Cell purification and transplantation of NSγ mice. (A-E) NSγ mice were transplanted with ALDH^br CD34⁺ cells prepared from CB that had been depleted of mature, lineage-marked cells (Lin^neg CB). Total CB has a low content of CD34⁺ progenitors (A), and these progenitors are enriched in Lin^neg CB (B). Enzyme-inhibited Lin^neg cells (treated with diethyl aminobenzaldehyde) were used to establish baseline fluorescence for ALDH and to establish sorting gates to purify ALDH^br CD34⁺ cells (C). ALDH^br CD34⁺ cells were purified from lineage-depleted CB (D), and each cell preparation was purified twice before cell transplantation (E). (F-I) After transplantation, the human hematopoietic chimerism was monitored based on the number of human CD45⁺ cells detected per microliter of peripheral blood. Without human hematopoietic engraftment, the peripheral blood contained only murine CD45⁺ cells (F). With engraftment, human CD45⁺ cells are present within the peripheral blood (G). In short-term studies (≤ 10 weeks), the human CD45⁺ cells within the blood are predominantly CD19⁺ (H). Using these methods, a history of human hematopoietic engraftment could be established for each transplantation assay (I). One cohort of mice that were transplanted with progenitors derived from a single CB that were transplanted into multiple different mice at multiple different cell doses.