Assessment and Characterization of Hemopoietic Stem Cells
Hans A . Messner
Ontario Cancer Institute and Institute of Medical Science, Department of Medicine, University of Toronto,
Toronto, Ontario, Canada
Key Words. Hemopoietic stem cells Cytokines Cytokine receptors
Abstract. The adequate production of blood cells is
sustained by pluripotent hemopoietic progenitors
whose behavior is influenced by a permissive hemo-
poietic microenvironment. Hemopoietic progenitors
have in common the expression of CD34 surface mol-
ecules, but are heterogeneous with respect to other
properties. It is commonly accepted that primitive
progenitors, considered to be candidates for marrow
repopulating cells, are HLA-DR-, without express-
ing lineage-specific determinants. They are usually
quiescent with respect to their cell cycle status. In
addition to CD34, they may display adhesion mole-
cules on their surface and express receptors for lig-
ands such as c-kit, FLT2lFLK3 and various other
cytokines. Some of these are expressed constitutively,
while others emerge as the cells progress through
their regular maturation program. This process
appears to include a gradual reduction of their pro-
liferative capabilities as demonstrated by a progres-
sive loss of the length of their telomeric structures.
Introduction
The German pathologist Emst Neumann was
the first to associate bone marrow function with
hemopoiesis. After his initial report in 1868 [l],
a controversy arose whether blood cells are pro-
geny of pluripotent stem cells or cells restricted
to specific hemopoietic lineages. In his last pub-
lication in 1917 [2], he made the prediction that
this question may be resolved if methods could be
developed that would permit the growth of hemo-
poietic progenitors using similar culture tech-
niques as those employed for studies of microbial
organisms. During the past three decades, cell
Correspondence: Dr. H.A. Messner, Princess
Margaret Hospital, 500 Sherbourne Street, Toronto,
ONT M4X lK9, Canada.
Received September I , 199.5; accepted for pub-
lication September 1, 1995. OAlphaMed Press
1066-5099/95/$.5.00/0
STEM CELLS
1995;13(~~pp13):13-18
Signaling pathways Telomeres
culture studies of hemopoietic progenitors have
indeed played a major role in advancing
knowledge about blood cell production.
Assay Systems for Hemopoietic
Progenitor Cells
The first experiments designed to investi-
gate the nature of hemopoiesis maintaining stem
cells were performed in rodents. Pioneering stud-
ies by Lorenz et al. [ 3 ] showed that bone marrow
function in lethally-irradiated mice and guinea
pigs can be restored by injecting suspensions of
hemopoietic cells obtained from normal animals.
These marrow-repopulating cells were also
found to circulate in the peripheral blood [4] as
demonstrated in experiments performed on para-
biotic animals with one normal and one lethally-
irradiated partner. The observation in mice that
irradiated recipients of normal hemopoietic cells
developed macroscopically visible hemopoietic
foci in their spleen led to the development of
the first quantitative assay for cells with radio-
protective effect [5]. These spleen colonies orig-
inated from individual pluripotent progenitors
with high proliferative potential and ability to
self-renew. Although it is now clear that these
spleen colony forming cells (CFU-S) are likely
not identical to the most primitive marrow-
repopulating progenitor population, the assay
served as a valuable tool to investigate early
events in hemopoiesis, and much of our current
knowledge is based on principles derived from
experiments using the CFU-S assay. One of the
several contributions made possible by this assay
resulted from studies of spleen colony forma-
tion by animals with macrocytic anemia that
were characterized by mutations of either the W
or steel locus [6, 71. Colony formation in both
14
types of animals was severely reduced or absent,
suggesting that early hemopoietic events were
genetically controlled. It also became obvious
that the different genetic defects interfered with
the function of different cell populations.
Mutations of the W locus were associated with
the inability of CFU-S to form spleen colonies,
while mutations of the steel locus resulted in the
generation of a defective microenvironment. The
Wand steel loci as well as mutants of both have
now been characterized at the molecular level
[8- 151. The W locus gives rise to a surface recep-
tor that is encoded by the proto-oncogene c-kit.
It binds a ligand (kit ligand or KL) encoded by
the steel locus.
The assessment of human hemopoietic pro-
genitors became feasible through the development
of suitable culture assays. While cell popula-
tions identified by these assays may not ful-
fill the criteria for radioprotective bone
marrow-repopulating progenitor cells, they
have served as surrogate markers to identify
regulatory principles of human hemopoiesis.
