Human Placenta-Derived Mesenchymal Stromal-Like Cells Enhance Angiogenesis via T Cell-Dependent Reprogramming of Macrophage Differentiation.

Peripheral arterial disease (PAD) is a leading cause of limb loss and mortality worldwide with limited treatment options. Mesenchymal stromal cell (MSC) therapy has demonstrated positive effects on angiogenesis in preclinical models and promising therapeutic efficacy signals in early stage clinical studies; however, the mechanisms underlying MSC-mediated angiogenesis remain largely undefined. Here, we investigated the mechanism of action of human placenta-derived MSC-like cells (PDA-002) in inducing angiogenesis using mice hind limb ischemia model. We showed that PDA-002 improved blood flow and promoted collateral vessel formation in the injured limb. Histological analysis demonstrated that PDA-002 increased M2-like macrophages in ischemic tissue. Analysis of the changes in functional T cell phenotype in the draining lymph nodes revealed that PDA-002 treatment was associated with the induction of cytokine and gene expression signatures of Th2 response. Angiogenic effect of PDA-002 was markedly reduced in Balb/c nude mice compared with wild type. This reduction in efficacy was reversed by T cell reconstitution, suggesting T cells are essential for PDA-002-mediated angiogenesis. Furthermore, effect of PDA-002 on macrophage differentiation was also T cell-dependent as a PDA-002-mediated M2-like macrophage skewing was only observed in wild type and T cell reconstituted nude mice, but not in nude mice. Finally, we showed that PDA-002-treated animals had enhanced angiogenic recovery in response to the second injury when PDA-002 no longer persisted in vivo. These results suggest that PDA-002 enhances angiogenesis through an immunomodulatory mechanism involving T cell-dependent reprogramming of macrophage differentiation toward M2-like phenotype. Stem Cells 2017;35:1603-1613.

Angiogenic properties of human placenta-derived adherent cells and efficacy in hindlimb ischemia.

OBJECTIVE: Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population from full-term placental tissue and were previously shown to possess anti-inflammatory and immunomodulatory properties. PDACs (formulated as PDA-002) are in clinical trials for peripheral arterial disease with diabetic foot ulcer. In the current study, we examined their angiogenic and tissue reparative properties. METHODS: The effects of PDACs on survival and tube formation of human umbilical vein endothelial cells (HUVECs) were tested using conditioned media and noncontact coculture. Angiogenic effects were assessed in the chick chorioallantoic membrane assay. Hindlimb ischemia (HLI) was induced in mice and rats by femoral artery transection, and blood flow and blood vessel density were monitored in vivo by laser Doppler and angiography in the ischemic and control limbs. Tissue damage and regeneration in HLI were examined in histologic sections of quadriceps muscle stained with hematoxylin and eosin, and newly synthesized blood vessels were detected by indoxyl-tetrazolium staining for alkaline phosphatase. RESULTS: PDACs enhanced the survival of serum-starved HUVECs and stimulated HUVEC tube formation, and in the chick chorioallantoic membrane assay, PDACs stimulated blood vessel formation. In HLI, intramuscular administration of PDACs resulted in improved blood flow and vascular density, and in quadriceps muscle, tissue regeneration and increased numbers of blood vessels were observed. CONCLUSIONS: PDACs exhibited various activities consistent with angiogenesis and tissue repair, supporting the continued investigation of this cell therapy as treatment for vascular disease-related indications.

Placenta Derived Adherent Cell (PDAC) Interaction and Response on Extracellular Matrix Isolated from Human Placenta t.

A method was developed to isolate extracellular matrix from the human placenta (pECM). The isolated material is composed primarily of collagen, in addition to, elastin, fibronectin, laminin, and glycosaminoglycans (GAGs). The pECM is isolated as a water insoluble paste. This paste can be molded into sheets, tubes, and other 3-D structures that are stable at room temperature. This report describes the interaction of the pluripotent progenitor cells (PDACs) with the isolated pECM. The stem cells used in this study are of human placental origin (placenta derived adherent cells or PDACs) and have a phenotype described as CD200+, CD105+, CD10+, CD34-, and CD45-. The PDACs bind to and proliferate on the pECM, and are stimulated to secrete soluble fibronectin. They actively assemble the soluble fibronectin into a complex network of detergent-insoluble extracellular matrix fibrils. While proliferating on the pECM, PDACs secrete key cytokines at levels well above that observed on tissue-treated tissue culture plates. These cytokines included monocyte chemoattractant protein (MCP-1), IL-6, and IL-8, all of which are important participants in wound healing processes. These results suggest the feasibility of designing a combination product of pECM with PDACs to augment repair processes in nonhealing deep wounds and in diabetic ulcers.

The Mechanism of Cell Interaction and Response on Decellularized Human Amniotic Membrane: Implications in Wound Healing.

