Deryl L. Troyer

Human umbilical cord matrix stem cells : Preliminary characterization and effect of transplantation in a rodent model of parkinson's disease

The umbilical cord contains an inexhaustible, noncontroversial source of stem cells for therapy. In the U.S., stem cells found in the umbilical cord are routinely placed into bio‐hazardous waste after birth. Here, stem cells derived from human umbilical cord Whartons Jelly, called umbilical cord matrix stem (UCMS) cells, are characterized. UCMS cells have several properties that make them of interest as a source of cells for therapeutic use. For example, they 1) can be isolated in large numbers, 2) are negative for CD34 and CD45, 3) grow robustly and can be frozen/thawed, 4) can be clonally expanded, and 5) can easily be engineered to express exogenous proteins. UCMS cells have genetic and surface markers of mesenchymal stem cells (positive for CD10, CD13, CD29, CD44, and CD90 and negative for CD14, CD33, CD56, CD31, CD34, CD45, and HLA‐DR) and appear to be stable in terms of their surface marker expression in early passage (passages 4–8). Unlike traditional mesenchymal stem cells derived from adult bone marrow stromal cells, small populations of UCMS cells express endoglin (SH2, CD105) and CD49e at passage 8. UCMS cells express growth factors and angiogenic factors, suggesting that they may be used to treat neurodegenerative disease. To test the therapeutic value of UCMS cells, undifferentiated human UCMS cells were transplanted into the brains of hemiparkinsonian rats that were not immune‐suppressed. UCMS cells ameliorated apomorphine‐induced rotations in the pilot test. UCMS cells transplanted into normal rats did not produce brain tumors, rotational behavior, or a frank host immune rejection response. In summary, the umbilical cord matrix appears to be a rich, noncontroversial, and inexhaustible source of primitive mesenchymal stem cells.

Matrix cells from Wharton's jelly form neurons and glia.

We have identified an easily attainable source of primitive, potentially multipotent stem cells from Whartons jelly, the matrix of umbilical cord. Whartons jelly cells have been propagated in culture for more than 80 population doublings. Several markers for stem cells, including c‐kit (CD117), and telomerase activity are expressed in these cells. Treatment with basic fibroblast growth factor overnight and low‐serum media plus butylated hydroxyanisole and dimethylsulfoxide induced Whartons jelly cells to express a neural phenotype. Within several hours of this treatment, Whartons jelly cells developed rounded cell bodies with multiple neurite‐like extensions, similar to the morphology of neural stem cells. Neuron‐specific enolase (NSE), a neural stem cell marker, was expressed in these cells, as shown by immunocytochemistry. Immunoblot analysis showed similar levels of NSE expression in both untreated and induced Whartons jelly cells. After 3 days, the induced Whartons jelly cells resembled bipolar or multipolar neurons, with processes that formed networks reminiscent of primary cultures of neurons. The neuron‐like cells in these cultures stained positively for several neuronal proteins, including neuron‐specific class III β‐tubulin, neurofilament M, an axonal growth‐cone‐associated protein, and tyrosine hydroxylase. Immunoblot analysis showed increasing levels of protein markers for mature neurons over time postinduction. Markers for oligodendrocytes and astrocytes were also detected in Whartons jelly cells. These exciting findings show that cells from the matrix of umbilical cord have properties of stem cells and may, thus, be a rich source of primitive cells. This study shows their capacity to differentiate into a neural phenotype in vitro.

Concise Review: Wharton's Jelly-Derived Cells Are a Primitive Stromal Cell Population