Two different types of culture assays are
currently available. The first group of assays
evaluates early progenitors by their ability to
form colonies when cultured under appropriate
conditions in semisolid medium. Based on the
cellular composition of individual colonies, one
can identify clonogenic cells with differing
potential. The most primitive known colony
forming cells (CFU-Blast) give rise to colonies
composed of cells with blast-like morphology
116, 171. These cells have sustained not only a
primitive morphological phenotype but also the
ability to produce secondary multi- and single
lineage colonies. Unfortunately, serial recloning
attempts of secondary colonies do not lead to
maintenance of hemopoiesis. It is at the moment
not clear whether this observation indicates lim-
ited self-renewal of clonogenic blast cells or
whether it reflects culture conditions that do not
support sustained growth. Pluripotent progenitor
cells (CFU-granulocyte-erythroid-macrophage-
megakaryocyte [GEMM]) identified by the pro-
duction of multilineage colonies are considered to
represent a more mature progenitor population
with little or no self-renewal [18-201. Among
CFU-GEMM there is also a small subpopulation
of pluripotent cells that gives rise to colonies
which contain myeloid and lymphoid elements
[21]. CFU-blasts and CFU-GEMM represent
a relatively small component of clonogenic
Hemopoietic Stem Cells
progenitor cells. They are observed at a respec-
tive frequency of 1-2 and 10-30 per lo’
mononuclear bone marrow cells. The majority
of clonogenic progenitors is restricted to pro-
duce progeny of a single hemopoietic lineage
(CFU-meg, burst-forming units-erythroid
[BFU-El, CFU-GM).
Colony assays have been instrumental in
identifying conditions that are critical for the
growth of various progenitor populations. Some
of the necessary growth factors such as M-CSF,
GM-CSF and G-CSF were initially purified chem-
ically. Newer molecular techniques have facili-
tated the cloning of genes encoding these and
other cytokines that influence hemopoiesis. Some
of these factors produced by recombinant tech-
niques are now introduced into clinical practice.
Typically, many of the growth factors display
pleiotrophic effects and interact with more than
one target cell population. While these factors
promote proliferation and maturation of progen-
itor cells, none of these substances have thus far
caused the sustained expansion of hemopoietic
stem cell clones.
Self-renewal of stem cells seems to require
the coexistence of a supportive microenviron-
ment. Originally described by Dexter [22-241, a
number of long-term conditions are now avail-
able that maintain hemopoiesis in cultures o f
murine and human bone marrow and peripheral
blood for a number of months. Cells that are able
to support the proliferation of primitive hemo-
poietic progenitors are not necessarily species-
specific. It is for instance, possible to observe the
long-term production of blood cells when human
progenitors are seeded on preformed murine or
porcine stromal layers [25,26]. A small subpop-
ulation of progenitor cells invades the established
stromal layer and forms foci of cobblestones.
These are typically composed of cells with prim-
itive blast-like morphology. Unlike cells observed
in blast colonies, cells within a cobblestone focus
continue to produce blood cells for more than
two to three months. However, even these cul-
ture systems are not able to maintain hemopoiesis
indefinitely. This phenomenon may either reflect
limiting culture conditions or alternatively, a
natural process of senescence.
It is currently not understood whether cells
generated in long-term cultures are able to func-
tion as marrow-repopulating cells in man.
Automated systems using multiple cytokines, lay-
ers of stromal cells and finely tuned provision of
Messner
nutrients and oxygen are now under development
by a number of groups to test whether human
marrow-repopulating cells can be grown in cul-
ture and expanded sufficiently to facilitate the
generation of a functioning bone marrow graft.
Characterization of Early Hemopoietic
Progenitors
Hemopoietic progenitors are characterized
by a number of properties. The most likely can-
didates for human hemopoietic stem cells are
characterized by the CD34 antigen [27] without
coexpression of HLA-DR molecules or molecules
associated with specific hemopoietic lineages
[281. These cells excrete the dye rhodamine 123 at
a rapid rate and are considered to be rhodamine-
low by flow cytometry [29]. In addition, CD34'
cells carry on their surface various adhesion mole-
cules that are part of the integrin family and medi-
ate the interaction with hemopoietic stroma [30].
