In VitroCell. Dev.BM.

--Anima137:73-78, February2001
9 2001 Societyfor In VitroBiology
1071-2690/01 $10.00+0.00

PROLIFERATION OF HUMAN HEMATOPOIETIC BONE MARROW CELLS IN

SIMULATED MICROGRAVITY

P. ARTUR PLETT,~ STACY M. FRANKOVITZ, RAFAT ABONOUR, ANDCHRISTIE M. ORSCHELL-TRAYCOFF

Division of Hematology/Oncology and Indiana Elks Cancer Research Center, Department of Medicine,
Indiana University School of Medicine, Indianapolis, Indiana 46202

SUMMARY
Expansion and/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major
obstacle in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian
cells in microgravity (p~-g) may reduce proliferation and differentiation of these cells. We investigated the application of
these findings to the field of stem cell biology in the hopes of expanding HSC with minimal loss of hematopoietic function.
To this end, BM CD34 + cells were cultured for 4-6 d in rotating wall vessels for simulation of p~-g, and assessed for
expansion, cell cycle activation, apoptosis, and hematopoietic potential. While CD34 + cells cultured in normal gravity
(l-g) proliferated up to threefold by day 4-6, cells cultured in ~-g did not increase in number. As a possible explanation
for this, cells cultured in simulated tx-g were found to exit Go/G1 phase of cell cycle at a slower rate than 1-g controls.
When assayed for primitive hematopoietic potential in secondary conventional 1-g long-term cultures, cells from initial
t~-g cultures produced greater numbers of cells and progenitors, and for a longer period of time, than cultures initiated
with 1-g control cells. Similar low levels of apoptosis and adhesion molecule phenotype in ~-g and 1-g-cultured cells
suggested similar growth patterns in the two settings. These data begin to elucidate the effects of g-g on proliferation of
human hematopoietic cells and may be potentially beneficial to the fields of stem cell biology and somatic gene therapy.

Key words: bone marrow; stem cells; CD34+; cell culture; cell cycle; ex vivo expansion.

INTRODUCTION Simulated microgravity (tx-g) may provide a methodology for re-

Cell cycle activation and proliferation of hematopoietic stem cells
(HSC) in vitro, essential criteria for retroviral-mediated gene trans-
fer and expansion of HSC, invariably lead to loss of primitive func-
tion and an accompanying increase in differentiation and lineage
commitment, (Sutherland et al., 1991; Verfaillie, 1992, 1993; Hen-
schler et al., 1994; Traycoff et al., 1995, 1996, 1998; Gothot et al.,
1998; Humeau et al., 1999) with limited maintenance or expansion
of HSC (Bhatia et al., 1997; Matsunaga et al., 1998; Griscelli et
al., 1999). It is possible that rapid induction of in vitro proliferation
may predispose HSC to undergo differentiation rather than self-
