1. Art and Diseño

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834
CORRESPONDENCE
IN VITRO ASSAYS FOR PRIMITIVE HEMATOPOIETIC CELLS
To the Editor
In their recent report in Blood, Weilbaecher et al’ presented
data indicating that limiting-dilution analysis (LDA) for long-term
reconstitution of long-term bone marrow cultures (LTBMC) can
serve as a quantitative measure of hematopoietic stem cell (HSC)
activity. Aside from their unfortunate denotation of ‘long-term
culture initiating cells’ with the nondescriptive term ‘CFU-Dex,’we
are pleased that Weilbaecher et a1 used our LDA assaf’ in their
laboratory, including the replating studies: and supported our
earlier conclusions, that the onset and longevity of clonal expansion in LTBMC is a function of HSC primitiveness.24 We would
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CORRESPONDENCE
have enjoyed it if these investigators had properly acknowledged
our time-consuming efforts to establish an in vitro assay, which
allows statistically sound frequency analysis of CFU-S day 12 and
more primitive stem cells with marrow repopulating ability, MRA?
Instead, the original study’ was used as a trivial citation in their
Material and Methods section. In the Discussion the investigators
admitted that we also described “an LDA assay” using microLTBMC, but unfairly discredited it by stating that specificity and
overall activity of stem cells during our separation protocols were
not determined. We feel that this critique does not reduce the
originality and impact of our LDA assay in showing frequency
correlations of cobblestone area-forming cells (CAFC) and in vivo
defined HSC subsets.
In addition to the doubts we have with regard to the originalityof
their study, we have various scientific reservations over the significance and presentation of the data in the report of Weilbaecher et
al. A minor point is their claim that the CFU-Dex “colonies consist
primarily of maturing myeloid and erythroid cells,” which is fully
contradicted by their results. More serious is the wide range of
CFU-Dex frequencies per cell fraction, which sharply contrasts
with the high reproducibility of the CAFC assay.*Inthe scanning of
wells for “proliferating colonies consisting of more than 30 cells,”
proliferation is not determined by any means. The investigators
have not observed that the ‘typical Dexter colony’ as they score it
may persist for up to 2 weeks without any proliferation occurring,
an event largely similar to that occurring in semisolid colony assays.
Due to this, frequency analysis using CFU-Dex will consistently
overestimate the number of proliferating clones as soon as the first
clones have matured, which depends on the culture time and the
relative proportion of HSC with transient and long-lived clonal
properties in a particular fraction. In our assay’” we used a
primitive and proliferation-dependent LDA endpoint, ie, wells
were scanned every 1 to 3 days and scored positive if containing at
least one cobblestone area, the presence of which we reported to
be strictly associated with replatable CFU-G/M. To establish a
relationship between CAFC and CFU-Dex in our laboratory we
have scored both parameters in an LDA assay using unfractionated
and sorted bone marrow cells. The data that are presented in Table
1 indicate that CFU-Dex frequencies are 1.5 to 4-fold higher than
CAFC frequencies after 1 week of culture, and therefore are less
suitable as a quantitative in vitro indicator of the CFU-S day-12
incidence and marrow repopulating ability of a graft as demonstrated by a regression study? Table 1 also shows that stem cells
with the potential for long-term hematopoietic engraftment of a
stromal layer in vitro do not necessarily display high CFU-S day-12
activity as is suggested by the data of Weilbaecher et a].
The report of Weilbaecher et a1 may impress the unprepared
reader with a 1,300-fold enrichment for Thy’”Lin-Sca-l+cells;
however, only one extremely low frequency for CFU-Dex day 28 in
unfractionated marrow is used throughout that was derived from
one experiment (ie, the 1in 110,OOO in their Tables 2,3, and Fig 3).
The investigators admit that “not enough colonies were found in
other experimentsto calculate the response frequencies” at day 28.
The enrichment is further expanded by their best separation on
ThpLin-Sca-1’ cells, since this frequency (as high as 1in 80) even
falls outside the range that the investigators give for this cell
population (1 in 85 to 230).