They also express receptors encoded by genes
such as c-kit [31], FLT2FLK3 [32], receptors for
various hemopoietic growth factors as well as
nuclear transcription factors such as GATAl and
GATA2 [ 3 3 ] .The expression of these compo-
nents is not static but may vary with the devel-
opmental status of the cell. This was demonstrated
in a model studying the expression of various
hemopoiesis-related genes on embryonic stem
cells derived from the inner-cell mass of murine
blastocysts [34] and on their embryoid body-form-
ing progeny. Some of the genes appear to be
expressed constitutively. These include c-kit,
FLT2/FLK3, CD34, KL, c-myb, GATAl, as well
as the receptors for erythropoietin, and the a sub-
units for the interleukin 3 (IL-3), IL-6 and
GM-CSF receptors. The level of expression, how-
ever, may vary over time. In contrast, the expres-
sion of other genes is only observed during later
stages of embryoid body development. These
include the genes for the receptors of G-CSF,
IL-7, the a subunit of the IL-5 receptor and the P
subunit common to the IL-3, GM-CSF and IL-5
receptors. A similar time-related expression of
various genes has been observed in a study using
lineage- cells, elutriated at various speeds, and
sorted for differences in c-kit expression [35].
The expression of different receptors on
the surface of hemopoietic progenitors permits
the interaction with various regulatory elements
present in their environment. These include
15
short-range interactions with stromal cells which
require direct cell-to-cell contact and long-range
interactions with cells such as T lymphocytes
and monocytes that communicate over long dis-
tances through production of hormone-like
cytokines.
Cytokines and Signaling
Investigations conducted in the past decade
have resulted in the description of multiple
cytokines that interact with various hemopoi-
etic progenitors and cloning of their genes. As
shown for human bone marrow, some cytokines
are expressed constitutively [36] and seem to
be required for the production of hemopoietic
cells under steady-state conditions. Others
including G-CSF, GM-CSF and IL-3 are pro-
duced by various cell types, in particular T lym-
phocytes and monocytes in response to changing
demands. These cytokines may represent emer-
gency mechanisms that increase the blood cell
production beyond steady-state levels. In addi-
tion to the well-described positive signals, there
are also cytokines produced that exercise
inhibitory effects. One of these examples is
transforming growth factor-p (TGF-P). Detailed
studies have indicated that this molecule may
interfere at multiple levels with the production
of blood cells. TGF-P reduces the production
of KL by stromal cells and shortens the survival
of c-kit transcripts in hemopoietic progenitors
[37]. Both mechanisms lead to decreased pro-
liferation. The interaction of cells with other
inhibitory molecules may display a similar level
of complexity.
The bioavailability of cytokines in vivo may
be modulated by the presence of soluble recep-
tors in the peripheral circulation. Soluble
cytokine receptors may remove excess quanti-
ties of cytokines and thus reduce the signal.
Alternatively, they may represent chaperones that
prevent, through transient binding, the inappro-
priate destruction of the respective cytokine [38].
The effect of cytokines is mediated by
respective receptors which subsequently acti-
vate various signaling pathways. The receptors
present on hemopoietic progenitors belong to
two main categories. One group of receptors
displays tyrosine kinase (TK) activity. These
receptors have an extracellular domain that is
responsible for the binding of the respective
16
cytokine, while the intracellular TK domain
mediates the transduction of the mitogenic sig-
nal. Ligand binding induces the formation of
receptor dimers or multimers activation of TK
function and physical association of the TK por-
tion of the receptor with other substrates. As
shown for the c-kit receptor, binding of KL
results in the dimerization autophosphorylation
of the receptor and complex formation with
phosphatidylinositol-3'-kinase,phospholipase
C, and other substrates [39-411. In addition, KL
has also been shown to induce serine phosphory-
lation of MAP and RAF-I kinases. The activa-
tion of a single receptor type may result in the
activation of multiple pathways. It is of note
that the same set of substrates may be activated
by other cytokines. Signaling therefore, repre-
sents a complex interaction of multiple
cytokines, multiple receptors and multiple intra-
cellular substrates. The sum total of the infor-
mation is then likely responsible for the
behavior of the cells. This will include the inter-
action with various nuclear proteins that may
function as transcription factors.
A second class comprises the receptors of
the hemopoietic superfamily which include
receptors for GM-CSF, G-CSF, erythropoietin,
IL-2, IL-3, IL-4, IL-5, IL-6 and IL-7. It lacks
the intrinsic TK domain. Some of these recep-
tors require the association with other proteins
such as gpl30 for high affinity ligand binding.