renewal, such that reduction in the rate of in vitro proliferation may
favor self-renewal divisions. This notion is supported by several
studies documenting better retention of HSC function in stromal
cell-based cultures compared to cultures devoid of stromal ele-
ments (Verfaillie, 1992, 1993; Sutherland et al., 1993; Henschler
et al., 1994; Koller et al., 1995). This observation has been attri-
buted to a reduced rate of proliferation in stromal-based cultures,
due to either lower concentrations of stimulatory cytokines or in-
hibitory factors elucidated by stromal cells. Regardless of the rea-
sons behind the reduced proliferation in stromal cell-based cul-
tures, these data suggest that a reduction in the rate of proliferation
of HSC in vitro may in turn reduce differentiation, and, subsequent-
ly, favor self-renewal of these cells.
1To whom correspondence should be addressed at Indiana University
School of Medicine, 1044 West Walnut Street, R4-202, Indianapolis, Indiana
46202-5121. E-mail: pplett@iupui.edu
ducing the overall proliferation rate of HSC in vitro, thereby in-
creasing self-renewal of HSC in lieu of differentiation. This hypoth-
esis is supported by several studies investigating the growth of dif-
ferent cell systems in t~-g and Earth's gravity (l-g). These studies
were initiated to examine space flight-associated anemia, loss of
bone density, and abrogated immune function observed in humans
after return from space (Kimzey, 1975; Tavassoli, 1982; Taylor,
1987; Atkov and Bednenkom, 1992; Meehan et al., 1992; Fulford,
1993). The majority of space flight studies of hematopoiesis have
been in whole organisms, making it difficult to assess direct effects
on hematopoiesis excluding secondary effects caused by physiolog-
ical changes from t~-g. However, cell-based studies performed in a
rotating wall vessel (RWV; Synthecon Inc., Houston, TX) bioreactor
all point to reduced proliferation of ceils grown in simulated Ix-g
(Hughes-Fulford and Lewis, 1996; Carmeliet et al., 1997; Smileym
et al., 1997). The RWV bioreactor rotates the vessel wall and cell
culture media contained within at the same speed, thereby contin-
uously randomizing the gravitational vector and holding the cells
relatively motionless in the fluid, simulating some aspects of tx-g
(Schwarz et al., 1992; Tsao et al., 1992). Some studies, looking at
osteoblast (Carmeliet et al., 1997) and bone marrow (BM)-derived
macrophage (Armstrong et al., 1995) growth in RWV bioreactors
have, in addition to documenting reduced rates of proliferation of
these ceils, even reported reduced differentiation of cells in simu-
lated p~-g. Studies with CD34 § BM cells, subjected to p~-g aboard
space flight, also demonstrated reduced proliferation of these cells
compared to ground controls (Davis et al., 1996). Data are accu-

