Identification of progenitor/stem cell populations that differentiate specifically towards superficial zone articular chondrocytes is an unmet challenge for cartilage tissue engineering. Using fluorescence activated cell sorting (FACS) analysis we found a characteristic pattern of "side population" (SP) stem cells identified by the Hoechst33342 dye. We established micromass cultures from this population of cells and tested their chondrogeneic potential. Control (untreated) cultures were minimally stained for alcian blue-a marker of chondrogenesis. However, with BMP-7 treatment, alcian blue staining was increased. Superficial zone protein-a specific marker for articular cartilage superficial zone chondrocytes-increased with BMP-7 and/or TGF-β1 treatment in SP micromass cultures. Our results demonstrate the presence of stem/progenitor cells in the SP fraction isolated from the surface zone of bovine cartilage and have the ability to specifically differentiate towards the superficial zone articular chondrocyte.

The CD34 ؉ CD38 ؊ phenotype identifies a population in the bone marrow that is enriched in the steady state for hematopoietic stem cells (HSCs). Following ex vivo culture of CD34 ؉ cells, HSC content is difficult to measure since committed CD34 ؉ CD38 ؉ progenitors down-regulate CD38 surface expression during culture. In this study, we sought to define the phenotype of human HSCs following ex vivo culture under conditions that support the expansion of human cells capable of repopulating non-obese diabetic/ severe combined immunodeficiency (SCID)-repopulating cells (SRCs) . IntroductionCell surface expression of the CD34 antigen is a reliable indicator of enrichment for hematopoietic progenitor and stem cells. 1,2 However, cells within the CD34 ϩ compartment are heterogeneous and include committed CD34 ϩ CD38 ϩ progenitors that lack stem cell activity. 3,4 The CD34 ϩ CD38 Ϫ fraction makes up 1% to 10% of the CD34 ϩ population and is highly enriched for both extended long-term culture-initiating cells (ELTC-ICs) and the most primitive assayable cells that are capable of repopulating nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (SCID-repopulating cells [SRCs]). [3][4][5][6][7] Using fluorescence-activated cell sorting to collect steady-state cord blood (CB) CD34 ϩ CD38 Ϫ cells, Bhatia et al 4 demonstrated a frequency of 1 SRC per 617 CB CD34 ϩ CD38 Ϫ cells and no detectable SRCs within the CD34 ϩ CD38 ϩ fraction. 4 Bhatia et al 8 also showed, via a limiting dilution analysis, that purified human CB CD34 ϩ CD38 Ϫ cells could be cultivated ex vivo with cytokines resulting in a 2-to 4-fold increase in SRCs at day 4 followed by loss of SRCs by day 9. A subsequent study by Glimm and Eaves 9 further demonstrated that self-renewal divisions occur within primitive CB-repopulating cells during 5-day cytokine suspension cultures. Other studies have suggested that human CB SRCs can be maintained ex vivo in liquid suspension cultures from 1 to 12 weeks. [10][11][12] Conversely, ex vivo culture of adult (bone marrow [BM], peripheral blood) sources of human CD34 ϩ stem cells with cytokines, with and without stroma, has been reproducibly associated with the loss of primitive repopulating cells over time. [13][14][15][16][17] Investigations into the proliferative capacity and SRC content of purified human BM CD34 ϩ CD38 Ϫ cells have been more limited in part due to a lack of culture conditions that support the maintenance or expansion of adult hematopoietic stem cells (HSCs). 8,18 Recent studies have indicated that expression of CD38 antigen on CD34 ϩ CD38 ϩ hematopoietic progenitors may be downmodulated during ex vivo culture with cytokines, calling into question the reliability of the CD34 ϩ CD38 Ϫ phenotype as an indicator of HSC content during or after culture. 19,20 As evidence that the CD34 ϩ CD38 Ϫ phenotype did not correlate with primitive stem cell content, Dorrell et al 19 demonstrated that surface expression of myeloid maturation antigens, CD33 and CD13, increased more than 2-fold on CB CD34 ϩ CD38 ...

Polychromatic flow cytometry offers the unprecedented ability to investigate multiple antigens per cell. Unfortunately, unwanted spectral overlaps and compensation problems increase when more than four colors are used, but these problems can be minimized if staining combinations are chosen carefully. We used an empiric approach to design, test and identify six-color T cell immunophenotyping reagent panels that can be expanded to include three or more functional or other markers in the FITC, PE, and APC channels without significant spectral limitations. Thirty different six-color T cell surface antigen reagent panels were constructed to identify major T cell subsets and maturational subtypes as defined by CCR7 and CD45RA expression, while excluding monocytes, B and non-viable cells. Staining performance of each panel was compared on cryopreserved cells from a single healthy donor recorded on a multiparameter cell sorter. Ten of the thirty reagent panels offered reliable resolution of T cell major and maturational surface markers. Of these, two panels were selected that showed the least spectral overlap and resulting background increase in the FITC, PE, and APC channels. These channels were left unoccupied for inclusion of additional phenotypic or functional markers, such as cytokines. Careful reagent titration and testing of multiple candidate panels are necessary to ensure quality results in multiparametric measurements. ' 2008 International Society for Advancement of Cytometry Key terms polychromatic flow cytometry; T cell immunophenotyping; fluorochrome conjugated antibody; compensation POLYCHROMATIC flow cytometry allows for detailed measurements even with small sample sizes and has recently been advanced by the development of new instrumentation, reagents and data analysis tools. Despite these significant improvements, it can be difficult to derive meaningful results when reagent combinations are expanded to include eight or more fluorescent markers. This is because unwanted spectral overlaps and measurement errors worsen as the number of fluorochromes used to label coordinately expressed cell markers increase (1-3). Using appropriate controls, software compensation algorithms can correct spillover problems post acquisition. However, due to the increased number of spectral overlaps in polychromatic reagent combinations, even properly compensated data can exhibit unwanted spreading into other measurement channels, complicating data analysis and interpretation (3,4). For example, dimly expressed markers, such as cytokines, are difficult to measure in channels where spreading in properly compensated data increases the background in the cytokine measurement channel. Such data spread in effect masks low intensity events; a problem not usually apparent when only a few stains are used simultaneously.A variety of sources contribute to this error in compensated data and are partially corrected by newer digital electronic configurations that collect and store data

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