Uma Lakshmipathy

PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages.

We compared the transcriptomes of marrow-derived mesenchymal stem cells (MSCs) with differentiated adipocytes, osteocytes, and chondrocytes derived from these MSCs. Using global gene-expression profiling arrays to detect RNA transcripts, we have identified markers that are specific for MSCs and their differentiated progeny. Further, we have also identified pathways that MSCs use to differentiate into adipogenic, chondrogenic, and osteogenic lineages. We identified activin-mediated transforming growth factor (TGF)-beta signaling, platelet-derived growth factor (PDGF) signaling and fibroblast growth factor (FGF) signaling as the key pathways involved in MSC differentiation. The differentiation of MSCs into these lineages is affected when these pathways are perturbed by inhibitors of cell surface receptor function. Since growth and differentiation are tightly linked processes, we also examined the importance of these 3 pathways in MSC growth. These 3 pathways were necessary and sufficient for MSC growth. Inhibiting any of these pathways slowed MSC growth, whereas a combination of TGF-beta, PDGF, and beta-FGF was sufficient to grow MSCs in a serum-free medium up to 5 passages. Thus, this study illustrates it is possible to predict signaling pathways active in cellular differentiation and growth using microarray data and experimentally verify these predictions.

Concise Review: MicroRNA Expression in Multipotent Mesenchymal Stromal Cells

Mesenchymal stem cells, or multipotent mesenchymal stromal cells (MSC), isolated from various adult tissue sources have the capacities to self‐renew and to differentiate into multiple lineages. Both of these processes are tightly regulated by genetic and epigenetic mechanisms. Emerging evidence indicates that the class of single‐stranded noncoding RNAs known as microRNAs also plays a critical role in this process. First described in nematodes and plants, microRNAs have been shown to modulate major regulatory mechanisms in eukaryotic cells involved in a broad array of cellular functions. Studies with various types of embryonic as well as adult stem cells indicate an intricate network of microRNAs regulating key transcription factors and other genes, which in turn determine cell fate. In addition, expression of unique microRNAs in specific cell types serves as a useful diagnostic marker to define a particular cell type. MicroRNAs are also found to be regulated by extracellular signaling pathways that are important for differentiation into specific tissues, suggesting that they play a role in specifying tissue identity. In this review, we describe the importance of microRNAs in stem cells, focusing on our current understanding of microRNAs in MSC and their derivatives.

Creation of Engineered Human Embryonic Stem Cell Lines Using phiC31 Integrase

It has previously been shown that the phage‐derived phiC31 integrase can efficiently target native pseudo‐attachment sites in the genome of various species in cultured cells, as well as in vivo. To demonstrate its utility in human embryonic stem cells (hESC), we have created hESC‐derived clones containing expression constructs. Variant human embryonic stem cell lines BG01v and SA002 were used to derive lines expressing a green fluorescent protein (GFP) marker under control of either the human Oct4 promoter or the EF1α promoter. Stable clones were selected by antibiotic resistance and further characterized. The frequency of integration suggested candidate hot spots in the genome, which were mapped using a plasmid rescue strategy. The pseudo‐attP profile in hESC differed from those reported earlier in differentiated cells. Clones derived using this method retained the ability to differentiate into all three germ layers, and fidelity of expression of GFP was verified in differentiation assays. GFP expression driven by the Oct4 promoter recapitulated endogenous Oct4 expression, whereas persistent stable expression of GFP expression driven by the EF1α promoter was seen. Our results demonstrate the utility of phiC31 integrase to target pseudo‐attP sites in hESC and show that integrase‐mediated site‐specific integration can efficiently create stably expressing engineered human embryonic stem cell clones.

Qualification of Embryonal Carcinoma 2102Ep As a Reference for Human Embryonic Stem Cell Research

As the number of human embryonic stem cell (hESC) lines increases, so does the need for systematic evaluation of each lines characteristics and potential. Comparisons between lines are complicated by variations in culture conditions, feeders, spontaneous differentiation, and the absence of standardized assays. These difficulties, combined with the inability of most labs to maintain more than a few lines simultaneously, compel the development of reference standards to which hESC lines can be compared. The use of a stable cell line as a reference standard offers many advantages. A line with a relatively unchanging hESC‐like gene and protein expression pattern could be a positive control for developing assays. It can be used as a reference for genomics or proteomics studies, especially for normalizing results obtained in separate laboratories. Such a cell line should be widely available without intellectual property restraints, easily cultured without feeders, and resistant to spontaneous changes in phenotype. We propose that the embryonal carcinoma (EC) line 2102Ep meets these requirements. We compared the protein, gene, and microRNA expression of this cell line with those of hESC lines and alternative reference lines such as the EC line NTERA‐2 and the karyotypically abnormal hESC line BG01V. The overall expression profiles of all these lines were similar, with exceptions reflecting the germ cell origins of EC. On the basis of global gene and microRNA expression, 2102Ep is somewhat less similar to hESC than the alternatives; however, 2102Ep expresses more hESC‐associated microRNAs than NTERA‐2 does, and fewer markers of differentiated fates.