73
74 PLETT ET AL.
mulating which suggest that hypogravity may have dampening ef-
fects on BM hematopoiesis by inhibition of cell cycle progression
(Lange et al., 1994; Cogoli-Greuter et al., 1996), while hypergravity
may act to accelerate BM hematopoietic cell proliferation through
enhanced cell cycle progression (Burton and Smith, 1969; Lange et
al., 1994; Cogoli and Cogoli-Greuter, 1997). These studies point to
the possibility that p~-g may inhibit proliferation and differentiation
of HSC, which may subsequently lead to enhanced self-renewal of
these cells.
To test this hypothesis, we initiated short-term (4-6 d) cultures
of adult human BM CD34 § ceils in RWV bioreactors held either
stationary (1-g controls) or rotated to produce simulated t~-g. Cells
from RWV cultures were compared to similar cells cultured in our
conventional 1-g stromal ceil-free culture system initiated in tissue
culture plates. Cultured cells were examined for their rate of entry
into active phases of cell cycle, then harvested after 4 - 6 d and
examined for their cellular expansion, hematopoietic potential, re-
tention of primitive cell phenotype, apoptotic cell content, and ad-
hesion molecule status. We report that ceils cultured initially in
simulated p~-g exit G~/G~ phases of cell cycle at a slower rate and
maintain a greater degree of hematopoietic potential than cells cul-
tured in 1-g. Additionally, }x-g conditions did not affect expression
of adhesion molecules and induction of apoptosis, which were sim-
ilar to those observed in l - g cultures.
MATERIALS AND METHODS
Collection and fractionation of human BM CD34 § cells. Human BM as-
pirates were obtained from normal adult volunteers after receiving informed
consent according to guidelines established by the Human Investigation Com-
mittee of the Indiana University School of Medicine.
Low-density BM cells were separated over Ficoll/Hypaque (Pharmacia,
Piscataway, NJ). CD34 § isolation was performed using magnetic cell sepa-
ration columns (Mihenyi Biotec GmbH, Bergisch Gladbach, Germany) and
antibodies recognizing the CD34 epitope QBEND/10 according to the man-
ufacturer's procedure. Purity of selected CD34' fraction was assessed by flow-
cytometric analysis using a phycoerythrin (PE)-conjugated monoclonal anti-
body recognizing a different CD34 epitope (HPCA-2, Becton Dickinson Im-
munocytometry Systems [BDIS], San Jose, CA). Purity of CD34+-selected
samples ranged from 80 to 92%.
Simulated gravity and ]-g cell culture conditions. Simulated p~-gconditions
we~ produced using RWV. The RWV is a cell culture vessel designed to
provide a low-shear cell culture system, which upon vertical rotation pro-
duces solid body rotation of the RWV and the cell culture medium. The solid
body rotation randomizes the gravitational vector and leaves cells in a state
of constant free-fall, mimicking some aspects of tL-g (Schwarz et al., 1992;
Tsao et al., 1992). The RWV used in these experiments were disposable 10-
ml vessels designed for the RCCS-4D rotator base. This vessel contains two
luer lock syringe ports, a central pot't, and a silicon membrane on one side
of the vessel that provides gas exchange. CD34 + cells were seeded in the
RWV and rotated at 8 rpm, as per manufacturer's instructions for lymphoid
suspension cells. 1-g controls consisted of cells cultured in either a conven-
tional culture system (24-well cell culture-treated flat bottom plates; Costar,
Corning, NY), designated 1-g Dish, or in static RWV (referred to as 1-g
RWV), incubated with the silicon membrane side up. p~-g RWV, 1-g RWV
and 1-g Dish cultures were all initiated with identical cell number/ml/cm2.
Cells were cultured for 4-6 d in complete medium (IMDM--1% penicillin
[100 U/m1], streptomycin [100 I-~g/ml],and 2 mM L-glutamine [Biowhittaker,
Walkerville, MD], and 10 % fetal bovine serum [FBS; Hyclone, Logan, UT]),
supplemented with four cytokines as follows: stem cell factor (SCE 100
ng/ml), interleukin-3 (IL-3, 100 ng/ml), IL-6 (100 ng/ml), and megakaryocyte
growth and development factor (MGDE 50 ng/ml). Growth factors were de-
livered in combination at initiation of culture and every 48 h thereafter. All
cytokines were a kind gift of Amgen (Thousand Oaks, CA).
Long-term hematopoietic cell cultures. Secondary long-term hematopoietic