Here, the literature was reviewed to evaluate whether a population of mesenchymal stromal cells derived from Whartons jelly cells (WJCs) is a primitive stromal population. A clear case can be made for WJCs as a stromal population since they display the characteristics of MSCs as defined by the International Society for Cellular Therapy; for example, they grow as adherent cells with mesenchymal morphology, they are self‐renewing, they express cell surface markers displayed by MSCs, and they may be differentiated into bone, cartilage, adipose, muscle, and neural cells. Like other stromal cells, WJCs support the expansion of other stem cells, such as hematopoietic stem cells, are well‐tolerated by the immune system, and they have the ability to home to tumors. In contrast to bone marrow MSCs, WJCs have greater expansion capability, faster growth in vitro, and may synthesize different cytokines. WJCs are therapeutic in several different pre‐clinical animal models of human disease such as neurodegenerative disease, cancer, heart disease, etc. The preclinical work suggests that the WJCs are therapeutic via trophic rescue and immune modulation. In summary, WJCs meet the definition of MSCs. Since WJCs expand faster and to a greater extent than adult‐derived MSCs, these findings suggest that WJCs are a primitive stromal cell population with therapeutic potential. Further work is needed to determine whether WJCs engraft long‐term and display self‐renewal and multipotency in vivo and, as such, demonstrate whether Whartons jelly cells are a true stem cell population.

Immune Properties of Human Umbilical Cord Wharton's Jelly-Derived Cells

Cells isolated from Whartons jelly, referred to as umbilical cord matrix stromal (UCMS) cells, adhere to a tissue‐culture plastic substrate, express mesenchymal stromal cell (MSC) surface markers, self‐renew, and are multipotent (differentiate into bone, fat, cartilage, etc.) in vitro. These properties support the notion that UCMS cells are a member of the MSC family. Here, the immune properties of UCMS cells are characterized in vitro. The overall hypothesis is that UCMS cells possess immune properties that would be permissive to allogeneic transplantation. For example, UCMS cells will suppress of the proliferation of “stimulated” lymphocytes (immune suppression) and have reduced immunogenicity (e.g., would be poor stimulators of allogeneic lymphocyte proliferation). Hypothesis testing was as follows: first, the effect on proliferation of coculture of mitotically inactivated human UCMS cells with concanavalin‐A‐stimulated rat splenocytes was assessed in three different assays. Second, the effect of human UCMS cells on one‐way and two‐way mixed lymphocyte reaction (MLR) assays was determined. Third, the expression of human leukocyte antigen (HLA)‐G was examined in human UCMS cells using reverse transcription‐polymerase chain reaction, since HLA‐G expression conveys immune regulatory properties at the maternal‐fetal interface. Fourth, the expression of CD40, CD80, and CD86 was determined by flow cytometry. Fifth, the cytokine expression of UCMS cells was evaluated by focused gene array. The results indicate that human UCMS cells inhibit splenocyte proliferation response to concanavalin A stimulation, that they do not stimulate T‐cell proliferation in a one‐way MLR, and that they inhibit the proliferation of stimulated T cells in a two‐way MLR. Human UCMS cells do not inhibit nonstimulated splenocyte proliferation, suggesting specificity of the response. UCMS cells express mRNA for pan‐HLA‐G. UCMS cells do not express the costimulatory surface antigens CD40, CD80, and CD86. UCMS cells express vascular endothelial growth factor and interleukin‐6, molecules previously implicated in the immune modulation observed in MSCs. In addition, the array data indicate that UCMS cells make a cytokine and other factors that may support hematopoiesis. Together, these results support previous observations made following xenotransplantation; for example, there was no evidence of frank immune rejection of undifferentiated UCMS cells. The results suggest that human UCMS will be tolerated in allogeneic transplantation.

Stem cells in the umbilical cord.

Stem cells are the next frontier in medicine. Stem cells are thought to have great therapeutic and biotechnological potential. This will not only to replace damaged or dysfunctional cells, but also rescue them and/or deliver therapeutic proteins after they have been engineered to do so. Currently, ethical and scientific issues surround both embryonic and fetal stem cells and hinder their widespread implementation. In contrast, stem cells recovered postnatally from the umbilical cord, including the umbilical cord blood cells, amnion/placenta, umbilical cord vein, or umbilical cord matrix cells, are a readily available and inexpensive source of cells that are capable of forming many different cell types (i.e., they are “multipotent”). This review will focus on the umbilical cord-derived stem cells and compare those cells with adult bone marrow-derived mesenchymal stem cells.