Longevity of Hemopoietic Stem Cells
Murine recipients of bone marrow appear
to maintain a normal production of blood cells
for the duration of their lives. As shown in genet-
ically-marked recipients [42, 43], hemopoiesis
after transplantation is usually maintained by a
limited number of clones. These clones can be
sustained beyond the life span of the animals and
used to repopulate second generations of irradi-
ated hosts. However, serial transfer experiments
have demonstrated the eventual exhaustion of
stem cells resulting in the failure to repopulate
lethally-irradiated hosts [44].
Hemopoiesis in human bone marow trans-
plant (BMT) recipients, likewise, does not
appear to return to the status displayed by a nor-
mal bone marrow when evaluated for the pres-
ence of clonogenic progenitors and the number
of hemopoietically active clones. The frequency
Hemopoietic Stem Cells
of clonogenic pluripotent and single lineage
progenitors is reduced to a level of 25% to 40%
of that observed in normal individuals even 10
or more years after the transplant [45]. The
majority of these progenitors remains in cell
cycle compared to normal individuals which
demonstrate the presence of progenitors in a
resting phase. The need for sustained prolifera-
tion by all available progenitors may depend
upon the reduced number of clones that con-
tributes to the production of blood cells after
BMT [46]. Little information is available with
respect to the reestablishment of a reserve of
bone marrow repopulating cells after BMT. In at
least three cases, it was possible to utilize mar-
row obtained from the original BMT recipient to
engraft the original donor after treatment with
ablative therapy. The kinetics of blood cell
recovery suggest that the grafts were established
from transfused cells. However, it is not known
whether long-term maintenance of hemopoiesis
in these cases was derived from the transplanted
cell pool or from regenerating endogenous cells.
A recent development has drawn attention
to the fact that cells indeed may be characterized
by an internal clock that determines the potential
to undergo divisions. As determined in cultures
of fibroblasts [47, 481, the number of tandem
repeats in telomeric structures may be reduced
significantly over time. On average, 30 to 40
base pairs may be eliminated during each divi-
sion. The progressive loss of the length of
telomeres eventually leads to cessation of cell
division. Recent reports have indicated that
telomeric structures of hemopoietic cells of dif-
ferent origin differ in their length [49, 501. Fetal
liver cells contain a larger number of tandem
repeats compared to cord blood cells and bone
marrow-derived progenitor cells. When placed
into long-term culture, fetal liver cells display a
higher proliferative potential and produce the
largest number of CD34' cells over time com-
pared to cord blood and marrow cells. Similar to
fibroblast cultures, hemopoietic progenitors may
eventually reach a critical telomeric length that
is inconsistent with further proliferation. This
hypothesis is readily testable in serial transfer
experiments. The importance of the length of
telomeric structures was further supported
through recent observations in patients with var-
ious malignancies [5 13. Eighty-five percent of
454 patients with primary tumors demonstrated
telomerase activities that led to the elongation of
Messner
telomeric structures. Whether this mechanism
is important f o r the development of hemopoi-
etic m a l i g n a n c i e s remains to b e d e t e r m i n e d .
The fine line between immortality a n d malig-
nancy requires careful consideration if manip-
u l a t i o n s of h e m o p o i e t i c s t e m c e l l s are
contemplated.
References
1 Neumann E. Ueber die bedeutung des knochen-
marks fuer die blutbildung. Centralblatt fuer die
medicinischen Wissenschaften 1868;44.
2 Neumann E. Blut und pigmente. Verlag von
Gustav Fischer, Jena, 1917.
3 Lorenz E, Uphoff DE, Reid TR et al.
Modification of irradiation injury in mice and
guinea pigs by bone marrow infusions. J Natl
Cancer Inst 1951;12:197-201.
4 Brecher G, Cronkite EP. Postradiation parabiosis
and survival in rats. Proc Soc Exp Biol Med
19.5 I ;77:2Y2-294.
5 Till JE, McCulloch EA. A direct measurement
of the radiation sensitivity of normal mouse bone
marrow cells. Radiat Res 1961;14:213-222.
6 McCulloch EA, Siminovitch L, Till JE. Spleen
colony formation in anemic mice of genotype
W,”. Science 1964;144:844-846.
7 McCulloch EA, Russell ES, Siminovitch L et al.
The cellular basis of the genetically determined
hemopoietic defect in anemic mice of genotype
sl/sld. Blood 1965;26:399-410.
8 Chabot B, Stephenson DA, Chapman VM et al.
The proto-oncogene c-kit encoding a transmem-
brane tyrosine kinase receptor maps to the mouse
W locus. Nature 1988;335:88-89.