cultures (LTC) were initiated with equal numbers of cells (ranging from 1 •
104 to 4 X 104) harvested at day 4-6 from the initial culture in 1-g or b~-g.
Cultures were established in 1 ml of complete medium in flat-bottomed 48-
well plates, as previously described (Brandt et al., 1990; Srour et al., 1993).
At initiation and every 48 h thereafter, secondary cultures were supplemented
with four cytokines, as described above. At weekly intervals, the cultures
were demidepopulated, by removal of half the cells, followed by the addition
of fresh medimn and cytokines. Hm'vested cells were enumerated and assayed
for their hematopoietic progenitor cell content.
Hematopoietic progenitor cell assays. Cells from secondary LTC (3 • 10:~
to 1 • 105 cells/ml) were suspended in duplicate in plastic 35-ram tissue
culture dishes containing 1 ml 30% FBS, 5 X 10 5 mol/L 2-mercaptoethanol,
1.1% methylcellulose, 100 ng/ml SCF, 25 ng/ml IL-3, 25 ng/ml IL-6, 25
ng/ml GM-CSE and 2 U/ml erythropoietin in IMDM. Cultures were incubated
in 100% humidified 5% C02 in air at 37 ~ C. After 14 d, BFU-E, CFU-GM,
and CFU-GEMM were enumerated using an inverted microscope. All colony
types were identified according to previously reported criteria (Bran& et al.,
1988).
Flow cytometric assays. Fresh cells or cells from secondary LTC were
washed with phosphate-buffered saline (PBS) with 1% FBS (Hyclone) and
stained using a combination of anti-CD34 PE-Cy5 (Dako, Carpinteria, CA),
and one of the following PE-conjugated antibodies against CDlla, CD44,
CD49d, CD49e, CD54, CD62L, CXCR4, and CD95 (Pharmingen, San Jose,
CA), and CD43 (Aneell, Bayport, MN). Samples were acquired on a FACScan
or FACSCalibur (BDIS) and analyzed using CellQuest software (BDIS).
Apoptosis analysis. Apoptosis was determined by staining the cells with a
combination of anti-CD34 PE, Annexin-V fluorescein isothiocyanate in 1%
FBS-Dulbecco modified Eagle medium (DMEM) at 4~ C for 15 rain. Cells
were washed and resuspended in 1% FBS-DMEM, followed by the addition
of 10 Ixl of 10 ng/ml propidium iodide (PI)-PBS and incubated for 2-3 rain
to discriminate live/dead cells. Samples were then acquired on FACScan
(BDIS) and analyzed with CellQuest software (BDIS).
Cell cycle analysis. Fresh or cultured CD34 + cells were stained for cell
cycle analysis as previously described (Srour et al., 1992). Briefly, cells were
stained with equal volumes of staining buffer (0.1 mg/ml PI + 0.6% Nonidet
P-40 in PBS) and 2 mg/ml ribonuclease, agitated, and incubated on ice for
30 rain in the dark. Samples were acquired on a FACScan flow cytometer
(BDIS).
Statistical analysis. Differences between the groups were established by
paired t-test. Where applicable, mean + standard error of multiple mea-
surements is reported, as indicated.
RESULTS
Expansion o f B M CD34 § cells in simulated tx-g. Expansion of BM
CD34 + ceils was assessed after 4~5 d of culture in simulated p~-g
(~-g RWV), and compared to cells grown in a static RWV (1-g
RWV) as well as a conventional cell culture dish (1-g Dish). Figure
1 shows that in the presence of SCF, IL-3, IL-6, and MGDF, total
cellular expansion was threefold in the 1-g Dish, and 1.5-fold in 1-
g RWV, while in simulated tx-g total cell number remained the same
as input. When CD34 expression was examined, cells cultured in
simulated p~-g retained a slightly higher percentage of CD34 § cells
after 4 - 6 d, but absolute number of CD34 § cells was lowest in
simulated ~x-g cultures.
Cell cycle progression in simulated Ix-g. Our initial hypothesis
stated that simulated p~-g may prove beneficial for culturing HSC
by reducing the rate of proliferation, which may, in turn, favor self=
renewal or maintenance of primitive phenotype. We investigated this
notion by analyzing the rate of entry into active phases of cell cycle
during the first 2 d of culture in simulated p~-g. Figure 2 shows that
indeed, cells cultured in simulated ~-g exited GJG1 slower than
cells cultured in either of the 1-g cultures.
Apoptosis analysis o f cells cultured in simulated tx-g. To ensure
that the reduced proliferation and cell cycle progression seen in ~-
g cultures were not due to increased cell death, the degree of ap-
optosis was analyzed among CD34 § cells cultured in the three dif-
 STEM CELL GROWTH IN MICROGRAVITY 75

