Mesenchymal stem cells (MSCs) with their multilineage developmental plasticity comprise a promising tool for regenerative cell-based therapy. Despite important biological properties, which the MSCs from different sources share, the differences between them are poorly understood. Hence, it is required to assign a molecular signature to each of these MSC populations, based on stem cell related genes and early lineage or developmental markers. Understanding their propensity to differentiate to different lineages is fundamental for the development of successful cell-based therapies. Culture expansion of MSCs is a prerequisite, since high absolute numbers of stem cells are required to attain a clinical dose. Here, we compared the different culture conditions for long-term expansion of human MSCs isolated from the Wharton's jelly (WJ) of the umbilical cord while preserving their stem cell characteristics and differentiation potential. We find that DMEM-KO and DMEM-F12 are superior as compared to the other media tested in supporting the in vitro expansion of the WJ-MSCs. We studied the gene expression profile of WJ and bone marrow-derived MSCs (BM-MSCs) both at early and late passages using Human Stem Cell Pluripotency Array, and our data revealed differences at the transcriptional level between the two MSC types. Compared to BM-MSCs, WJ-MSCs had higher expression of undifferentiated human embryonic stem cell (hES) markers like NANOG, DNMT3B, and GABRB3, pluripotent/stem cell markers, as well as some early endodermal markers both at early and late passages. To conclude, WJ-MSCs possess properties of true stem cells, which they retain even after extended in vitro culturing.

Multipotent mesenchymal stromal cells (MSCs) from Wharton's jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.

IntroductionMesenchymal stromal/stem cells (MSCs) for clinical use have largely been isolated from the bone marrow, although isolation of these cells from many different adult and fetal tissues has been reported as well. One such source of MSCs is the Whartons Jelly (WJ) of the umbilical cord, as it provides an inexhaustible source of stem cells for potential therapeutic use. Isolation of MSCs from the umbilical cord also presents little, if any, ethical concerns, and the process of obtaining the cord tissue is relatively simple with appropriate consent from the donor. However, a great majority of studies rely on the use of bovine serum containing medium for isolation and expansion of these cells, and porcine derived trypsin for dissociating the cells during passages, which may pose potential risks for using these cells in clinical applications. It is therefore of high priority to develop a robust production process by optimizing culture variables to efficiently and consistently generate MSCs that retain desired regenerative and differentiation properties while minimizing risk of disease transmission.MethodsWe have established a complete xeno-free, serum-free culture condition for isolation, expansion and characterization of WJ-MSCs, to eliminate the use of animal components right from initiation of explant culture to clinical scale expansion and cryopreservation. Growth kinetics, in vitro differentiation capacities, immunosuppressive potential and immunophenotypic characterization of the cells expanded in serum-free media have been compared against those cultured under standard fetal bovine serum (FBS) containing medium. We have also compared the colony-forming frequency and genomic stability of the large scale expanded cells. Secretome analysis was performed to compare the angiogenic cytokines and functional angiogenic potency was proved by Matrigel assays.ResultsResults presented in this report identify one such serum-free, xeno-free medium for WJ expansion. Cells cultured in serum-free, xeno-free medium exhibit superior growth kinetics and functional angiogenesis, alongside other MSC characteristics.ConclusionsWe report here that WJ-MSCs cultured and expanded in Mesencult XF, SF Medium retain all necessary characteristics attributed to MSC for potential therapeutic use.Electronic supplementary materialThe online version of this article (doi:10.1186/scrt477) contains supplementary material, which is available to authorized users.

MSCs are promising candidates for stem cell therapy and regenerative medicine. Umbilical cord is the easiest obtainable biological source of MSCs and the Wharton's jelly of the umbilical cord is a rich source of fetus-derived stem cells. However, the use of MSCs for therapeutic application is based on their subsequent large-scale in vitro expansion. A fast and efficient protocol for generation of large quantities of MSCs is required to meet the clinical demand and biomedical research needs. Here we have optimized conditions for scaling up of WJ-MSCs. Low seeding density along with basic fibroblast growth factor (bFGF) supplementation in the growth medium, which is DMEM-KO, resulted in propagation of more than 1 x 10(8) cells within a time period of 15 days from a single umbilical cord. The upscaled WJ-MSCs retained their differentiation potential and immunosuppressive capacity. They expressed the typical hMSC surface antigens and the addition of bFGF in the culture medium did not affect the expression levels of HLA-DR and CD 44. A normal karyotype was confirmed in the large-scale expanded WJ-MSCs. Hence, in this study we attempted rapid clinical-scale expansion of WJ-MSCs which would allow these fetus-derived stem cells to be used for various allogeneic cell-based transplantations and tissue engineering.

