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Mesenchymal stem cells (MSCs) are highly useful in a variety of cell therapies owing to their multipotential differentiation capability. MSCs derived from umbilical cord blood are generally isolated by their plastic adherence without using specific cell surface markers and examined for their osteogenic,adipogenic,and chondrogenic differentiation properties retrospectively. Here,we report 2 subpopulations of MSCs,separated based on aldehyde dehydrogenase (ALDH) activity. MSCs with a high ALDH activity (Alde-High) proliferated more than those with a low ALDH activity (Alde-Low). Alde-High MSCs had a greater ability to differentiate than Alde-Low MSCs in in vitro culture. Transplantation of Alde-High MSCs into fractured mouse femurs enabled early repair of tissues and rapid bone substitution. Alde-High MSCs were also more responsive to hypoxia than Alde-Low MSCs,with the upregulation of Flt-1,CXCR4,and Angiopoietin-2. Thus,MSCs with a high ALDH activity might serve as an effective therapeutic tool for healing fractures within a short period of time. View Publication

Since the first report of derivation of human embryonic stem cell (hESC) lines in 1998,many progresses have been achieved to reliably and efficiently derive,maintain,and differentiate this therapeutically promising cell type. This chapter introduces some basic and widely recognized methods that we use in our hESC core laboratory. Specifically,it includes methods for (1) deriving hESC lines without using enzyme and antibody to isolate the inner cell mass; (2) sustaining hESC self-renewal under feeder-dependent,feeder-conditioned,and defined conditions as well as pluripotency validation and quality control assays; and (3) inducing hESC differentiation to trophoblast with BMP4. View Publication

High aldehyde dehydrogenase (ALDH) activity has recently been used to identify tumorigenic cell fractions in many cancer types. Herein we hypothesized that a subpopulation of cells with cancer stem cells (CSCs) properties could be identified in established human osteosarcoma cell lines based on high ALDH activity. We previously showed that a subpopulation of cells with high ALDH activity were present in 4 selected human osteosarcoma cell lines,of which a significantly higher ALDH activity was present in the OS99-1 cell line that was originally derived from a highly aggressive primary human osteosarcoma. Using a xenograft model in which OS99-1 cells were grown in NOD/SCID mice,we identified a highly tumorigenic subpopulation of osteosarcoma cells based on their high ALDH activity. Cells with high ALDH activity (ALDH(br) cells) from the OS99-1 xenografts were much less frequent,averaging 3% of the entire tumor population,compared to those isolated directly from the OS99-1 cell line. ALDH(br) cells from the xenograft were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDH(lo) cells),generating new tumors with as few as 100 cells in vivo. The highly tumorigenic ALDH(br) cells illustrated the stem cell characteristics of self-renewal,the ability to produce differentiated progeny and increased expression of stem cell marker genes OCT3/4A,Nanog and Sox-2. The isolation of osteosarcoma CSCs by their high ALDH activity may provide new insight into the study of osteosarcoma-initiating cells and may potentially have therapeutic implications for human osteosarcoma. View Publication

Transforming growth factor (TGF)-beta superfamily proteins play a key role in the regulation of human embryonic stem cells (hESCs). Those of the TGFbeta/activin/nodal branch seem to support self-renewal and pluripotency,whereas those of the bone morphogenic protein (BMP) branch induce differentiation. In contrast to this generalization,we found that hESC remained undifferentiated after knockdown of SMAD4 with inducible short hairpin RNA interference,although the knockdown inhibited TGFbeta signaling and rendered the cells nonresponsive to BMP-induced differentiation. Moreover,the rapid differentiation of hESC after pharmacological inhibition of TGFbeta/activin/nodal receptor signaling was restricted after SMAD4 knockdown. These results suggest that TGFbeta/activin/nodal signaling supports the undifferentiated phenotype of hESC by suppressing BMP activity. During long-term culture,SMAD4 knockdown cell populations became less stable and more permissive to neural induction,a situation that was rescued by re-establishment of SMAD4 expression. These results suggest that SMAD4 is not required for maintenance of the undifferentiated state of hESC,but rather to stabilize that state. View Publication

AIM: To determine whether transcriptional reprogramming is capable of reversing the developmental aging of normal human somatic cells to an embryonic state. MATERIALS & METHODS: An isogenic system was utilized to facilitate an accurate assessment of the reprogramming of telomere restriction fragment (TRF) length of aged differentiated cells to that of the human embryonic stem (hES) cell line from which they were originally derived. An hES-derived mortal clonal cell strain EN13 was reprogrammed by SOX2,OCT4 and KLF4. The six resulting induced pluripotent stem (iPS) cell lines were surveyed for telomere length,telomerase activity and telomere-related gene expression. In addition,we measured all these parameters in widely-used hES and iPS cell lines and compared the results to those obtained in the six new isogenic iPS cell lines. RESULTS: We observed variable but relatively long TRF lengths in three widely studied hES cell lines (16.09-21.1 kb) but markedly shorter TRF lengths (6.4-12.6 kb) in five similarly widely studied iPS cell lines. Transcriptome analysis comparing these hES and iPS cell lines showed modest variation in a small subset of genes implicated in telomere length regulation. However,iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines. In order to verify these results in an isogenic background,we generated six iPS cell clones from the hES-derived cell line EN13. These iPS cell clones showed initial telomere lengths comparable to the parental EN13 cells,had telomerase activity,expressed embryonic stem cell markers and had a telomere-related transcriptome similar to hES cells. Subsequent culture of five out of six lines generally showed telomere shortening to lengths similar to that observed in the widely distributed iPS lines. However,the clone EH3,with relatively high levels of telomerase activity,progressively increased TRF length over 60 days of serial culture back to that of the parental hES cell line. CONCLUSION: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However,the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine. View Publication