ACELAGRAFT™ (Celgene Cellular Therapeutics, Cedar Knolls, NJ) was developed as a decellularized and dehydrated human amniotic membrane product (DDHAM). The product has demonstrated potential as a wound healing product with several ongoing preclinical and clinical studies in the area of acute and chronic ulcers. Although the mechanism of action of such a decellularized product has not been examined, a detailed study of the ability of fibroblasts to interact with DDHAM and subsequent cellular responses are presented. These studies indicate that the composition of DDHAM is that of an extracellular matrix (ECM)-like material with high collagen content, retaining key bioactive molecules, such as fibronectin, laminin, glycosaminoglycans (GAGs), and elastin. No cytokines or growth factors were identified as one might expect in a nondecellularized amniotic membrane product. Cell assays show that fibroblasts can recognize fibronectin in DDHAM and bind to it via typical integrin-fibronectin interactions. Fibroblasts secrete fibronectin and can actively assemble the soluble fibronectin into a complex extracellular matrix on DDHAM. Fibroblasts are also stimulated by DDHAM to secrete key proinflammatory(IL-1 and IL-6) and chemotactic cytokines or chemokines (proand IL-8) involved in regulating and enhancing wound repair processes. Microarray gene expression studies on fibroblasts bound to DDHAM show increased expression of key wound healing cytokines. Together, these studies provide insight into the mechanisms by which DDHAM may augment the wound healing process.

Modulation of Cell Attachment, Proliferation, and Angiogenesis by Decellularized, Dehydrated Human Amniotic Membrane in In Vitro Models.

BACKGROUND: Decellularized, dehydrated human amniotic membrane (DDHAM) is an extracellular matrix devoid of cells, cell debris, and growth factors. This study examines the effect of cell attachment to the DDHAM and the induced cellular responses. MATERIALS AND METHODS: The cell types employed in this study were human dermal fibroblasts (HDF), human epithelial keratinocytes (HEK), and human dermal microvascular endothelial cells (HDMEC), all of which play critical roles in the wound healing process. Further, the DDHAM was compared to a dehydrated human amnion/chorion membrane (dHACM), which contains and releases biological entities including growth factors and cytokines. The HDF and HEK were cultured on the DDHAM and the dHACM, and cell imaging and proliferation assays were performed to evaluate cell attachment to and the ability to proliferate on the DDHAM relative to the dHACM. In addition, the effect of soluble factors released by the DDHAM and the dHACM on cell survival, attachment, and proliferation were examined. The authors also evaluated the effect of soluble factors produced by culturing cells on the DDHAM in in vitro functional assays, including cell survival and endothelial cell migration in a wound closure angiogenesis assay. RESULTS: The HDF and HEK cells readily attached to and proliferated on the DDHAM, while the dHACM did not support cell attachment and proliferation when cultured under the same conditions. Soluble factors secreted when HDF were cultured on the DDHAM enhanced both endothelial cell and keratinocyte survival and endothelial cell migration in a wound closure assay. CONCLUSIONS: Although DDHAM is only an extracellular matrix and serves primarily as a scaffold, it has sufficient cues to allow for cell attachment and proliferation. Further, the biological entities released as a consequence of cell attachment promote cell survival and migration.

Characterization and ex vivo Expansion of Human Placenta-Derived Natural Killer Cells for Cancer Immunotherapy.

Recent clinical studies suggest that adoptive transfer of donor-derived natural killer (NK) cells may improve clinical outcome in hematological malignancies and some solid tumors by direct anti-tumor effects as well as by reduction of graft versus host disease (GVHD). NK cells have also been shown to enhance transplant engraftment during allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies. The limited ex vivo expansion potential of NK cells from peripheral blood (PB) or umbilical cord blood (UCB) has however restricted their therapeutic potential. Here we define methods to efficiently generate NK cells from donor-matched, full-term human placenta perfusate (termed Human Placenta-Derived Stem Cell, HPDSC) and UCB. Following isolation from cryopreserved donor-matched HPDSC and UCB units, CD56+CD3- placenta-derived NK cells, termed pNK cells, were expanded in culture for up to 3 weeks to yield an average of 1.2 billion cells per donor that were >80% CD56+CD3-, comparable to doses previously utilized in clinical applications. Ex vivo-expanded pNK cells exhibited a marked increase in anti-tumor cytolytic activity coinciding with the significantly increased expression of NKG2D, NKp46, and NKp44 (p < 0.001, p < 0.001, and p < 0.05, respectively). Strong cytolytic activity was observed against a wide range of tumor cell lines in vitro. pNK cells display a distinct microRNA (miRNA) expression profile, immunophenotype, and greater anti-tumor capacity in vitro compared to PB NK cells used in recent clinical trials. With further development, pNK may represent a novel and effective cellular immunotherapy for patients with high clinical needs and few other therapeutic options.