Mark L. Weiss

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.

A Comparison of Human Bone Marrow–Derived Mesenchymal Stem Cells and Human Umbilical Cord–Derived Mesenchymal Stromal Cells for Cartilage Tissue Engineering

Bone marrow-derived mesenchymal stem cells (BMSCs) have long been considered the criterion standard for stem cell sources in musculoskeletal tissue engineering. The true test of a stem cell source is a side-by-side comparison with BMSCs. Human umbilical cord-derived mesenchymal stromal cells (hUCMSCs), one such candidate with high potential, are a fetus-derived stem cell source collected from discarded tissue (Whartons jelly) after birth. Compared with human BMSCs (hBMSCs), hUCMSCs have the advantages of abundant supply, painless collection, no donor site morbidity, and faster and longer self-renewal in vitro. In this 6-week study, a chondrogenic comparison was conducted of hBMSCs and hUCMSCs in a three-dimensional (3D) scaffold for the first time. Cells were seeded on polyglycolic acid (PGA) scaffolds at 25 M cells/mL and then cultured in identical conditions. Cell proliferation, biosynthesis, and chondrogenic differentiation were assessed at weeks 0, 3, and 6 after seeding. At weeks 3 and 6, hUCMSCs produced more glycosaminoglycans than hBMSCs. At week 6, the hUCMSC group had three times as much collagen as the hBMSC group. Immunohistochemistry revealed the presence of collagen types I and II and aggrecan in both groups, but type II collagen staining was more intense for hBMSCs than hUCMSCs. At week 6, the quantitative reverse transcriptase polymerase chain reaction (RT-PCR) revealed less type I collagen messenger RNA (mRNA) with both cell types, and more type II collagen mRNA with hBMSCs, than at week 3. Therefore, it was concluded that hUCMSCs may be a desirable option for use as a mesenchymal cell source for fibrocartilage tissue engineering, based on abundant type I collagen and aggrecan production of hUCMSCs in a 3D matrix, although further investigation of signals that best promote type II collagen production of hUCMSCs is warranted for hyaline cartilage engineering.

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.

Musculoskeletal tissue engineering with human umbilical cord mesenchymal stromal cells

Multipotent mesenchymal stromal cells (MSCs) hold tremendous promise for tissue engineering and regenerative medicine, yet with so many sources of MSCs, what are the primary criteria for selecting leading candidates? Ideally, the cells will be multipotent, inexpensive, lack donor site morbidity, donor materials should be readily available in large numbers, immunocompatible, politically benign and expandable in vitro for several passages. Bone marrow MSCs do not meet all of these criteria and neither do embryonic stem cells. However, a promising new cell source is emerging in tissue engineering that appears to meet these criteria: MSCs derived from Whartons jelly of umbilical cord MSCs. Exposed to appropriate conditions, umbilical cord MSCs can differentiate in vitro along several cell lineages such as the chondrocyte, osteoblast, adipocyte, myocyte, neuronal, pancreatic or hepatocyte lineages. In animal models, umbilical cord MSCs have demonstrated in vivo differentiation ability and promising immunocompatibility with host organs/tissues, even in xenotransplantation. In this article, we address their cellular characteristics, multipotent differentiation ability and potential for tissue engineering with an emphasis on musculoskeletal tissue engineering.

Effect of initial seeding density on human umbilical cord mesenchymal stromal cells for fibrocartilage tissue engineering.

Cells derived from Whartons jelly from human umbilical cords (called umbilical cord mesenchymal stromal cells herein) are a novel cell source for musculoskeletal tissue engineering. In this study, we examined the effects of different seeding densities on seeding efficiency, cell proliferation, biosynthesis, mechanical integrity, and chondrogenic differentiation. Cells were seeded on non-woven polyglycolic acid (PGA) meshes in an orbital shaker at densities of 5, 25, or 50 million cells/mL and then statically cultured for 4 weeks in chondrogenic medium. At week 0, initial seeding density did not affect seeding efficiency. Throughout the 4-week culture period, absolute cell numbers of the 25 and 50 million-cells/mL (higher density) groups were significantly larger than in the 5 million-cells/mL (lower density) group. The presence of collagen types I and II and aggrecan was confirmed using immunohistochemical staining. Glycosaminoglycan and collagen contents per construct in the higher-density groups were significantly greater than in the lower-density group. Constructs in the high-density groups maintained their mechanical integrity, which was confirmed using unconfined compression testing. In conclusion, human umbilical cord cells demonstrated the potential for chondrogenic differentiation in three-dimensional tissue engineering, and higher seeding densities better promoted biosynthesis and mechanical integrity, and thus a seeding density of at least 25 million cells/mL is recommended for fibrocartilage tissue engineering with umbilical cord mesenchymal stromal cells.

Combination Treatment of Human Umbilical Cord Matrix Stem Cell-Based Interferon-Beta Gene Therapy and 5-Fluorouracil Significantly Reduces Growth of Metastatic Human Breast Cancer in SCID Mouse Lungs

Umbilical cord matrix stem (UCMS) cells that were engineered to express interferon-beta (IFN-β) were transplanted weekly for three weeks into MDA 231 breast cancer xenografts bearing SCID mice in combination with 5-fluorouracil (5-FU). The UCMS cells were found within lung tumors but not in other tissues. Although both treatments significantly reduced MDA 231 tumor area in the SCID mouse lungs, the combined treatment resulted in a greater reduction in tumor area than by either treatment used alone. These results indicate that a combination treatment of UCMS-IFN-β cells and 5-FU is a potentially effective therapeutic procedure for breast cancer.

