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Fig. 3. Potential of day-11 and day-12 embryonic haematopoietic tissues to
produce/expand definitive HSC/RUs as assessed by an organ culture approach.
(A) Cultured day-11 tissues, (B) cultured day-12 AGM region and YS, (C)
cultured day-12 liver. Note that among day-11 tissues (A) only the AGM region
was able to expand HSC/RUs and among day-12 tissues (B) only the YS was able
to expand HSC/RUs, suggesting two consecutive waves of HSC
generation/expansion and liver colonisation, firstly from the AGM and
secondly, the next day, from the YS. 12 d.p.c. embryonic liver was not capable
of maintaining HSCs: a dramatic decrease in number of HSC/RUs was observed
after 3 days in culture (see Discussion). Numbers of HSC/RUs in tissues are
estimated using a limiting dilution method and presented as described in
Materials and Methods. Numbers of recipient mice (RM) and the dilutions (D)
used were as follows. Day 11 tissues (A): (AGM, uncultured) 27RM, 2D; (AGM,
3-5 days in culture) 41RM; 8D; (AGM, 7-9 days in culture) 14RM, 3D; (YS
uncultured) 25RM, 3D; (YS 3-5 days in culture) 20RM, 6D; (YS 7-9 days in
culture) 15RM,3D; (liver, uncultured) 23RM, 2D; (liver, 3-5 days in culture)
29RM, 9D; (liver, 7-9 days in culture) 14RM, 4D. Day 12 tissues (B): (AGM,
uncultured) 25RM, 7D; (AGM, 3-5 days in culture) 31RM, 7D; (AGM, 7-9 days in
culture) 14RM, 3D; (uncultured YS) 39RM, 8D; (YS, 3-5 days in culture) 17RM,
4D; (YS, 7-9 days in culture) 11RM, 2D; (liver, uncultured) 37RM, 5D. Day 12
liver (C): (3-5 days in culture) 14RM, 3D; (7-9 days in culture) 24RM; 6D.
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markers. In some
reconstituted mice the percentage of Ly5.1+, Mac- 1+ cells was very low (data
not shown). (B) Y-specific PCR analysis of blood samples. Each line on the gel
represents an individual mouse. The level of reconstitution was assessed with
reference to results of PCR using standard dilutions of male DNA in female
DNA. Arrowheads point to the mice reconstituted at the level of close to 10%
or more.