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藤椅

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As originally indicated by Friedenstein (1), one of the most prominent prop-
erties of MSCs is their ability to generate colonies after they are plated at low
density. In this chapter we describe what has become a traditional assay of 7 X8 C. ?( U: q2 `1 K
MSCs for colony- forming units (CFU assay) in which the cells are plated at low
density in large plates and discrete colonies counted after 2 or 3 wk. When used 9 D7 d3 G, z5 K. Q0 i( B4 C
for assay of human MSCs, each colony is generated by a single cell. When used * h8 f! X% ]7 K4 f6 {4 f& K8 B
with rat or mouse MSCs, however, single cells can generate more than one colony
because the cells can detach as they expand and re-seed the plate  (8,9). In this 4 D7 H1 y* o* K! f# M  E8 I
chapter we also describe a refined assay in which single MSCs are plated into indi-/ v: ^) l" [: O& {3 A" ^
vidual wells of a microtiter plate with a protocol that ensures that each observed ' ]% j* \7 x* v& p0 L& e
colony was generated by a single cell (Sc-CFU assay). With the Sc-CFU assay it is
possible to distinguish the colony forming potential of 2 distinct kinds of MSCs # r7 B" O0 I" A/ Y
present in early passage cultures: (a) spindle shaped cells that are rapidly selfrepli-. S/ c  R  d0 {$ o  L
cating (RS-cells) that predominant in the first few days after plating the cells at low
density, and (b) broader, slowly replicating cells (SRcells) that predominate as colo-
nies or cultures become confluent. The RS-cells can also be distinguished from 3 ~$ t" X& Q- R7 c
SR-cells by their lower forward scatter (FSlo
) and lower side scatter (SSlo
) of light, / d" b9 ~* a1 I) z7 j' L8 \( m
but the assays of FS/SS are difficult to standardize. Therefore the CFU assays are
more useful in estimating the proportion of early progenitors in different prepa-
rations of MSCs. To develop an improved assay for CFUs, we employed a fluo-
rescent flow cytometer with an automated cell sorter (FACSVantage SE with
Clonesort accessory; Becton-Dickinson) to plate single cells into individual * j) g0 I( I0 ~. F/ m
wells of a 96-well microtiter plate. We then incubated the samples in complete
medium for 10–14 d and assayed visible colonies by staining the plates with
Crystal Violet. As indicated in Figure 1A, the single-cell CFU assay (sc-CFU) 7 I& \0 O. t& K- {% W8 F. y) M
had a smaller variation than the standard CFU assay (Figure 1A and 1B). The
average coefficient of variation  (10) was 4.52 for the sc-CFU and 14.6 for the ! C! @/ R5 Y+ ?  T6 d4 f1 Y
standard CFU assay. Therefore, the sc-CFU assay was about 3 times more repro-! H4 w5 O! @0 N! N
ducible. Also, the sc-CFU assay detected important differences not detected by . F# S; f8 f8 x9 B. d% G. L
the standard assay (Figure 1B) between cultures initially plated at 50 or 100 + I4 ~# h2 W; g7 y1 W% b) j+ R
cells/cm23 z$ |+ W! f5 G: n
and cultures plated 500 or 1,000 cells/cm2* `3 P; W% u* E7 J
. The lower values obtained ! e/ S! ?9 M4 u6 y6 g0 ]7 Y* w
with the sc-CFU assay for cultures plated at the higher density are consistent with   N& H3 p! \! O) h- Y
previous observations that cultures plated at higher density show a rapid decrease
in the number of multipotential and rapidly self-renewing cells (RS cells)
(9,11–13).8 u9 y+ e4 _/ s" N! Z6 d3 b
2 Materials
2.1 Reagents
1.  αMinimum Essential medium (αMEM) without deoxyribonucleotides
(GIBCO/BRL).