Furthermore, in their discussion the investigators state that
establishing assay systems that can be used to quantify HSC
requires information about the proportion of HSC and non-HSC
precursors responding in the assay. However, they present semiquantitative data based on earlier reports, and even a report under
review, that do not allow correlations with the CFU-Dex frequencies. Interestingly, the Thy’”Lin-Sca-1’ population that is claimed
835
Table 1. CAFC and CFU-Dex Frequencies(1/f) in NBM and in a
Low-DensityFraction From Day-6 Post 5-Fluorouracil Marrow With
Low Affinity for Wheat Germ Agglutinin
CAFC
CFU-Dex
Day
NBM
WGA-dim
NBM
WGA-dim
Relative
CAFC
Contribution
7
14
21
28
41
467
1.885
8,333
39,217
588,235
2,136
207
137
150
654
425
857
4,630
15,083
147,059
1,942
159
62
56
159
0.0
0.6
3.7
15.7
54.0
Frequencies were calculated using Poisson statistics on the percentage of negativewells and are expressed as the number of cells required
to generate one cobblestonearea (CAFC assay) or 1 clone containingat
least 30 cells (CFU-Dex). The relative contribution values are the
percentages of CAFC activity that the indicated fraction contributes
relative to the 100% CAFC activity from NBM. These data were
calculated by dividing the frequency response of NBM by the CAFC
frequency of the fraction and multiplying by the percentagethat this cell
fraction represented in NBM. The WGA-dim fraction was sorted from a
1.069-1.078 g/mL light-density fraction of day-6 post 5-fluorouracil
bone marrow in a light scatter window that included all lymphocytes
and blast cells, and represents a 0.4% fraction of 5-fluorouracil day-6
marrow and a 0.04% fraction of NBM. Incidence of CFU-S-12: 27.5 per
I O 5 NBM; 84.6 per I O 5 WGA-dim cells.
to give rise to day-12 spleen colonies and thymus reconstitution
“with almost unit efficiency” has been shown to be heterogeneous
for long-term repopulating capacities! The compelling evidence
that CFU-S day-12 and CAFC day-10 activity can be largely
separated from CAFC day-28 and in vivo MRA and LTRA
(references 2, 7, 8) is just ignored by the investigators. Unlike the
investigators state, Thy’”Lin-Sca-l+ cells are not the only cells
generating colonies that continued to proliferate after 3 weeks,
since ThfB+M+ clones were also detected at week 4. In our
opinion, separation of functionally different HSC subsets is far
more the issue when defining the LDA micro-LTBMC assay than
the enrichment of a single cell fraction over unseparated marrow.
Finally, Weilbaecher et a1 comment that our enriched HSC
populations were 10- to 30-fold lower in response frequency than
the values they obtained for the Thy’”Lin-Sca-1’ fraction. Although we consider this item to be irrelevant for the establishment
of the CAFC assay, it can be easily seen from our publication’ that
their calculation is wrong: our rhodamine-123 dull fraction contained 1 CAFC day 28 in 500 cells plated. This corresponds to 1
CFU-Dex day 28 in about 187 cells according to the ratio of CAFC
and CFU-Dexon day 28 in Table 1of this letter. Since their highest
CFU-Dex day-28 range is 1 in 85 to 230, the absolute frequencies
are therefore truly comparable. In summary, we have found no
evidence in the report of Weilbaecher et a1 to suggest that the
CFU-Dex assay offers advantages over the existing CAFC assay.