We used cre/loxP-based genetic lineage tracing analysis to test a previously proposed hypothesis that in vitro cultured adult pancreatic ␤-cells undergo epithelial-mesenchymal transition (EMT) to generate a highly proliferative, differentiation-competent population of mesenchymal islet "progenitor" cells. Our results in the mouse that are likely to be directly relevant to the human system show that adult mouse ␤-cells do not undergo EMT in vitro and that the mesenchymal cells that arise in cultures of adult pancreas are not derived from ␤-cells. We argue that these cells most likely originate from expansion of mesenchymal cells integral to the heterogeneous pancreatic islet preparations. As such, these mesenchymal "progenitors" might not represent the best possible source for generation of physiologically competent ␤-cells for treatment of diabetes. Diabetes 56:699 -702, 2007 E pithelial-mesenchymal transition (EMT) plays an essential role in embryonic development and tumorigenesis (1). Recently, it has been suggested that human pancreatic ␤-cells may undergo EMT when cultured in vitro (2-4). According to this model, the EMT is accompanied by the drastic downregulation of ␤-cell insulin expression and transformation of ␤-cells into rapidly replicating human islet progenitor cells (hIPCs) that can be generated in virtually unlimited quantities. The investigators hypothesized that in the future, it might be possible to induce efficient differentiation of the hIPCs via mesenchymal-epithelial transition into ␤-cells producing physiological levels of insulin. Because redifferentiation of hIPCs could provide an abundant source of ␤-cells for transplantation into patients with type 1 and type 2 diabetes, this would represent a major advance. Moreover, a conclusive demonstration of ␤-cell EMT would signify a fundamental discovery in islet cell biology, with important basic science implications.Given that islet preparations contain islet and non-islet epithelial and mesenchymal cells, a full substantiation of the EMT hypothesis requires a conclusive demonstration that ␤-cells, and not the other cell types intrinsic to islet preparations, give rise to mesenchymal "progenitors." This can be best accomplished through a direct lineage tracing of the cellular progeny of in vitro cultured ␤-cells. To carry out such tracing, we used a well-established transgenic mouse cre/loxP-based system (5). Our results provide compelling evidence against EMT of ␤-cells and suggest that abundant populations of mesenchymal cells in pancreatic cultures are not derived from ␤-cells. RESEARCH DESIGN AND METHODSTransgenic animals. The rat insulin promoter (RIP)-cre/EG mice were obtained by crossing RIP-cre recombinase mice containing a 668-bp fragment of the rat insulin II gene promoter (stock #0 003573; The Jackson Laboratory, Bar Harbor, ME) with double-reporter Z/EG transgenic mice (60). In the RIP-cre/EG mice, the cre-mediated ␤-cell-specific recombination results in positioning of enhanced green fluorescent protein (EGFP) gene, under control...

Insulin injections alleviate hyperglycemia in most patients with diabetes. However, they do not provide dynamic control of glucose homeostasis. Consequently, patients with longterm diabetes commonly develop life-threatening complications such as cardiovascular and kidney disease, neuropathy, and blindness. It has recently been shown that sustained independence from insulin injections can be achieved by transplantation of pancreatic islets into patients with diabetes [1]. Unfortunately, practical application of this clinical protocol is severely hampered by the shortage of islets available for transplantation. If functional β-cells and islets could be generated ex vivo, present severe islet shortage could be overcome. Another possible approach for restoration of islet cell mass is enhancement of endogenous regenerative capacity of endocrine pancreas. Recent results in rodents and humans suggest that pancreas has an extensive capacity to regenerate after injury [2][3][4]. In fact, it has been hypothesized that diabetes might result from β-cell destruction overpowering β-cell regeneration and that even a long time after the onset of

Contrary to the belief of the chemistry community, intense charge transfer is observed between meta-oriented donor–acceptor moieties in ultrasmall single-benzenic fluorophores. Red emission in any solvent has so far been reported with molecular fluorophores having the lowest molecular weight (MW) of 252.5 Da; however, we have achieved the same with a meta-fluorophore having an MW of only 203.1 Da. Red emission in a nonpolar solvent has so far been reported with a fluorophore having a minimum MW of 464.5 Da, but we have achieved the same with a meta-fluorophore having an MW of only 255.3 Da. Intense Stokes (260 nm) and solvatochromic (160 nm) shifts, high magnitudes of fluorescence quantum yield (>0.4), and excited-state lifetime (>16 ns) have been obtained in these single-benzenic meta-fluorophores, and these values are comparatively much higher in comparison to corresponding o-/p-fluorophores. These extraordinary meta-fluorophores have been employed to measure subcellular nanopolarity in live stem cells toward cost-effective white fluorescent ink and white light-emitting diodes (LEDs) in solid state.

The smallest fluorophore reported in literature to emit in the red has a MW > 250 Da. Hereby, we report a unique way to obtain red, orange emission in ultrasmall fluorophores (DEAMB, MW = 207 Da), (DEAB, MW = 177 Da), respectively. In this exceptional concept, we explore charge transfer between meta-oriented donor–acceptor moieties in a single-benzenic fluorophore (meta-fluors). Unlike existing red-emitting small-fluorophores, our meta-fluors exhibit an intense Stokes shift (225 nm), solvatochromic shift (175 nm), and excited-state lifetime (21 ns), quite suitable for fluorescence multiplexing. Thus, from size, MW, emission maximum, Stokes shift, solvatochromic shift, excited-state lifetime, etc., points of view meta-fluors are highly advanced in comparison to para-analogues. These two meta-fluors could be employed successfully to image and measure nanopolarity of the perinuclear membrane of live stem cell. These exceptional observations will open up a new branch of developing galaxy of ultrasmall meta-fluors for various applications.