40

4
m~-g- RWV /
9 u-g - RWV
.............................
] 30
20
10
I-A-1 ~ 30 . . . . . . . . . . . . . . . . . .

................../ # 0

9~ 2
~J

b~
o , 7 /
1 . . . . . 5 19 26 36 57 86

Days post c u l t u r e
0 r
-- ~ 91 - g D i s h
TOTAL
- -m - 1 - g R W V
FIG. 1. Expansion of BM CD34 § cells in simulated }x-g. BM CD34' cells
A u-g RWV
were cultured for 4-6 d under the three conditions described in "Materials
and Methods" and then enumerated and examined for CD34 expression by
flow cytometric analysis. Data are mean + SEM fold expansion of total cells
and total CD34" cells (n = 5). Inset shows the percentage of cells expressing 25
A
CD34. *P = 0.03 when compared to 1-g Dish.
2o
90

85 %
"<,<.N....
-G
"~":,'.NN-
x'4,
,'q: ~N...,..
.
O

9 "g
l-g
Dish

" R-gWV. . . .
.

RWV
.
___

- --
. .
rate = - 8 . 6 1
r2 =

rate =-8.43
r2 = 0.99
rate = -6.71
r2 = 0.99
0.98
10!
,~ 15

z go ,.
o~ "4'4.'
~ 75 ...............................................
70
65 D/~Y0 DA'Y1
FIG. 2. Cell cycle progression of BM CD34 + cells in simulated ~-g. Cell
cycle status of BM CD34 + cells assessed at time 0 and 24 and 48 h after
initiation of 1-g and jx-g cultures. Aliquots of cultured cells were removed
from the cultures at the indicated time points and examined for cell cycle
position by PI staining, and flow eytometry. To illustrate differences in the
rate of exit from GJG~, linear regression analysis was performed and indi-
cated as "rate" in the figure. R2 = correlation coefficient for the regression/
Results are expressed as the mean of nine experiments. For ease of presen-
tation, error bars were omitted from the figure. w -< 0.05 when p~-g RWV
is compared to either 1-g Dish or 1-g RWV at 24 h.
. . . . . .
~ 0
5 19 26 36 57 86
Days post c u l t u r e
FIG. 3. Progenitor potential of BM CD34 + cells cultured in simulated ~-
"~"" g. Representative experiment (of a total of four) showing the production of
total cells (A) and total clonogenie progenitor cells (BEU + CFU-GM + CFU-
GEMM) (B) in secondary long-term cultures initiated with equal numbers of
cells derived from primary jx-g or 1-g cultures. Cells were initially cultured
' D/~Y2 in 1-g and p.-g for 4-6 d, after which an equal number of bulk cells was
removed and used to initiate secondary LTC in conventional 1-g Dish cul-
tures. Secondary cultures were maintained with the addition of SCE, IL-3,
IL-6, and MGDE every 48 h, and assayed weekly for progenitor potential as
described in "Materials and Methods." Culture was terminated at day 86,
since colony production ceased at this time. Total production of elonogenie
progenitors at every time point was calculated using the formula x = (number
of clonogenie cells per euhure) x (2)", where x is the number of total elon-
ogenie progenitors in culture and n is equal to the number of previous de-
midepopulations.