Differentiating Human Multipotent Mesenchymal Stromal Cells Regulate microRNAs: Prediction of microRNA Regulation by PDGF During Osteogenesis

OBJECTIVEnHuman multipotent mesenchymal stromal cells (MSC) have the potential to differentiate into multiple cell types, although little is known about factors that control their fate. Differentiation-specific microRNAs may play a key role in stem cell self-renewal and differentiation. We propose that specific intracellular signaling pathways modulate gene expression during differentiation by regulating microRNA expression.nnnMATERIALS AND METHODSnIllumina mRNA and NCode microRNA expression analyses were performed on MSC and their differentiated progeny. A combination of bioinformatic prediction and pathway inhibition was used to identify microRNAs associated with platelet-derived growth factor (PDGF) signaling.nnnRESULTSnThe pattern of microRNA expression in MSC is distinct from that in pluripotent stem cells, such as human embryonic stem cells. Specific populations of microRNAs are regulated in MSC during differentiation targeted toward specific cell types. Complementary mRNA expression analysis increases the pool of markers characteristic of MSC or differentiated progeny. To identify microRNA expression patterns affected by signaling pathways, we examined the PDGF pathway found to be regulated during osteogenesis by microarray studies. A set of microRNAs bioinformatically predicted to respond to PDGF signaling was experimentally confirmed by direct PDGF inhibition.nnnCONCLUSIONnOur results demonstrate that a subset of microRNAs regulated during osteogenic differentiation of MSCs is responsive to perturbation of the PDGF pathway. This approach not only identifies characteristic classes of differentiation-specific mRNAs and microRNAs, but begins to link regulated molecules with specific cellular pathways.

Development and Characterization of a Clinically Compliant Xeno-Free Culture Medium in Good Manufacturing Practice for Human Multipotent Mesenchymal Stem Cells

Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for Crohns disease, multiple sclerosis, graft‐versus‐host disease, type 1 diabetes, bone fractures, cartilage damage, and cardiac diseases. Despite remarkable progress in clinical trials, most applications still use traditional culture media containing fetal bovine serum or serum‐free media that contain serum albumin, insulin, and transferrin. The ill‐defined and variable nature of traditional culture media remains a challenge and has created a need for better defined xeno‐free culture media to meet the regulatory and long‐term safety requirements for cell‐based therapies. We developed and tested a serum‐free and xeno‐free culture medium (SFM‐XF) using human bone marrow‐ and adipose‐derived MSCs by investigating primary cell isolation, multiple passage expansion, mesoderm differentiation, cellular phenotype, and gene expression analysis, which are critical for complying with translation to cell therapy. Human MSCs expanded in SFM‐XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of MSCs expanded in traditional serum‐containing culture medium (SCM). To monitor global gene expression, the transcriptomes of bone marrow‐derived MSCs expanded in SFM‐XF and SCM were compared, revealing relatively similar expression profiles. In addition, the SFM‐XF supported the isolation and propagation of human MSCs from primary human marrow aspirates, ensuring that these methods and reagents are compatible for translation to therapy. The SFM‐XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to serum‐containing media for the production of large scale, functionally competent MSCs for future clinical applications.

Novel live alkaline phosphatase substrate for identification of pluripotent stem cells.

