A phase I study was performed to determine the safety and tolerability of injecting autologous CD34؉ cells into five patients with liver insufficiency. The study was based on the hypothesis that the CD34 ؉ cell population in granulocyte colony-stimulating factor (G-CSF)-mobilized blood contains a subpopulation of cells with the potential for regenerating damaged tissue. We separated a candidate CD34؉ stem cell population from the majority of the CD34 ؉ cells (99%) by adherence to tissue culture plastic. The adherent and nonadherent CD34؉ cells were distinct in morphology, immunophenotype, and gene expression profile. Reverse transcription-polymerase chain reactionbased gene expression analysis indicated that the adherent CD34؉ cells had the potential to express determinants consistent with liver, pancreas, heart, muscle, and nerve cell differentiation as well as hematopoiesis. Overall, the characteristics of the adherent CD34 ؉ cells identify them as a separate putative stem/progenitor cell population. In culture, they produced a population of cells exhibiting diverse morphologies and expressing genes corresponding to multiple tissue types. Encouraged by this evidence that the CD34 ؉ cell population contains cells with the potential to form hepatocyte-like cells, we gave G-CSF to five patients with liver insufficiency to mobilize their stem cells for collection by leukapheresis. Between 1 ؋ 10 6 and 2 ؋ 10 8 CD34 ؉ cells were injected into the portal vein (three patients) or hepatic artery (two patients). No complications or specific side effects related to the procedure were observed. Three of the five patients showed improvement in serum bilirubin and four of five in serum albumin. These observations warrant further clinical trials.

These results identify adherent CD34+ stem cells as candidate fetal microchimeric cells, which are capable of sustaining the fetal cell population in the long term and have the ability to colonize multiple tissues and organs.

Summary. Microarrays provide a powerful tool for the study of haemopoietic stem and progenitor cells (HSC). Because of the low frequency of HSC, it is rarely feasible to obtain enough mRNA for microarray hybridizations, and amplification will be necessary. Antisense RNA (aRNA) amplification is reported to give high-fidelity amplification, but most studies have used only qualitative validation. Before applying aRNA amplification to the study of HSC, we wished to determine its fidelity and reproducibility, and whether statistically significant results can be obtained. We found that aRNA amplification introduced biases into relative RNA abundance. However, these biases were extremely consistent, and valid comparisons could be made, if amplified RNA was compared with amplified RNA. By applying this method to the effect of interferon-c and tumour necrosis factor-a on normal primary CD34 + HSC, biologically significant differences could be detected, including potential mechanisms for resistance of CD34 + cells to CD95-mediated apoptosis and evidence of the differentiating effects of the cytokines. Differences of twofold or less were detected, and most of these differences attained statistical significance after triplicate experiments. These data demonstrate that aRNA amplification can be used with microarray hybridization to study the transcriptional profiles of small numbers of primary CD34 + HSC.