Expression of early transcription factors Oct-4, Sox-2 and Nanog by porcine umbilical cord (PUC) matrix cells

BackgroundThree transcription factors that are expressed at high levels in embryonic stem cells (ESCs) are Nanog, Oct-4 and Sox-2. These transcription factors regulate the expression of other genes during development and are found at high levels in the pluripotent cells of the inner cell mass. The downregulation of these three transcription factors correlates with the loss of pluripotency and self-renewal, and the beginning of subsequent differentiation steps. The roles of Nanog, Oct-4 and Sox-2 have not been fully elucidated. They are important in embryonic development and maintenance of pluripotency in ESCs. We studied the expression of these transcription factors in porcine umbilical cord (PUC) matrix cells.MethodsCells were isolated from Whartons jelly of porcine umbilical cords (PUC) and histochemically assayed for the presence of alkaline phosphatase and the presence of Nanog, Oct-4 and Sox-2 mRNA and protein. PCR amplicons were sequenced and compared with known sequences. The synthesis of Oct-4 and Nanog protein was analyzed using immunocytochemistry. FACS analysis was utilized to evaluate Hoechst 33342 dye-stained cells.ResultsPUC isolates were maintained in culture and formed colonies that express alkaline phosphatase. FACS analysis revealed a side population of Hoechst dye-excluding cells, the Hoechst exclusion was verapamil sensitive. Quantitative and non-quantitative RT-PCR reactions revealed expression of Nanog, Oct-4 and Sox-2 in day 15 embryonic discs, PUC cell isolates and porcine fibroblasts. Immunocytochemical analysis detected Nanog immunoreactivity in PUC cell nuclei, and faint labeling in fibroblasts. Oct-4 immunoreactivity was detected in the nuclei of some PUC cells, but not in fibroblasts.ConclusionCells isolated from PUC express three transcription factors found in pluripotent stem cell markers both at the mRNA and protein level. The presence of these transcription factors, along with the other characteristics of PUC cells such as their colony-forming ability, Hoechst dye-excluding side population and alkaline phosphatase expression, suggests that PUC cells have properties of primitive pluripotent stem cells. Furthermore, PUC cells are an easily and inexpensively obtained source of stem cells that are not hampered by the ethical or legal issues associated with ESCs. In addition, these cells can be cryogenically stored and expanded.

A/C magnetic hyperthermia of melanoma mediated by iron(0)/iron oxide core/shell magnetic nanoparticles: a mouse study

BackgroundThere is renewed interest in magnetic hyperthermia as a treatment modality for cancer, especially when it is combined with other more traditional therapeutic approaches, such as the co-delivery of anticancer drugs or photodynamic therapy.MethodsThe influence of bimagnetic nanoparticles (MNPs) combined with short external alternating magnetic field (AMF) exposure on the growth of subcutaneous mouse melanomas (B16-F10) was evaluated. Bimagnetic Fe/Fe3O4 core/shell nanoparticles were designed for cancer targeting after intratumoral or intravenous administration. Their inorganic center was protected against rapid biocorrosion by organic dopamine-oligoethylene glycol ligands. TCPP (4-tetracarboxyphenyl porphyrin) units were attached to the dopamine-oligoethylene glycol ligands.ResultsThe magnetic hyperthermia results obtained after intratumoral injection indicated that micromolar concentrations of iron given within the modified core-shell Fe/Fe3O4 nanoparticles caused a significant anti-tumor effect on murine B16-F10 melanoma with three short 10-minute AMF exposures. We also observed a decrease in tumor size after intravenous administration of the MNPs followed by three consecutive days of AMF exposure 24 hrs after the MNPs injection.ConclusionsThese results indicate that intratumoral administration of surface modified MNPs can attenuate mouse melanoma after AMF exposure. Moreover, we have found that after intravenous administration of micromolar concentrations, these MNPs are capable of causing an anti-tumor effect in a mouse melanoma model after only a short AMF exposure time. This is a clear improvement to state of the art.