Lentiviruses are capable of infecting many cells irrespective of their cycling status,stably inserting DNA copies of the viral RNA genomes into host chromosomes. This property has led to the development of lentiviral vectors for high-efficiency gene transfer to a wide variety of cell types,from slowly proliferating hematopoietic stem cells to terminally differentiated neurons. Regardless of their advantage over gammaretroviral vectors,which can only introduce transgenes into target cells that are actively dividing,lentiviral vectors are still susceptible to chromosomal position effects that result in transgene silencing or variegated expression. In this chapter,various genetic regulatory elements are described that can be incorporated within lentiviral vector backbones to minimize the influences of neighboring chromatin on single-copy transgene expression. The modifications include utilization of strong internal enhancer-promoter sequences,addition of scaffold/matrix attachment regions,and flanking the transcriptional unit with chromatin domain insulators. Protocols are provided to evaluate the performance as well as the relative biosafety of lentiviral vectors containing these elements. View Publication

Determining how normal and leukemic stem cells behave in vivo,in a dynamic and noninvasive way,remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes,which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context,we evaluated the possibility to use gene transfer-free labeling technologies. The lipophilic dye 3,3,3',3' tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate [DiI],DiD,DiR,and PKH26) can give rise to microenvironmental contamination,including when used in suboptimal concentration,after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually,we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non-genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches. View Publication

UNLABELLED: Human stem cells from adult sources have been shown to contribute to the regeneration of muscle,liver,heart,and vasculature. The mechanisms by which this is accomplished are,however,still not well understood. We tested the engraftment and regenerative potential of human umbilical cord blood-derived ALDH(hi)Lin(-),and ALDH(lo)Lin(-) cells following transplantation to NOD/SCID or NOD/SCID beta2m null mice with experimentally induced acute myocardial infarction. We used combined nanoparticle labeling and whole organ fluorescent imaging to detect human cells in multiple organs 48 hours post transplantation. Engraftment and regenerative effects of cell treatment were assessed four weeks post transplantation. We found that ALDH(hi)Lin(-) stem cells specifically located to the site of injury 48 hours post transplantation and engrafted the infarcted heart at higher frequencies than ALDH(lo)Lin(-) committed progenitor cells four weeks post transplantation. We found no donor derived cardiomyocytes and few endothelial cells of donor origin. Cell treatment was not associated with any detectable functional improvement at the four week endpoint. There was,however,a significant increase in vascular density in the central infarct zone of ALDH(hi)Lin(-) cell-treated mice,as compared to PBS and ALDH(lo)Lin(-) cell-treated mice. CONCLUSIONS: Our data indicate that adult human stem cells do not become a significant part of the regenerating tissue,but rapidly home to and persist only temporarily at the site of hypoxic injury to exert trophic effects on tissue repair thereby enhancing vascular recovery. View Publication

The pressing demand to elucidate the biology of human embryonic stem (ES) cells and to realize their therapeutic potential has greatly promoted the progresses in the optimization of the culture systems used for this highly promising cell type. These progresses include the characterization of exogenous regulators of pluripotency and differentiation,the development of animal-free,defined,and scalable culture systems,and some pioneering efforts to establish good manufactory practice facilities to derive and expand clinical-grade human ES cells and their derivatives. All of these advancements appear to be also applicable to the derivation and culture of human induced pluripotent stem cells,an ES cell-like cell type derived from somatic cells via reprogramming. This review attempts to summarize these progresses and discuss some of the remaining challenges. View Publication

We have derived 30 human embryonic stem cell lines from supernumerary blastocysts in our laboratory. During the derivation process,we have studied new and safe method to establish good quality lines. All our human embryonic stem cell lines have been derived using human foreskin fibroblasts as feeder cells. The 26 more recent lines were derived in a medium containing serum replacement instead of fetal calf serum. Mechanical isolation of the inner cell mass using flexible metal needles was used in deriving the 10 latest lines. The lines are karyotypically normal,but culture adaptation in two lines has been observed. Our human embryonic stem cell lines are banked,and they are available for researchers. View Publication

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support,but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study,five separate laboratories,each with experience in human embryonic stem cell culture,used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods,with propagation in the presence of Knockout Serum Replacer,FGF-2,and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment,death,and differentiated morphology by phase contrast microscopy,for growth by serial cell counts,and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems,only the control and those based on two commercial media,mTeSR1 and STEMPRO,supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment,cell death,or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study,and the lack of success with other formulations from academic groups compared to previously published results,include: the complex combination of growth factors present in the commercial preparations; improved development,manufacture,and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. View Publication

BACKGROUND: Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47,CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. DESIGN AND METHODS: We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. RESULTS: We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47,higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression,arguing against a direct reciprocal relationship. CONCLUSIONS: We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated,in part,by increased CD44-cytoskeleton binding. View Publication