitotically expansive in a collagen hydrogel delivery vehicle, and they secreted the immunomodulatory and neurotrophic paracrine factors interleukin (IL)‐6, IL‐8, monocyte chemoattractant protein 1 (MCP‐1), and vascular endothelial growth factor (VEGF). cPMSCs also stimulated the growth of complex neural networks when co‐cultured with SH‐SY5Y cells, a neuroblastoma cell line used to model neuron growth in vitro. cPMSCs are analogous to human PMSCs. They meet the criteria to be defined as MSCs and represent a potential regenerative therapy option for neurological disorders in dogs with their robust growth in collagen hydrogel, stimulation of neural network formation, and secretion of potent paracrine factors.

Placenta as a Source of Stem Cells for Regenerative Medicine

The development of effective cell transplantation therapies is currently the focus of biomedical research worldwide, and numerous cell types and sources have been explored for applications in regenerative medicine. The placenta is a unique organ of fetomaternal origin that plays an important role in fetal development, and multiple types of placenta-derived stem cells show great promise for application in regenerative medicine. This review concisely summarizes the recent proceedings on the characterization, biological properties, and applications of mesenchymal stem cells that have been isolated from various anatomic regions of the placenta and at different gestational ages. In addition, this review summarizes recent studies that have isolated amniotic epithelial cells, hematopoietic stem cells, and trophoblast stem cells from placental tissue.

Fetal surgical repair with placenta-derived mesenchymal stromal cell engineered patch in a rodent model of myelomeningocele

PURPOSE The purpose of this study is to determine the feasibility of fetal surgical repair of myelomeningocele (MMC) in a rodent model using human placental mesenchymal stromal cells (PMSCs) seeded onto extracellular matrix (ECM) and to characterize the resulting changes in spinal cord tissue. METHODS Fetal rodents with retinoic acid (RA) induced MMC underwent surgical repair of the MMC defect using an ECM patch on embryonic age (EA) 19 and were collected via caesarean section on EA 21. Various seeding densities of PMSC-ECM and ECM only controls were evaluated. Cross-sectional compression (width/height) and apoptotic cell density of the lumbosacral spinal cord were analyzed. RESULTS 67 dams treated with 40mg/kg of RA resulted in 352 pups with MMC defects. 121 pups underwent MMC repair, and 105 (86.8%) survived to term. Unrepaired MMC pups had significantly greater cord compression and apoptotic cell density compared to normal non-MMC pups. Pups treated with PMSC-ECM had significantly less cord compression and demonstrated a trend towards decreased apoptotic cell density compared to pups treated with ECM only. CONCLUSION Surgical repair of MMC with a PMSC-seeded ECM disc is feasible with a postoperative survival rate of 86.8%. Fetal rodents repaired with PMSC-ECM have significantly less cord deformity and decreased histological evidence of apoptosis compared to ECM only controls.

High density placental mesenchymal stromal cells provide neuronal preservation and improve motor function following in utero treatment of ovine myelomeningocele

PURPOSE The purpose of this study was to determine whether seeding density of placental mesenchymal stromal cells (PMSCs) on extracellular matrix (ECM) during in utero repair of myelomeningocele (MMC) affects motor function and neuronal preservation in the ovine model. METHODS MMC defects were surgically created in 33 fetuses and repaired following randomization into four treatment groups: ECM only (n = 10), PMSC-ECM (42 K cells/cm2) (n = 8), PMSC-ECM (167 K cells/cm2) (n = 7), or PMSC-ECM (250-300 K cells/cm2) (n = 8). Motor function was evaluated using the Sheep Locomotor Rating Scale (SLR). Serial sections of the lumbar spinal cord were analyzed by measuring their cross-sectional areas which were then normalized to normal lambs. Large neurons (LN, diameter 30-70 μm) were counted manually and density calculated per mm2 gray matter. RESULTS Lambs treated with PMSCs at any density had a higher median SLR score (15 [IQR 13.5-15]) than ECM alone (6.5 [IQR 4-12.75], p = 0.036). Cross-sectional areas of spinal cord and gray matter were highest in the PMSC-ECM (167 K/cm2) group (p = 0.002 and 0.006, respectively). LN density was highest in the greatest density PMSC-ECM (250-300 K/cm2) group (p = 0.045) which positively correlated with SLR score (r = 0.807, p < 0.0001). CONCLUSIONS Fetal repair of myelomeningocele with high density PMSC-ECM resulted in increased large neuron density, which strongly correlated with improved motor function. TYPE OF STUDY Basic science. LEVEL OF EVIDENCE N/A.