Wharton’s jelly or bone marrow mesenchymal stromal cells improve cardiac function following myocardial infarction for more than 32 weeks in a rat model: a preliminary report

The therapeutic effect of mesenchymal stromal cells (MSCs) following myocardial infarction (MI) is small. This may be due to differences in cellular sources and donor age, route of administration, in vitro cellular manipulations and the short time course of follow up in many animal studies. Here, we compared MSCs from two different sources (adult bone marrow or Whartons jelly from umbilical cord) for their long-term therapeutic effect following MI in a rat model to evaluate the effect of donor age. MSCs (or control infusions) were given intravenously 24-48 hr after myocardial ischemia (MI) induced by coronary artery ligation. Cardiac function was assessed by ultrasound at time points starting from before MSC infusion through 68 weeks after MI. A significant improvement in ejection fraction was seen in animals that received MSCs in time points 25 to 31 wks after treatment (p < 0.01). These results support previous work that show that MSCs can cause improvement in cardiac function and extend that work by showing that the beneficial effects are durable. To investigate MSCs cardiac differentiation potential, Whartons jelly MSCs were co-cultured with fetal or adult bone-derived marrow MSCs. When Whartons jelly MSCs were co-cultured with fetal MSCs, and not with adult MSCs, myotube structures were observed in two-three days and spontaneous contractions (beating) cells were observed in fiveseven days. The beating structures formed a functional syncytium indicated by coordinated contractions (beating) of independent nodes. Taken together, these results suggest that MSCs given 24-48 hr after MI have a significant and durable beneficial effect more than 25 weeks after MI and that MSC treatment can home to damaged tissue and improve heart function after intravenous infusion 24-48 hrs after MI, and that WJCs may be a useful source for off-the-shelf cellular therapy for MI.

Derivation and characterization of embryonic stem cells lines derived from transgenic Fischer 344 and Dark Agouti rats.

Rat embryonic stem cell (ESC) lines are not widely available, and there are only 2 lines available for distribution. Here, ESC lines were derived and characterized from Fischer 344 (F344) rats that express marker transgenes either β-galactosidase or human placental alkaline phosphatase (AP), nontransgenic F344 rats, and from Dark Agouti (DA) rats. The ESC lines were maintained in an undifferentiated state as characterized by colony morphology, expression of Oct4, Nanog, Sox-2, Cdx2, and Stella, staining for AP, and stage-specific embryonic antigen-1. Pluripotency was demonstrated in vitro by differentiation to embryoid bodies, followed by embryonic monsters. The Cdx2 expression by ESCs was unexpected and was confirmed via reverse transcriptase-polymerase chain reaction, immunocytochemistry. Pluripotency of ESCs was demonstrated in vivo by production of teratoma after an injection into F344 nontransgenic rats, and by an injection of male DA ESCs into F344 or Sprague-Dawley rat blastocysts and the generation of chimeric rats and germline contribution. ESCs from both F344 and DA contributed to chimeric rats, and one DA ESC line was proved to be germline competent. ESC sublines were created by transfection with a plasmid expressing enhanced green fluorescent protein (eGFP) under the control of a beta actin promoter and cytomegalovirus enhancer (pCX-eGFP) or by transfection with a plasmid expressing GFP under the control of a 3.1 kb portion of the rat Oct4 promoter (pN1-Oct4-GFP). In pN1-Oct4-GFP sublines, GFP gene expression and fluorescence were shown to be correlated with endogenous Oct4 gene expression. Therefore, these new ESC lines may be useful for tissue engineering and transplantation studies or for optimizing culture conditions required for self-renewal and differentiation of rat ESCs. While they made chimeric rats, further work is needed to confirm whether the transgenic F344 rat ESCs described here are germline-competent ESCs.

Promising cellular therapeutics for prevention or management of graft-versus-host disease (a review)

Graft-versus-host disease (GVHD) frequently occurs following allogeneic hematopoietic stem cell transplantation. The primary treatment for GVHD involves immune suppression by glucocorticoids. If patients become refractory to steroids, they have a poor prognosis. Therefore, there is a pressing need for alternative therapies to treat GVHD. Here, we review clinical data which demonstrate that a cellular therapy using mesenchymal stromal cells (MSCs) is safe and effective for GVHD. Since MSCs derived from bone marrow present certain limitations (such as time lag for expansion to clinical dose, expansion failure inxa0vitro, painful and invasive bone marrow MSC isolation procedures), alternative sources of MSCs for cellular therapy are being sought. Here, we review data which support the notion that MSCs derived from Whartons jelly (WJ) may be a safe and effective cellular therapy for GVHD. Many laboratories have investigated the immune properties of these discarded MSCs with an eye towards their potential use in cellular therapy. We also review data which support the notion that the licensing of MSCs (meaning the activation of MSCs by prior exposure to cytokines such as interferon-γ) may enhance their effectiveness for treatment of GVHD. In conclusion, WJCs can be collected safely and painlessly from individuals at birth, similar to the collection of cord blood, and stored cryogenically for later clinical use. Therefore, WJCs should be tested as a second generation, off-the-shelf cell therapy for the prevention or treatment of immune disorders such as GVHD.