1. Instead of the CD34+ Isolation kit, the AC133 Isolation kit (Miltenyi Biotech) can be used for the purification of hemopoietic progenitors. Also, instead of IMDM supplemented with insulin, transferring, and selenium, other liquid media, such as Stem Span (Stem Cell Technologies) can be used. There is no reason to use FCS for the expansion of hemopoietic stem cells. However, the addition of FCS may increase the FceRIa expression on cord blood-derived mast cells.

2. Although they may morphologically look mature (Fig. 4), when cord blood-derived mast cells in serum-free medium almost lack FceRI expression, the addition of FCS (15) or IL-4 (14) increases FceRIa expression but suppresses the proliferation of mast cells derived from progenitors.

3. Peripheral blood progenitors derived from elderly people do not give rise to mast cell colonies, while they give rise to hemopoietic colonies consisting of myeloid and erythroid lineages. Thus, donors for peripheral mast cell progenitors are recommended to be less than 40 yr of age. Progenitors obtained from neonates (cord blood) and infants can create large mast cell colonies (12,13).

Listeria Giemsa Stain
Fig. 4. (A) May-Grünwald Giemsa stain and (B) antichymase immunostaining of a typical cord blood-derived mast cell colony. Cord blood-derived CD34 +cells were cultured in methylcellulose for 6 wk and further cultured with SCF, IL-6, and IL-4 for 4 wk (magnification X60)

4. Plating more than 103 CD34+ cells or 106 lin-MNCs per 3 mL of methylcellulose medium at the beginning of culture may result in logarithmic increase in macrophage colonies secreting various cytokines such as GM-CSF in an autocrine manner. It also may result in a profound loss of mast cell colonies.

5. Before 4 wk of culture, agitation of methylcellulose medium may result in pro found growth of adherent macrophages on the culture plate bottom and may sup-

press the growth of mast cells. Fresh methylcellulose medium should be softly layered over the old medium.

6. After 15 wk of culture, the addition of FCS or IL-4 do not induce further expression of FceRIa or chymase messenger ribonucleic acid (mRNA) in cord blood-derived mast cells.

7. After terminal maturation (after 20 wk of culture), mast cells lose some granular compound-related mRNA, such as histidine decarboxylase and chymase. However, they can survive in this state for years without proliferation.

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