cultures (LTC) in our conventional culture system. Figure 3A shows

ferent culture conditions for 4 - 6 d. The percentage of CD34 § cells that total cellular production over the life of secondary culture was
undergoing apoptosis ( A n n e x i n - V + ) , ranging from 0.3 to 3 % of total greatest for the culture initiated with cells cultured initially in ~ - g
CD34 + ceils, was not statistically different in the w-g compared to RVW. Of interest is that cellular production p e a k e d on day 36 a n d
1-g cultures. Additionally, no differences in th e expression of the 57 of secondary. LTC initiated with 1-g Dish a n d 1-g R W V cells,
proapoptotic receptor CD95 (Fas) or the antiapoptotic protein Bcl- respectively, while cellular production was continuing to increase
2 were f o u n d on CD34 § cells propagated in the three different cul- in the LTC initiated with ~ - g R W V cells. Production of total CFU
ture conditions (data not shown). (Fig. 3B) was also greatest in LTC initiated with v,-g R W V cells,
Hematopoietic potential of cells cultured in simulated tx-g. T h e a n d p e a k e d later in t h e s e cultures t h a n in either of the cultures
hematopoietic potential of cells cultured in the three different short- initiated with 1-g ceils.
term culture conditions was e x a m i n e d by a s s e s s i n g the ability of Adhesion molecule expression of cells cultured in simulated Ix-g.
t h e s e cells to initiate and m a i n t a i n secondary long-term s u s p e n s i o n A d h e s i o n molecules ~ e b e c o m i n g increasingly important as poten-
76 PLETT ET AL.
120
100
8 8o
~ 6o
N 4O
~ 2o
CD11a CD43 CD44 CD49d CD49e CD54 CD62L CXCR4
FIG. 4. Expression of adhesion molecules on BM CD34+ cells after 5 d
of culture in 1-g and p,-g conditions. Fresh or day 5 cultured cells were
phenotyped, using antibodies against CD34 and one of the adhesion mole-
cules listed in the figure, and the expression of each adhesion molecule on
CD34 + cells calculated. Data are mean + SEM (n - 3).
tial markers to predict hematopoietic potential and homing of fresh
and ex vivo expanded HSC (Dercksen et al., 1995; Zanjani et al.,
1999). In light of this, and of data documenting changes in adhesion
molecule expression in lymphocytes cultured in t*-g (Pellis et al.,
1997), we investigated the adhesion molecule status of BM CD34 §
cells before and after short-term culture in ~-g RWV, 1-g RWV, or
1-g Dish. While no consistent differences in adhesion molecule
expression were observed among ex vivo expanded cells cultured
in the tx-g and l-g conditions, expression of CD62L and CXCR4
appeared to be lower on expanded CD34 § ceils, regardless of the
culture conditions, compared to fresh CD34 § cells, while expression
of C D l l a was slightly higher (Fig. 4).
DISt:USSIt)N
Several studies investigating the behavior of different biological
cell systems in simulated ~-g documented reduced proliferation
(Davis et al., 1996; Carmeliet et al., 1997; Smileym et al., 1997)
and in some cases, reduced differentiation (Armstrong et al., 1995;
Carmeliet et al., 1997). In the field of HSC biology, reducing the
overall proliferation rate of HSC in vitro may provide better reten-
tion of primitive cell function. In this article, we report that human
BM CD34 + cells cultured for 4-6 d in simulated p,-g, exhibit re-
duced proliferation coupled with better maintenance of HSC func-
tion compared to cells cultured in 1-g. These results do not appear
to be due to an incompatibility of hematopoietic cell growth in IX-
g, as cells propagated in Ix-g possessed similar viability, apoptotic
cell content, and cell surface adhesion molecule expression as cells
grown in 1-g conditions.
This is the first report, to our knowledge, describing growth and
expansion of human BM CD34 § cells in simulated Ix-g. These re-
suits, generated using the RWV bioreactor, reveal that culture with
SCE IL-3, IL-6, and MGDF in Ix-g does not induce expansion of
the cells, due possibly, to a slower entry of BM CD34 § cells into
active phase of cell cycle during the first 2 d of culture. An increase
in the length of the cell cycle has been postulated to explain some
of the observed hematologic changes seen in whole organisms dur-
ing space flight, such as reduction in erythropoiesis (Burton and
Smith, 1969; Lange et al., 1994) as well as reduced proliferation
and reactivity of immune ceils (reviewed in Criswell-Husak [1991]).
The reduced proliferation of BM CD34 § cells in our tx-g RWV
cultures suggests that this technology may indeed mimic some as-
pects of true Ix-g and its effects on the proliferation rate of primary
cells.
It is unlikely that cells remained totally quiescent in the Ix-g
RWV since entry into cell cycle was documented in these cultures.
Ongoing experiments are designed to examine this issue more com-
pletely through the use of cell tracking dyes coupled with cell sur-