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板凳

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2. Fetal bovine serum (Atlanta Biologicals)—selected for enhanced cell growth 2 N" p; [" ~$ T, K: B$ D( g
and low alkaline phosphatase (AP) activity.
  3. Penicillin 10,000 units/mL/ 10,000 µg/mL Streptomycin (GIBCO/BRL – Cat#
15140-122).
4.  l-glutamine (200 mM) (GIBCO/BRL).0 L  j, N6 G" E7 H  D3 h
  5.  Phosphate Buffered Saline (PBS), 1× (GIBCO/BRL).% b7 s, E2 o5 _- p/ s0 w4 @
  6.  0.25% Trypsin in 1 mM ethylene diamine tetraacetic acid (EDTA) (Sigma).: W  a: z( p. U
  7.  0.4% Trypan Blue (Sigma).
8. Crystal Violet (Sigma).
9. Methanol (100%).
10.  Annexin V-FITC (Sigma).
2.2 Equipment and Supplies) {5 q7 j( @* S5 [  G: \; n2 w
  1.  5-, 10-, and 25-mL sterile serological pipets.
  2.  Sterile transfer (Pasteur) pipets.
  3.  50-mL sterile conical centrifuge tubes.# o& O* w1 {' n
  4.  15-mL sterile conical centrifuge tubes.
5. 10-cm (58 cm2) and/or 15-cm (152 cm2) sterile tissue culture dishes or T75 8 v  g  P7 M0 \
(75 cm2)  flasks.
  6.  Laminar Flow Hood (Biosafety Cabinet).! {: P4 z$ g/ c( M4 L
7. 70% ethanol.
8. Water bath (37 °C).
  9.  Humid Incubator (37 °C) with CO2 source.
10.  Vacuum aspiration source.
11. Hemocytometer.' O2 K; L& h6 C' I7 H* ^) O. l
12.  Centrifuge with swinging bucket rotor.& g( W9 g, i4 |5 P( i) L# T
13.  Inverted Microscope (for checking culture confluence).* `* C  ~* x- s! V$ Z, Q: ~
2.3 Cell culture# J  Y- @) y: i: b" @7 X
1. Complete Culture Medium (CCM) with 18% FBS: 500 mL αMEM with or with-
out deoxyribonucleotides, 100 mL FBS, 5 mL 10,000 units/mL Penn/10,000 µg/
mL Strep (Gibco/BRL, Cat# 15140-122) for a final of 100 units Pen/mL and
100 ug/mL  Strep,  5 mL  200 mM l-glutamine, 29.2 mg/mL of 0.85% NaCl for a
final concentration of 2 mM l-glutamine (292 µg/mL).
2. 3.0% Crystal Violet in 100% Methanol4 j% a$ @& M3 ?8 j& o4 y0 w) }3 w7 E
3.0 g Crystal Violet in 100 mL 100% Methanol.
Filter through Whatman filter paper. Store at room temperature (RT)

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报纸

发表于 2010-3-9 18:28 |只看该作者

3 Methods
3.1  Colony Forming Unit—Fibroblast assay
1. Human MSCs are prepared as described previously in Chapter 1.
2. Expand MSCs cultures to 70–80% confluency and harvest with trypsin-EDTA
(as described previously).
3. Count the number of cells using a hemocytometer (see Note 1). To ensure cell
separation, a glass Pasteur pipet can be flamed at its tip to reduce its diameter, , `8 n$ R& T+ n) i# {! s
and the cells drawn through the narrowed pipet several times (see Note 2).
4. Dilute in complete culture medium, and plate at about 100 cells per 100-mm
tissue culture dish (Falcon) in complete culture medium.
5. Incubate for 10–14 d at 37 °C in 5% humidified CO2, and wash with PBS and
stain with 0.5% Crystal Violet in methanol for 5–10 min at room temperature.
6. Wash the plates with PBS twice and visible count colonies (Fig. 6.1).
3.2  Colony Forming Unit— Single Cell assay
Rapidly self-renewing population of MSCs are characterized by low forward scat-
ter (FSlo7 a6 o7 o& K; E" d& F
) and low side scatter (SSlo
) of light. The following experiments explain the 8 w$ O) Y# j' f
isolation of FSlo. X/ b( c! M: y
/SSlo
MSCs that are rapidly self-renewing. It is also a rapid, stand-
ardized assay for FS/SS useful to identify preparations of MSCs enriched for RS 3 J1 e/ Z' _* p) ?) i3 X
cells that will expand rapidly during subsequent passage in culture. The use of the 6 I# t4 Z9 F) n' H( f& p
assay should help to resolve discrepancies in data obtained by different laboratories % i% Q: K# w" M
with apparently similar preparations of MSCs.
3.2.1  Measurement of FS and SS# U9 D! S8 M1 z+ R% Z4 C* G( O
1. A closed stream flow cytometer (Epics XL/ADC; Beckman-Coulter) is stand-
ardized using microbeads with known diameters (7, 10, 15, and 20 microns;
Dynosphere Uniform Microspheres; Bangs Laboratories Inc., Fisher, IN).
2. The gains and voltages on the photomultiplier tubes are adjusted so that the
mean value of the FS peak for the 20 µ bead is about 650 and the peak of the SS
for the 7 µM bead about 450.# \1 S8 H0 T% C* n5 s  Y
3. With these settings, the standard deviation for FS of the largest bead should be 0 j; {0 n6 K4 F8 I
less than +/− 0.4% (n = 3) of the mean and the slope of FS on a linear scale of * w6 [, ^  v' k& N+ r0 o$ f
0 to 1,023 at least 41 (see Notes 3) (Fig. 6.2).
4.  For the assays, cells are lifted with trypsin/EDTA, washed with CCM by cen-
trifugation at 450  g for 10 min to neutralize trypsin, counted on a hemocytometer,