Alkaline phosphatases (AP) are a class of enzymes that hydrolyze phosphate containing molecules under alkaline conditions. In humans, there are primarily four different types of this enzyme; intestinal, placental, placental-like and non-tissue specific forms. The non-tissue specific isozyme of AP is expressed in liver, bone and kidney. A similar isozyme was identified in pluripotent stem cells when monoclonal antibodies, TRA-2-49/6E, recognizing determinants of human embryonal carcinoma (EC) cells showed specific reactivity to this isoform.1 AP is also known to be expressed at high levels in other pluripotent stem cell types such as embryonic germ cells (EG), embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC).2–5 Although definitive measures of pluripotency involves in-vitro tri-lineage differentiation and in-vivo teratoma formation, the most widely tested and validated panel for initial evaluation of ESC and iPSC consists of Stage Specific Embryonic Antigen SSEA4; Tumor Rejection Antigens TRA-1-60, TRA-1-81; AP; Oct4 and Nanog.6, 7 n nIn the case of murine ESC, AP positive colony forming in-vitro assay is used as a measure of pluripotency to demonstrate the ability of cells to single cell clone, attach and proliferate.8 A similar assay has been adapted for hESC where the sensitivity of the AP positive Colony forming assay to detect loss of pluripotent hESC has been found to be more sensitive than marker expression.9 More recently, the onset of AP positive colonies during early stages of reprogramming is used as an initial indicator of successful reprogramming of cells. Furthermore, in some instances the number of AP positive colonies is used as a mark of reprogramming efficiency.10 Nevertheless, this marker alone is not a definitive marker for the established iPSC clones. Additional marker evaluation is necessary to identify and qualify bona fide iPSCs.11 AP expression levels is a less sensitive measure to differentiate between undifferentiated and early differentiating cells since its expression level is reported to be varied depending on the lineage of differentiation.12 AP staining has been used as a fast and easy method that results in a specific chromogenic or fluorescent staining of the pluripotent stem cells. However, the current methods using AP staining require cell fixation and/or result in end products that accumulate within the cells. As a result, these AP stained colonies often lose their morphology and cannot be propagated any further. Inability to further culture selected pluripotent colonies identified using AP staining is a serious disadvantage of this methodology. An ideal solution would be an AP substrate that stains cells without altering the integrity or characteristics of stem cells thereby allowing further expansion of the stained colonies. n nHere in, we report the development and application of a novel fluorogenic live cell permeant substrate for AP (Live AP Stain). When incubated with cells for 20–30xa0min in basal culture media, this stain shows specific and robust staining of pluripotent cells such as human EC, murine and human ESC and iPSC with minimal or no staining of feeder cells and human fibroblasts. Stained colonies retain their morphology and preserve their cell health. The green fluorescence of the stained colonies is eliminated from cells 60–90xa0min after removal of the stain from the media. We have further utilized this stain in iPSC work flow to identify emerging iPSC clones during reprogramming of BJ human fibroblasts using CytoTune™; a Sendai-virus based non-integrating reprogramming method.13 Clones with robust AP staining were manually picked and propagated further. Expanded clones expressed other pluripotent markers, differentiated into cell types representative of the three germ layers and maintained a normal karyotype. These results indicate that AP Live Stain reported in this study does not alter the integrity or characteristics of the stained cells and is therefore an ideal tool to label early intermediates during iPSC generation or clonal populations of ESC for further selection and expansion.

Generation of Platform Human Embryonic Stem Cell Lines That Allow Efficient Targeting at a Predetermined Genomic Location

Bacteriophage recombinases can target specific loci in human embryonic stem cells (hESCs) at high efficiency, allowing for long-term expression of transgenes. In the present work, we describe a retargeting system where we used phiC31 integrase to target a plasmid to a pseudo-attP site in the cellular genome. The integration site was mapped and the chromosomal location evaluated for potential to be transcriptionally active in differentiated cells. The target plasmid, thus inserted, carried a wild-type R4 attB site that acts as a target for further integration of expression constructs. We engineered 2 hESC lines, BG01V and H9, to contain the target and showed that genetic elements such as promoter-reporter pairs can be inserted at the target efficiently and specifically. The retargeting construct has been adapted for complex element assembly using Multisite Gateway technology. Retargeted clones show sustained expression and appropriate regulation of the transgenes over long-term culture, upon random differentiation, and directed induction into neural lineages. The system described here represents a method to rapidly assemble complex plasmid-based assay systems, controllably insert them into the hESC genome, and have them actively express in undifferentiated as well as in differentiated cells.

Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1α) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5 and 3 of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1α or CMV early enhancer/chicken β actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1α-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain- and loss-of-function experiments, and human disease modeling using hESCs.

miRNA in pluripotent stem cells.

Embryonic stem cells and induced pluripotent stem cells are characterized by their ability to self-renew and differentiate into any cell type. The molecular mechanism behind this process is a complex interplay between the transcriptional factors with epigenetic regulators and signaling pathways. miRNAs are an integral part of this regulatory network, with essential roles in pluripotent maintenance, proliferation and differentiation. miRNAs are a class of small noncoding RNAs that target protein-encoding mRNA to inhibit translation and protein synthesis. Discovered close to 20 years ago, miRNAs have rapidly emerged as key regulatory molecules in several critical cellular processes across species. Recent studies have begun to clarify the specific role of miRNA in regulatory circuitries that control self-renewal and pluripotency of both embryonic stem cells and induced pluripotent stem cells. These advances suggest a critical role for miRNAs in the process of reprogramming somatic cells to pluripotent cells.