The neural crest of vertebrate embryos gives rise to a variety of differentiated cell types, including neuronal and non-neuronal cells of peripheral ganglia (sensory and autonomic), pigment cells, and mesectodermal derivatives. Neural crest cells were taken from quail embryos at the level encompassing mesencephalon and metencephalon and the developmental potentials were evaluated by culturing them as single cells on 3T3 feeder layers. Such conditions proved to be particularly favorable for survival, proliferation, and differentiation of quail neural crest cells. Two hundred and fortythree clones that contained from 1 to >20,000 cells were analyzed after 7-10 days of culture. Phenotype analysis pro- investigate the differentiation capacity of a given embryonic cell is to identify the phenotypes of its progeny. One way to achieve this in higher vertebrates is to label a progenitor cell with a stable transmittable marker in vivo-for example, with an inheritable retroviral marker, as was done in newborn rat retina (8). Alternatively, a single isolated precursor cell could be cultured under conditions permitting full expression of its proliferation and differentiation potentials. Clonal analysis of vertebrate NC cell development showed (5, 9-12) that the progeny of individual truncal NC cells varied qualitatively and quantitatively, indicating differences in commitment at the time the cells were cultured. In these experiments, only three phenotypes were considered-namely melanocytes, adrenergic cells, and "other" cells (expressing neither set of traits). To extend the analysis of the developmental potentialities of single NC cells in vitro, we used the system described by Green and coworkers (13,14) that was devised to culture and clone human keratinocytes. Cranial NC cells were seeded individually on feeder layers ofgrowth-inhibited 3T3 cells in a complex culture medium. Under these conditions, NC cells produced a wide variety of clones, in terms of their size and phenotypic properties, thus revealing the remarkable heterogeneity of these cells, many of which are pluripotent. MATERIALS AND METHODSThe NC was removed, over a length corresponding to mesencephalon and metencephalon from 9-to 13-somite quail embryos as described (15) 5325The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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To purify satellite cells directly from human muscle biopsies, we have developed a method based on size separation of dissociated cells by flow cytometry. Immediately after tryptic dissociation of human muscle biopsies and elimination of erythrocytes, microscopic observation and flow cytometry analysis of cell suspensions revealed two populations of cells differing in size and nucleocytoplasmic ratio. Clonal cultures of these two cell types with a manual procedure demonstrated that only the small cells were myogenic satellite cells. Flow cytometry-sorting and analysis of the small cell population showed that (1) all sorted cells contained desmin immediately after dissociation and plating; (2) more than 98% of the cells expressed the 5.1.H11 epitope after 2 weeks of proliferation in culture; and (3) 90% of the sorted cells were able to form myotubes when cultivated at low density or in clonal cultures. Thus, human muscle satellite cells can be directly purified from human muscle samples using flow cytometry.

Schwann-cell differentiation in clonal cultures of the neural crest, as evidenced by the anti-Schwann cell myelin protein monoclonal antibody (cell

SUMMARY1. A voltage-dependent proton current, IH' was studied in cultured myotubes obtained from biopsies of human muscle, using whole-cell recording with the patchclamp technique.2. With a pH. of 8-0 and a calculated pHi of 6-3, IH was activated at voltages more depolarized than -50 mV and its conductance reached its maximum value at voltages more depolarized than + 10 mV.3. Studies of the reversal potential ofIH during substitution of K+, Na+, Ca2+, Cl-, Cs+ and H+ in the extracellular solution indicated that protons were the major charge carriers of IH 4. IH was also activated during a voltage step to + 22 mV with a pHo of 7-3 and a calculated pHi of 7-3. 5. Acidification of the extracellular solution led to a shift towards depolarized voltages of the conductance-voltage relationship.6. Stationary noise analysis ofIH suggested that the elementary event underlyingIH was very small with a conductance of less than 0 09 pS.7. Extracellular application of various divalent cations blocked IH. The block by divalent cations was voltage dependent, being more efficient at hyperpolarized than at depolarized voltages. For Cd2 , the Michaelis-Menten constant (Km) for the block was 0-6 ,tM at -28 mV and 10-4 #4M at + 12 mV.8. Ca2+ was a less efficient blocker than Cd2+ but could block IH at physiological concentrations (the Km values for the block were 0 9 mm at -38 mV and 7-3 mm at -8 mV).9