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地板

发表于 2010-3-9 18:29 |只看该作者

6 Colony Forming Unit Assays for MSCs 870 e$ g  z4 s. k
suspended in PBS at 4 °C at a concentration of about 0.5 million cells/mL, and
then assayed shortly thereafter (see Notes 4).# H- g+ ]* v9 q( l
5.  Staining with Annexin V-FITC demonstrates that the events in the upper left of % {$ T3 J5 n3 c& H8 [  f
the plot are cell debris and dead cells (R1 in Fig. 6.3B). To obtain sub-fractions 2 s. j: B  c) Z, s; d8 M
of cells, the Annexin V+
events are gated out and four sub-populations were
defined on the basis of FS and SS (Fig. 6.3C).
6.  For single cell sorting the cells treated with the Annexin V-FITC and maintained * S# V/ i! _! B* a2 F
at 4 °C to prevent aggregation from the presence of calcium and reagent-induced & _, q9 W) U. I, F* D
toxicity.6 E1 b6 r1 E0 U& E
7.  To isolate distinct fractions on the basis of FS and SS (Fig. 6.3C), divided the & G9 d7 K5 q8 Q$ k. `6 z
Annexin V−" y* O- V2 t0 I! n
events into 4 quadrants on the basis of FS and SS.9 b7 `! [/ {7 M) l
Fig. 6.1  (A) Crystal violet stained plates of CFU-F assays performed on two different donors
(10). (B) Sc-CFU is more sensitive and reproducible than the traditional CFU assay: A: Sc-CFU # q. Q+ m7 }, g
assay of MSCs initially plated at varying densities and incubated for 10–11 d (mean+/−SD, n=2).
B: Standard CFU assay of MSCs plated at same densities and incubated for 13–14 d (mean+/-SD, 2 |# P0 E1 D2 ?9 F* w* f9 M0 L9 ]
n = 3 or 4) (See Color Plates)

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7楼

发表于 2010-3-9 18:30 |只看该作者

8. The cells analyzed by the above method can be sorted using a flow cytometer. : z0 v- W# n* O; N
Cell preparations are run through open stream FACS instrument (FACSVantage;
Becton, Dickinson, Franklin Lakes, NJ). To isolate distinct fractions on the
basis of FS and SS (Fig. 6.3), divide the AnnexinV–
events into four quadrants 5 t _7 L: v8 C$ V8 w
on the basis of FS and SS. Then offset the sort gates from the boundaries.