9 Geissler EN, Ryan MA, Housman DE. The domi-
nant white-spotting (w)locus of the mouse encodes
the c-kit proto-oncogene. Cell 1988;55: 185192.
10 Williams DE, Eisenman J , Baird A et al.
Identification of a ligand for the c-kit proto-onco-
gene. Cell 1990;63: 167-174.
I I Copeland MG, Gilbert DJ, Cho BC et al. Mast
cell growth factor maps near the steel locus on
mouse chromosome 10 and is deleted in a number
of steel alleles. Cell 1990;63:175-183.
I2 Flanagan JG, Leder P. The kit ligand: a cell sur-
face molecule altered in steel mutant fibroblasts.
Cell 19Y0;63:185194.
13 Zsebo KM, Williams DA, Geissler EN et al. Stem
cell factor is encoded by the Sl locus of the mouse
17
and is the ligand of the c-kit tyrosine kinase
receptor. Cell 1990;63:213-224.
14 Huang E, Nocka K, Beier DR et al. The hematopoi-
etic growth factor KL is encoded by the Sl locus
and is the ligand of the c-kit receptor, the gene
product of the W locus. Cell 1990;63:525-533.
15 Anderson DM, Lyman SD, Baird A et al.
Molecular cloning of mast cell growth factor, a
hematopoietin that is active in both membrane
bound and soluble forms. Cell 1990;63:235-243.
16 Nakahata T,Ogawa M. Identification in culture
of a class of hemopoietic colony-forming units
with extensive capability to self renew and gen-
erate multi-potential hemopoietic colonies. Proc
Natl Acad Sci USA 1982;79:3843-3847.
17 Keller GM, Phillips RA. Detection in vitro of a
unique multi-potent hemopoietic progenitor. J
Cell Physiol 1982;l(suppl):31-36.
18 Fauser AA, Messner HA. Granuloerythropoietic
colonies in human bone marrow, peripheral blood
and cord blood. Blood 1978;52:1243-1248.
19 Fauser AA, Messner HA. Identification of
megakaryocytes, macrophages, and eosinophils in
colonies of human bone marrow containing neu-
trophilic granulocytes and erythroblasts. Blood
1979;53:1023-1027.
20 Metcalf D, Johnson GR, Mandel TE. Colony for-
mation in agar by multipotential hemopoietic
cells. J Cell Physiol 1979;98:401-420.
21 Lim B, Jamal N , Tritchler D et al. G-6-PD
isoenzyme analysis of myeloid and lymphoid
cells in human multilineage colonies. Blood
1984;63: 1481-1487.
22 Dexter TM, Lajtha LG. Proliferation of hemo-
poietic stem cells in vitro. Br J Haematol
1974;28:525-530.
23 Dexter TM, Allen TD, Lajtha LG. Conditions
controlling the proliferation of hemopoietic stem
cells in vitro. J Cell Physiol 1977;91:335-344.
24 Gartner S, Kaplan HS. Long term culture of
human bone marrow cells. Proc Natl Acad Sci
USA 1980;77:4756-4759.
2 s Breems DA, Blokland EAW, Neben S et al.
Frequency analysis of human primitive hematopoi-
etic stem cell subsets using a cobblestone area
forming cell assay. Leukemia 1994;8: 1095.1 104.
26 Davis TA, Robinson DH, Lee KP et al. Porcine
brain microvascular endothelial cells support the
in vitro expansion of human primitive hematopoi-
etic bone marrow progenitor cells with a high
replating potential: requirement for cell-to-cell
interactions and colony-stimulating factors. Blood
l995:8S:175 1-1761.
18
27 Berenson RJ, Andrews RG, Bensinger WI et al.
Antigen CD34' marrow cells engraft lethally irra-
diated baboons. J Clin Invest 1988;81:951-955.
28 Sutherland HJ, Eaves HJ, Eaves CJ et al.
Characterization and partial purification of human
marrow cells capable of initiating long-term
hematopoiesis in vitro. Blood 1989;74:1563-1570.
29 Visser JWM, deVries P. Isolation of spleen-
colony forming cells (CFU-S) using wheat germ
agglutinin and rhodamine 123 labelling. Blood
Cells 1988;14:369-384.
30 Williams DA, Rios M, Stephens C e t al.
Fibronectin and VLA-4 in hematopoietic stem
cell-microenvironment interactions. Nature
199 I ;352:438-441.
31 Yarden Y, Kuang W-J, Yang-Feng T et al. Human
proto-oncogene c-kit: a new cell surface recep-
tor tyrosine kinase for an unidentified ligand.