R.E. PLOEMACHER
J.C.M. VAN DER LOO
J.P. VAN DER SLUIJS
Department of Cell Biology II
Erasmus University
Rotterdam, The Netherlands
REFERENCES
1. Weilbaecher K, Weissman I, Blume K, Heimfield S: Culture
of defined hematopoietic stem cells and other progenitors at
limiting dilution on Dexter monolayers. Blood 78:945,1991
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CORRESPONDENCE
836
2. Ploemacher RE, Van der Sluijs JP, Voerman JSA An in vitro
limiting dilution assay of long-term repopulating hemopoietic stem
cells of the mouse. Blood 74:2755,1989
3. Ploemacher RE, Van der Sluijs JP, Brons NHC, Voerman
JSA An in vitro limiting dilution assay of long-term repopulating
hemopoietic stem cells of the mouse. Exp Hematol 17:660, 1989
(abstr)
4. Van der Sluijs JP, De Jong, JP, Brons NHC, Ploemacher RE:
Marrow repopulating cells, but not CFU-S, establish long-term in
vitro hemopoiesis on a marrow-derived stromal layer. Exp Hematol
18:893,1990
5. Ploemacher RE, Van der Sluijs JP, Van Beurden CAJ, Baert
MRM, Chan P L Use of limiting-dilution type long-term marrow
cultures in frequency analysis of marrow repopulating and spleen
colony-forming hematopoietic stem cells in the mouse. Blood
78:2527,1991
6. Spangrude GJ, Johnson GR: Resting and activated subsets of
mouse multipotent hematopoietic stem cells. Proc Natl Acad Sci
USA 87:7433,1990
7. Ploemacher RE, Brons NHC: Cells with marrow and spleen
repopulating ability and forming spleen colonies on day 16,12, and
8 are sequentially ordered on the basis of increasing rhodamine 123
retention. J Cell Physiol136:531,1988
8. Jones RJ, Wagner JE, Celano P, Zicha MS, Sharkis SJ:
Separation of pluripotent haematopoietic stem cells from spleen
colony-forming cells. Nature 347:188,1990
RESPONSE
We have read the comments by Ploemacher et al. We would like
to acknowledge their thorough and important work entitled “An In
Vitro Limiting Dilution Assay of Long-term Repopulating Hematopoietic Stem Cells in the Mouse,” described in Ploemacher et al.’
We regret that Ploemacher et a1 did not feel that their important
contributions to this field was adequately acknowledged in Weilbaecher et a1.2 We are somewhat at a loss about how to respond to
the statement that we neglected to acknowledge a part of their
work that had not been published when we submitted our report.
However, we look forward to reading their newest work in the
November 15,1991 issue of Blood.3
We, among others, cloned bone marrow stromal cell lines that
could support progenitor cell proliferation and then used a limiting
dilution assay (LDA) to characterize different bone marrow
fraction^.'^ We had first learned of Ploemacher et al’s interesting
work from their poster presentation entitled “Limiting Dilution
Assay (LDA) of Long-Term Repopulating Hemopoietic Stem
Cells of the Mouse.”’ One of us (K.W.) presented a poster entitled
“In Vitro Assay for Highly Enriched Mouse Hematopoietic Stem
Cells” in the same poster session of the 1989 International Society
for Experimental Hematology meeting. We enjoyed interesting
and lively discussions with members of their group at that time and
were pleased that others were using the long-term bone marrow
culture and limiting dilution assays to study stem cell-enriched
populations. While each group used different cell separation
approaches, a common conclusion in both posters was that the
stem cell-enriched fractions gave a higher response frequency in
the LDA assay and in subsequent replating experiments.
We chose the name CFU-Dex, similar to the naming of CFU-S
or CFU-GM designations, to describe the progenitor cell type(s)
that can proliferate on a Dexter monolayer. We wanted to
acknowledge and honor T.M. Dexter’s original description of the
long-term bone marrow culture ~ystem.8.~
We had asked Dr Dexter
if he thought this terminology was appropriate and he agreed to the
naming of CFU-Dex.