face phenotyping. In addition, it does not appear that lack of pro-
liferation was simply due to an increase in cell death in Ix-g cul-
tures, since the percentage of CD34 + cells undergoing apoptosis
was not statistically higher in ~-g cultures than in the 1-g controls.
Additionally, expression of the proapoptotic marker, CD95, and the
antiapoptotic protein, Bcl-2, were not different between the three
culture conditions, suggesting that the low proliferation rate ob-
served in Ix-g cultures does not seem to be due to differential sur-
vival of the cells. Cell growth, in as much as was examined in these
studies, appeared to progress normally, albeit at a slower rate, in
t-u-g compared to 1-g cultures.
Reduction in proliferation of CD34 § cells in simulated Ix-g was
manifested as higher retention of primitive hematopoietic potential
as documented by enhanced ability of these cells to initiate and
maintain secondary long-term hematopoietic cultures. We have pre-
viously shown that the number of in vitro cell divisions is associated
with a concomitant loss of hematopoietic potential and an increase
in apoptosis of CD34 § ceils (Traycoff et al., 1998). Moreover, simply
traversing from Go to the G1 phase of cell cycle effects a substantial
loss in the capability of CD34 § cells to repopulate NOD/SCID mice
in vivo (Gothot et al., 1998). Similar loss in primitive cell function
associated with cell division has been shown by other investigators
using other systems (Berardi et al., 1995; Verfaillie and Miller,
1995; Young et al., 1996). Expansion of HSC in simulated tx-g may
provide a means of inducing HSC to undergo a limited number of
critical cell divisions, potentially sufficient for efficient gene transfer
while maintaining the highest degree of primitive hematopoietic cell
function.
It should be noted that there is a possibility that RWV culture
is not a favorable environment for hematopoietic cell growth, par-
tially evidenced by the reduced proliferation, and loss of hemato-
poietic function of cells cultured in the static RWV. However, given
that the levels of apoptosis in the 1-g and I-u-gRWV are very similar
to that seen in the 1-g Dish, and are very low, it is unlikely that
the overall decreased proliferation observed in the RWV system is
due to an incompatibility of hematopoietic cell growth in these ves-
sels. Furthermore, examination of ceils in secondary LTC showed
that cells cultured in l-g, despite the low proliferation in primary
cultures, did not retain the same degree of proliferative and clon-
ogenic capacity as cells cultured in tx-g RWV. Since cells would
settle to a higher concentration in the static RWV compared to the
rotated RWV, it is possible that differences in proliferation and
henmtopoietic potential in the two vessels could be related to dif-
ferences in local cell concentration during initial culture. This is
unlikely, however, since the cell number/ml/cm 2 was identical be-
tween the static RWV and the 1-g Dish, while the proliferation and
hematopoietic potential was different. Collectively, these data bring
into question the validity of maintaining RWV vessels in a static
position as adequate 1-g control cultures for simulated tx-g in the
rotating RWV.
Our results open a variety of questions regarding the basic mech-
anism by which this simulated Ix-g produces a change in the pro-
 STEM CELL GROWTH IN MICROGRAVITY
liferative capacity of ceils. Some studies have indicated that signal
transduction may be affected by tx-g (Cooper and Pellis, 1998).
Other data indicate that locomotion and cell adhesion is changed
when cells are exposed to p~-g (Pellis et al., 1997). Our resuhs
documenting reduced proliferation and slower cell cycle kinetics
can possibly be explained by data from Cogoli-Greuter et al. (1996)
in which they showed that lymphocytes exposed to simulated p~-g
for 3 d do not proceed through the cell cycle at a normal rate. These
authors went on to show that HeLa ceils exposed to hypergravity
have a reduced G~ phase of cell cycle, resulting in an increased
proliferation rate (Cogoli and Cogoli-Greuter, 1997). Whether
changes in the length of specific phases of cell cycle are responsible
for the slower cell cycle kinetics in our studies remain to be deter-
mined. Nevertheless, these studies are a first step in elucidating
the effects of ground-based simulated ~-g on the growth and pro-
liferation of BM CD34 § cells. Additionally, information gleaned
from these studies may provide a basis for application of this tech-
nology in developing improved protocols for ex vivo expansion and
gene transfer of HSC, which could have direct clinical applications
in BM transplantation.
ACKNOWLEDGMENT
This work was supported by National Aeronautics and Space Administra-
tion grant NAG-1586 to C. M. O.-T.
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