We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

Previous reports have demonstrated the growth of undifferentiated human embryonic stem (HES) cells on mouse embryonic fibroblast (MEF) feeders and on laminin- or Matrigel-coated plastic surfaces supplemented with MEF-conditioned medium. These xenosupport systems run the risk of cross-transfer of animal pathogens from the animal feeder, matrix, or conditioned medium to the HES cells, thus compromising later clinical application. Here we show that human fetal and adult fibroblast feeders support prolonged undifferentiated HES cell growth of existing cell lines and are superior to cell-free matrices (collagen I, human extracellular matrix, Matrigel, and laminin) supplemented with human or MEF feeder-conditioned medium. Additionally, we report the derivation and establishment of a new HES cell line in completely animal-free conditions. Like HES cells cultured on MEF feeders, the HES cells grown on human feeders had normal karyotypes, tested positive for alkaline phosphatase activity, expressed Oct-4 and cell surface markers including SSEA-3, SSEA-4, Tra 1-60, and GCTM-2, formed teratomas in severely combined immunodeficient (SCID) mice, and retained all key morphological characteristics. Human feeder#150;supported HES cells should provide a safer alternative to existing HES cell lines in therapeutic applications.

The human umbilical cord that originates from the embryo is an extra-embryonic membrane and the Wharton's jelly within it is a rich source of stem cells (hWJSCs). It is not definitely known whether these cells behave as human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSC) or both. They have the unique properties of high proliferation rates, wide multipotency, hypoimmunogenicity, do not induce teratomas and have anticancer properties. These advantages are important considerations for their use in cell based therapies and treatment of cancers. In a search for properties that confer these advantages we compared a detailed transcriptome profiling of hWJSCs using DNA microarrays with that of a panel of known hESCs, hMSCs and stromal cells. hWJSCs expressed low levels of the pluripotent embryonic stem cell markers including POUF1, NANOG, SOX2 and LIN28, thus explaining why they do not produce teratomas. Several cytokines were significantly upregulated in hWJSCs including IL12A which is associated with the induction of apoptosis, thus explaining their anticancer properties. When GO Biological Process analysis was compared between the various stem cell types, hWJSCs showed an increased expression of genes associated with the immune system, chemotaxis and cell death. The ability to modulate immune responses makes hWJSCs an important compatible stem cell source for transplantation therapy in allogeneic settings without immunorejection. The data in the present study which is the first detailed report on hWJSC transcriptomes provide a foundation for future functional studies where the exact mechanisms of these unique properties of hWJSCs can be confirmed.

Human embryonic stem (hES) cells are typically derived and serially propagated on inactivated murine embryonic fibroblast (MEF) feeders. The use of MEFs and other components of animal origin in the culture media for hES cell support substantially elevates the risk of contaminating these cell lines with infectious agents of animal origin thereby severely limiting their potential for clinical application. We have previously shown that it is possible to derive and establish new hES cell lines in a xeno-free culture system using human fetal muscle fibroblast feeders. In this report, we have comparatively evaluated a panel of 11 different human adult, fetal, and neonatal feeders for hES cell support and have ranked them as supportive and non-supportive. We report that two adult skin fibroblast cell lines established in-house from abdominal skin biopsies supported prolonged undifferentiated hES cell growth for over 30 weekly passages in culture. Furthermore, hES cell lines cultured on adult skin fibroblast feeders retain hES cell morphology and remain pluripotent. Also, differences in feeder support exist between human cell types and sources. The use of human adult skin feeders is convenient for hES cell support given the ease of obtaining skin biopsies.

Mesenchymal stem cells (MSCs) from bone marrow, adult organs and fetuses face the disadvantages of invasive isolation, limited cell numbers and ethical constraints while embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) face the clinical hurdles of potential immunorejection and tumorigenesis respectively. These challenges have prompted interest in the study and evaluation of stem cells from birth-associated tissues. The umbilical cord (UC) has been the most popular. Hematopoietic stem cells (HSCs) harvested from cord blood have been successfully used for the treatment of hematopoietic diseases. Stem cell populations have also been reported in other compartments of the UC viz., amnion, subamnion, perivascular region, Wharton's jelly, umbilical blood vessel adventia and endothelium. Differences in stemness characteristics between compartments have been reported and hence derivation protocols using whole UC pieces containing all compartments yield mixed stem cell populations with varied characteristics. Stem cells derived directly from the uncontaminated Wharton's jelly (hWJSCs) appear to offer the best clinical utility because of their unique beneficial properties. They are non-controversial, can be harvested painlessly in abundance, proliferative, possess stemness properties that last several passages in vitro, multipotent, hypoimmunogenic and do not induce tumorigenesis even though they have some ESC markers. hWJSCs and its extracts (conditioned medium and lysate) also possess anti-cancer properties and support HSC expansion ex vivo. They are thus attractive autologous or allogeneic agents for the treatment of malignant and non-malignant hematopoietic and non-hematopoietic diseases. This review critically evaluates their therapeutic value, the challenges and future directions for their clinical application.