In their letter, Ploemacher et a1 state that the endpoint determi-
nation of CAFC and of CFU-Dex are slightly different, which we
agree could lead to different and therefore not comparable
frequency calculations. This point is correct, therefore, a direct
comparison of absolute frequencies between WFA-dim and
Thy’”,Lin-,Sca+may not be valid. In their report, Ploemacher et al’
presented an impressive and thorough comparison and correlation
of CFU-S, CAFC, and CFU-GM of the stem cell-enriched bone
marrow fractions. In Weilbaecher et alz we did not attempt such a
complete frequency study, in part because none of the bone marrow
fractions we used contain all the CFU-S or CFU-GM activity.’”’2
It was our intention to show that the Thy’”,Lin-,Sca’ fraction,
which appears to contain all in vivo stem cell activity, is also highly
enriched for long-term CFU-Dex and replatable CFU-GM. No
other bone marrow fractions, and all of the other cell types were
tested, could contribute significantly to long-term Dexter colony
formation. Furthermore, quantitative analyses suggested we could
recover two thirds of the long-term CFU-Dex and 97% of the
replatable CFU-GM of unseparated bone marrow in the
Thy’”,Lin-,Sca’ fraction.
We hope that this letter has helped to clarify some of the points
raised by Ploemacher and colleagues.
KATHERINE WEILBAECHER
IRVING WEISSMAN
SyStemix
Palo Alto, CA and
Department of Pathology and the Howard Hughes Medical Institute
Stanford University
Stanford, CA
KARL BLUME
Bone Marrow Transplantation Program
Stanford University Hospital
Stanford, CA
SHELLY HEIMFELD
Bioimmunology Department
CeIlPro, Inc
Bothell, WA
REFERENCES
1. Ploemacher RE, van der Sluis JP, Voerman JS, Brons NH:
An in vitro limiting-dilution assay of long-term repopulating
hematopoietic stem cells in the mouse. Blood 74:2755,1989
2. Weilbaecher K, Weissman I, Blume K, Heimfeld S: Culture of
phenotypically defined hematopoietic stem cells and other progenitors at limiting dilution on Dexter monolayers. Blood 78:945,1991
3. Ploemacher RE, Van der Sluijs JP, Van Beurden CAJ, Baert
MRM: Use of limiting-dilution type long-term marrow cultures in
frequency analysis of marrow repopulating and spleen colony-forming
hematopoieticstem cells in the mouse. Blood 782527,1991
4. Whitlock C, Tidmarsh G, Muller-Sieberg CE, Weissman I:
Bone marrow stromal cell lines with lymphopoieticactivity express high
levels of a pre-B neoplasia associated molecule. Cell 48:1009,1987
5. Muller-Sieberg CE, Whitlock CA, Weissman I L Isolation of
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CORRESPONDENCE
two early B lymphocyte progenitors from mouse marrow: A
committed pre-pre B cell and a clonogenic Thy-1'" hematopoetic
stem cell. Cell 44:653,1986
6. Lipshitz DA, Robinson DH, Udupa KB: A micro method for
long-term in vitro culture of bone marrow cells. Exp Hematol
12291,1980
7. Ploemacher RE, Van der Sluijs JP, Brons NHC, Voennan
JSA An in vitro limiting dilution assay of long-term repopulating
hemopoietic stem cells of the mouse. Exp Hematol 17:660, 1989
(abstr)
8. Dexter TM, Allen TD, Lajtha LG: Conditions controlling the
proliferation of haemopoietic stem cells in vitro. J Cell Physiol
91:335,1976
037
9. Allen TD, Dexter TM: The essential cells of the hemopoietic
microenvironment. Exp Hematol12:517,1984
10. Muller-Sieburg CE, Townsend K, Weissman IL, Rennick D:
Proliferation and differentiationof highly enriched mouse hematopoietic stem cells and progenitor cells in response to defined
growth factors. J Exp Med 167:1825,1988
11. Heimfeld, S, Hudak, S, Weissman, I, Rennick, D: The in
vitro response of phenotypically defined mouse stem cells and
myeloerythroid progenitors to single or multiple growth factors.
Proc Natl Acad Sci USA 88:9902,1991
12. Spangrude GJ, Smith L, Uchida N, Ikuta K, Heimfeld S,
Friedman J, Weissman I: A perspective on mouse hematopoietic
stem cells. Blood 78:1395,1991
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1992 79: 834-837
In vitro assays for primitive hematopoietic cells [letter; comment]
RE Ploemacher, JC Van der Loo and JP Van der Sluijs
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