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9. The accuracy of sorting single cells into each well of a microtiter plated should ' L/ T0 \# O. w6 ]4 a" |, v
be verified routinely by sorting fluorescent beads (Flowchek; Beckman-
Coulter) into a test plate and examining the wells with an epifluorescence
microscope.
10. The microtiter plates with one MSC per well are incubated in 0.15 mL CCM 6 s0 K. w; k0 r2 \2 n9 s
with change of medium every 4–5 d./ T& n# d' P5 x, C. i9 s
11.  After 2 wk, the medium is removed and the wells washed with PBS.
12. Parallel plates could be used for assays of sc-CFUs, for differentiation into
osteoblasts, and for differentiation into adipocytes. For sc-CFU assays, the
samples were incubated with 1 mL Crystal Violet in methanol for 1 min,
washed with water, and colonies with diameters greater than 1 mm counted by 1 b3 v& F& X/ J; E. y" x
microscopy (4× phase contrast) (Fig. 6.4).7 S9 Z1 ?( i$ c$ x; W# k
13. For assay of differentiation, the microtiter plates were incubated in 0.15 mL per
well of αMEM and specific differentiation medium as described in Chapter 7.
14. Cells treated with the Annexin V-FITC were maintained at 4 °C to prevent ( D, o: P9 m5 r8 N8 @  ~
aggregation and death from the presence of calcium and reagent-induced
toxicity.
4 Notes
1.  Suspension density for cell counting should be between 0.5 million to 1 million cells per mL.
2.  The cells should be well disassociated./ G" X1 ~. \4 B* C% z: Z2 ]
3.  The variation in values for log (%G/%T) should be established against samples containing 0.5
or 1 million MSCs per mL when the following parameters were varied: (a) the flow rate was
250, 500 or 900 cells per second; (b) the FS was assayed with 67 or 122 volts and a gain of 2
or with 353 volts and a gain of 1; and (c) the peak for FS of the 20 micron bead was set at 550,
650, or 750; and (d) the peak for SS the 7 micron bead was set at 350, 450 or 550." P+ H/ ^( V" H: B
4.  Confluency of cells in culture is important and cells should be harvested when they are less
than 80% confluent.& y6 d8 O1 F0 a1 q. N
References
1. Friedenstein, A. J., Chailakhyan, R. K., Latsinik, N. V., Panasyuk, A. F., and Keiliss-Borok, I. 9 V/ t6 p+ n+ G9 N, {: u* Z
V. (1974) Stromal cells responsible for transferring the microenvironment of the hemopoietic 5 s3 j" X" \- u; c& j' E' l
tissues. Cloning in vitro and retransplantation in vivo Transplantation. 17, 331–340.0 {$ W2 k2 F$ W
2. Prockop, D. J. (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues Science.
276, 71–74.  _* U1 C* b2 ~# u0 P6 P( J
3. Colter, D. C., Class, R., DiGirolamo, C. M., and Prockop, D. J. (2000) Rapid expansion of recy-
cling stem cells in cultures of plastic-adherent cells from human bone marrow  PNAS.  97, 4 f# U- B% Y$ U1 s" K
3213–3218.0 q9 {) \6 G5 g! ^7 E5 H7 e
4. Smith, J. R., Pochampally, R., Perry, A., Hsu, S. C., and Prockop, D. J. (2004) Isolation of a 6 k! B0 \  y) h
highly clonogenic and multipotential subfraction of adult stem cells from bone marrow stroma ! z0 P- \$ g! v3 u5 p& K8 ]! K
Stem Cells. 22, 823–831.

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9楼

发表于 2010-3-9 18:30 |只看该作者

6 Colony Forming Unit Assays for MSCs 91
  5. Sekiya, I., Larson, B. L., Smith, J. R., Pochampally, R., Cui, J. G., and Prockop, D. J. (2002) ; Z; g* O8 N- {" b9 T5 S6 E4 w
Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the
yields of early progenitors and evaluate their quality Stem Cells. 20, 530–541.( T) }& m( r; q
  6. Prockop, D. J., Sekiya, I., and Colter, D. C. (2001) Isolation and characterization of rapidly
self-renewing stem cells from cultures of human marrow stromal cells  Cytotherapy.  3, # L* i( {- Z2 \) R
393–396./ J$ j' _$ g: n3 z5 D
  7. Colter, D. C., Sekiya, I., and Prockop, D. J. (2001) Identification of a subpopulation of rapidly ! |. U) p7 e4 t7 t5 I: J
self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells.
Proc. Natl. Acad Sci. U S A. 98, 7841–7845.
  8. Peister, A., Mellad, J. A., Larson, B. L., Hall, B. M., Gibson, L. F., and Prockop, D. J. (2004)
Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary
in surface epitopes, rates of proliferation, and differentiation potential. Blood. 103, 1662–1668.# \3 r1 [, Y+ t" j) P
  9. Javazon, E. H., Colter, D. C., Schwarz, E. J., and Prockop, D. J. (2001) Rat marrow stromal ' R( ^* k/ @6 {, ]5 t0 z) L
cells are more sensitive to plating density and expand more rapidly from single-cell-derived - T9 ]% q- x+ e+ o5 ]; R
colonies than human marrow stromal cells Stem Cells. 19, 219–225.% J0 g+ A9 X7 b
10. DiGirolamo, C. M., Stokes, D., Colter, D., Phinney, D. G., Class, R., and Prockop, D. J. (1999) ' q* P- [5 |& M! G) h
Propagation and senescence of human marrow stromal cells in culture: a simple colony-form-9 t4 I3 O  H; c. ]! L2 Q5 S
ing assay identifies samples with the greatest potential to propagate and differentiate Br. J. - l, s7 V2 y! |1 q# l
Haematol. 107, 275–281.

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