EMBO J 1987;6:3341-3351.
32 Rosnet 0, Schiff C, Pebusque M-J et al. Human
FLT3/FLK2 gene; cDNA cloning and expression
in hematopoietic cells. Blood 1993;82:1110-1119,
33 Orkin SH. Globin gene regulation and switch-
ing: circa 1990. Cells 1990;63:665-672.
34 McClanahan T, Dalrymple S, Barkett M et al.
Hematopoietic growth factor receptor genes as
markers of lineage commitment during in vitro
development of hematopoietic cells. Blood
1993;81:2903-29 1.5.
35 Orlic D, Anderson S , Biesecker L et al.
Pluripotent hematopoietic stem cells contain
higher levels of mRNA for c-kit, GATA-2, p4.5,
NF-E2, and c-myb and low levels or not mRNA
for c-,fms and the receptors for granulocyte
colony-stimulating factor and interleukins 5 and
7. Proc Natl Acad Sci USA 1995;92:4601-4605.
36 Cluitmans FMH, Esendam BHJ, Landegent JE et
al. Constitutive in vivo cytokine and hematopoietic
growth factor gene expression in the bone mar-
row and peripheral blood of healthy individuals.
Blood 1995;85:2038-2044.
31 Heinrich MC, Dooley DC, Keeble WW.
Transforming growth factor p 1 inhibits expres-
sion of the gene products for steel factor and its
receptor (c-kit). Blood 1995;85: 1769- 1780.
38 Heaney ML, Golde DW. Soluble hormone recep-
tors. Blood 1993;82:1945-1948.
39 Alai M, Mui ALF, Cutler RL et al. Steel factor
stimulates the tyrosine phosphorylation of the
Hemopoietic Stem Cells
proto-oncogene product pYS""", in human hemo-
poietic cells. J Biol Chem 1992;267:18021-18025.
40 Rottapel R, Reedijk M, Williams D et al. The
steel/w transduction pathway: kit autophos-
phorylation and its association with a unique
subset of cytoplasmic signaling proteins is
induced by t h e steel factor. M o l Cell Biol
1991;l l(supp1 6):3043-3051.
41 Duronio V, Welham MJ, Abraham S et al. ~ 2 1 ' " ~
activation via hemopoietin receptors and c-kit
requires tyrosine kinase activity but not tyrosine
phosphorylation of ~ 2 1 ' GTPase-activating
"~ pro-
tein. Proc Natl Acad Sci USA 1992;89:1587-1591.
42 Lemischka IR, Rauiet DH, Mulligan RC.
Developmental potential and dynamic behavior of
hematopoietic stem cells. Cell 1986;45:917-927.
43 Jordan CT, Lemischka IR. Clonal and system-
atic analysis of long-term hemopoiesis in the
mouse. Genes Dev 1990;4:220-232.
44 Harrison DE, Astle CM. Loss of stem cell repopu-
lating ability upon transplantation. Effects of donor
age, cell number and transplantation procedure.
J Exp Med 1982;156:1767-1779.
45 Messner HA, Curtis JE, Minden M D et al.
Clonogenic hemopoietic precursors in bone mar-
row transplantation. Blood 1987;70: 1425-1432.
46 Turhan AG, Humphries RK, Philip4 GL et al.
Clonal hematopoiesis demonstrated by X-lined
DNA poly-morphisms after allogeneic bone marrow
transplantation. N Engl J Med 1989;320:1655-1661.
47 Harley CB, Futcher BA, Fredier CW. Telomeres
shorten during aging of human fibroblasts. Nature
1990;345:458-460.
48 Allsopp RC, Vaziri H, Patterson C e t al.
Telomere length predicts replicative capacity of
human fibroblasts. Proc Natl Acad Sci USA
1992;89:10114-10118.
49 Lansdorp PM, Dragowska W, Mayan H.
Ontogeny-related changes in proliferative poten-
tial of human hematopoietic cells. J Exp Med
1993; 178:787-79 1.
50 Vaziri H, Dragowska W, Allsopp RC e t al.
Evidence for a mitotic clock in human hematopoi-
etic stem cells. Loss of telomeric DNA with age.
Proc Natl Acad Sci USA 1994,91:9857-9860.
51 Kim NW, Piatyszek MA, Prowse KR e t al.
Specific association of human telomerase activ-
ity with immortal cells and cancer. Science
19Y4;266:2011-2015.