Human mesenchymal stem cells (MSC) are non-controversial multipotent stem cells. Their presence in umbilical cord blood (UCB) has been debated in some studies and others report low counts per cord blood unit and poor proliferation rates. On the other hand, Wharton's jelly of human umbilical cords appears to be a rich source of human MSC. This study derived 13 human Wharton's jelly stem cell (WJSC) lines from 13 human umbilical cords (100%) and recovered 4.7 +/- 0.2 x 10(6) live WJSC/cm of cord before culture. Complex culture medium produced greater proliferation rates of the WJSC in culture compared with simple medium. The mean population doubling times were 24.47 +/- 0.33 to 26.25 +/- 0.50 h in complex medium. The stem-cell markers of the WJSC were retained for at least 10 passages in both media. After programmed machine freezing, the thaw-survival rates of WJSC were 85-90% and they could be differentiated into neurons. Given the high derivation efficiency, availability of large numbers of fresh live cells, high expansion capabilities, prolonged maintenance of stem-cell properties and differentiation potential, it is proposed that human WJSC may be frozen at the same time as UCB in cord blood banks for regenerative medicine purposes.

Although basic research on human embryonic stem cells (hESCs) at the laboratory bench has progressed with enviable speed there has been little head way in terms of its clinical application. A look at the Internet however shows several stem cell clinics worldwide offering direct transplantation of undifferentiated hESCs to patients for the cure of a variety of diseases before bona fide evidence-based results can be demonstrated from large controlled studies. This raises concern because reliable protocols have to be first developed to resolve the three major hurdles delaying clinical trials such as inadequate cell numbers, immunorejection and tumorigenesis. Cell expansion methods using bioreactors, rotary culture and mitotic agents have now been developed to generate stem cell derivatives in large numbers. The problem of immunorejection can now be overcome with the development of indirect and direct reprogramming protocols to personalize tissues to patients (human induced pluripotent stem cells, hiPSCs; nuclear transfer stem cells, NTSCs; induced neuronal cells, iN). However, hESC, hiPSC, and NTSCs being pluripotent have the disadvantage of teratoma formation in vivo which has to be carefully addressed so as to provide safe stem cell based therapies to the patient. This review addresses the issue of tumorigenesis and discusses approaches by which this concern may be overcome and at the same time emphasizes the need to concurrently explore alternative stem cell sources that do not confer the disadvantages of pluripotency but are widely multipotent so as to yield safe desirable tissues for clinical application as soon as possible.

The human umbilical cord (UC) is an attractive source of mesenchymal stem cells (MSCs) with unique advantages over other MSC sources. They have been isolated from different compartments of the UC but there has been no rigorous comparison to identify the compartment with the best clinical utility. We compared the histology, fresh and cultured cell numbers, morphology, proliferation, viability, stemness characteristics and differentiation potential of cells from the amnion (AM), subamnion (SA), perivascular (PV), Wharton’s jelly (WJ) and mixed cord (MC) of five UCs. The WJ occupied the largest area in the UC from which 4.61 ± 0.57 x 106 /cm fresh cells could be isolated without culture compared to AM, SA, PV and MC that required culture. The WJ and PV had significantly lesser CD40+ non-stem cell contaminants (26-27%) compared to SA, AM and MC (51-70%). Cells from all compartments were proliferative, expressed the typical MSC-CD, HLA, and ESC markers, telomerase, had normal karyotypes and differentiated into adipocyte, chondrocyte and osteocyte lineages. The cells from WJ showed significantly greater CD24+ and CD108+ numbers and fluorescence intensities that discriminate between MSCs and non-stem cell mesenchymal cells, were negative for the fibroblast-specific and activating-proteins (FSP, FAP) and showed greater osteogenic and chondrogenic differentiation potential compared to AM, SA, PV and MC. Cells from the WJ offer the best clinical utility as (i) they have less non-stem cell contaminants (ii) can be generated in large numbers with minimal culture avoiding changes in phenotype, (iii) their derivation is quick and easy to standardize, (iv) they are rich in stemness characteristics and (v) have high differentiation potential. Our results show that when isolating MSCs from the UC, the WJ should be the preferred compartment, and a standardized method of derivation must be used so as to make meaningful comparisons of data between research groups.