Method to isolate mesenchymal-like cells from Wharton's Jelly of umbilical cord

The umbilical cord is a noncontroversial source of mesenchymal-like stem cells. Mesenchymal-like cells are found in several tissue compartments of the umbilical cord, placenta, and decidua. Here, we confine ourselves to discussing mesenchymal-like cells derived from Whartons Jelly, called umbilical cord matrix stem cells (UCMSCs). Work from several laboratories shows that these cells have therapeutic potential, possibly as a substitute cell for bone marrow-derived mesenchymal stem cells for cellular therapy. There have been no head-to-head comparisons between mesenchymal cells derived from different sources for therapy; therefore, their relative utility is not understood. In this chapter, the isolation protocols of the Whartons Jelly-derived mesenchymal cells are provided as are protocols for their in vitro culturing and storage. The cell culture methods provided will enable basic scientific research on the UCMSCs. Our vision is that both umbilical cord blood and UCMSCs will be commercially collected and stored in the future for preclinical work, public and private banking services, etc. While umbilical cord blood banking standard operating procedures exist, the scenario mentioned above requires clinical-grade UCMSCs. The hurdles that have been identified for the generation of clinical-grade umbilical cord-derived mesenchymal cells are discussed.

Development of human umbilical cord matrix stem cell-based gene therapy for experimental lung tumors

Umbilical cord matrix stem (UCMS) cells are unique stem cells derived from Whartons jelly, which have been shown to express genes characteristic of primitive stem cells. To test the safety of these cells, human UCMS cells were injected both intravenously and subcutaneously in large numbers into severe combined immunodeficiency (SCID) mice and multiple tissues were examined for evidence of tumor formation. UCMS cells did not form gross or histological teratomas up to 50 days posttransplantation. Next, to evaluate whether UCMS cells could selectively engraft in xenotransplanted tumors, MDA 231 cells were intravenously transplanted into SCID mice, followed by intravenous transplantation of UCMS cells 1 and 2 weeks later. UCMS cells were found near or within lung tumors but not in other tissues. Finally, UCMS cells were engineered to express human interferon beta – designated ‘UCMS−IFN-β’. UCMS−IFN-β cells were intravenously transplanted at multiple intervals into SCID mice bearing MDA 231 tumors and their effect on tumors was examined. UCMS−IFN-β cells significantly reduced MDA 231 tumor burden in SCID mouse lungs indicated by wet weight. These results clearly indicate safety and usability of UCMS cells in cancer gene therapy. Thus, UCMS cells can potentially be used for targeted delivery of cancer therapeutics.

Transplantation of porcine umbilical cord matrix cells into the rat brain

Immune rejection of transplanted material is a potential complication of organ donation. In response to tissue transplantation, immune rejection has two components: a host defense directed against the grafted tissue and an immune response from the grafted tissue against the host (graft vs host disease). To treat immune rejection, transplant recipients are typically put on immunosuppression therapy. Complications may arise from immune suppression or from secondary effects of immunosuppression drugs. Our preliminary work indicated that stem cells may be xenotransplanted without immunosuppression therapy. Here, we investigated the survival of pig stem cells derived from umbilical cord mucous connective tissue (UCM) after transplantation into rats. Our data demonstrate that UCM cells survive at least 6 weeks without immune suppression of the host animals after transplantation into either the brain or the periphery. In the first experiment, UCM cells were transplanted into the rat brain and recovered in that tissue 2-6 weeks posttransplantation. At 4 weeks posttransplantation, the UCM cells engrafted into the brain along the injection tract. The cells were small and roughly spherical. The transplanted cells were positively immunostained using a pig-specific antibody for neuronal filament 70 (NF70). In contrast, 6 weeks posttransplantation, about 10% of the UCM cells that were recovered had migrated away from the injection site into the region just ventral to the corpus callosum; these cells also stained positively for NF70. In our second experiment, UCM cells that were engineered to constitutively express enhanced green fluorescent protein (eGFP) were transplanted. These cells were recovered 2-4 weeks after brain transplantation. Engrafted cells expressing eGFP and positively staining for NF70 were recovered. This finding indicates a potential for gene therapy. In the third experiment, to determine whether depositing the graft into the brain protected UCM cells from immune detection/clearance, UCM cells were injected into the tail vein and/or the semitendinosis muscle in a group of animals. UCM cells were recovered from the muscle or within the kidney 3 weeks posttransplantation. In control experiments, rat brains were injected with PKH 26-labeled UCM cells that had been lysed by repeated sonic disruption. One and 2 weeks following injection, no PKH 26-labeled neurons or glia were observed. Taken together, these data indicate that UCM cells can survive xenotransplantation and that a subset of the UCM cells respond to local signals to differentiate along a neural lineage.