CONCENTRATION, QUANTITA TION, AND IDENTIFICATIONOF HEMOPOIETIC STEM CELLS*

BY HELGARD NIEWISCH, HELMUT VOGEL, t AND GASTONE MATIOLI

DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF SOUTHERN CALIFORNIA,

SCHOOL OF MEDICINE, LOS ANGELES

Communicated by Seymour Benzer, October 16, 1967

Hemopoietic tissue contains "stem cells" capable of differentiating into a varietyof descendent cells (granulocytes, red cells, platelets, and their precursors) andcapable of self-renewal. Stem cells are defined only functionally as colony-formingunits (CFU) assayed by colonies formed in the spleen when injected into irradiatedsyngeneic mice.1 2

It has been shown3' 4 that each colony is a clone from a single stem cell. Turneret al.6 fractionated mouse bone-marrow suspensions by bovine serum albumingradient centrifugation and reported a 15-fold enrichment of stem cells in the densityfraction p = 1.050-1.055, but did not comment on the morphology of the cells.We have used spleen cells as the source of CFU, which can be prepared more

easily and in much larger quantities. The CFU from both marrow and spleenare functionally very similar, since both produce similar colonies in the spleen.CFU can be enriched by a combination of density gradient fractionation and trans-plantation.The assay for colony production can be improved by direct injection into the

spleen. The results, together with microscopic analysis of the cells, indicate aparticular cell type as being a CFU.

Materials and Methods.-Animals and irradiation procedure: Adult inbred mice, strain C3H(Bar Harbor) are used. A Co6n source is employed for the irradiation of the mice at a total bodydose of 960 rads at 60 rads per minute. This dose reduces the development of endogenous coloniesin noninjected control mice to an average of < 0.07 colonies per spleen 10 days postirradiation.

Preparation of spleen cells: Suspensions of isolated cells are prepared from the spleens of un-irradiated donor mice (15-20 for each experiment) by gently teasing the cells off the splenic cap-sule in small amounts of Hank's balanced salt solution (BSS) at 40C. This cell suspension ispassed through 4 or 5 layers of cheesecloth in order to remove cell clusters. The cells are washedby low-speed centrifugation in BSS, nucleated cells are counted in a hemocytometer, and 0.1 mlof a suspension containing 30-40 X 106 nucleated cells is injected intravenously into about 30-40C3H recipients ("first recipients") immediately after irradiation. Eleven days after injectionthese "first recipients" show growth of confluent colonies.

Concentration of CFU by density gradient centrifugation: A cell suspension is prepared from thespleens of the "first recipients" as described above, the final dilution containing 3-4 X 109 nu-cleated cells in 7-9 ml BSS. This volume is layered on a 60-ml linear gradient, with a densityrange of p = 1.015-1.085 prepared from 27% Ficoll (Pharmacia Fine Chemicals) in H20 with0.1 M NaCl, 0.022 M KCl, and 0.008 M sucrose. The pH is adjusted to 7.1 with NaHCO3. Thetube is spun at 11,000 rpm for 20 min in a Spinco SW 25.2 rotor at 40C. After centrifugation,various fractions are collected and their refractive index is checked with a Zeiss-Abbe refractometerto calculate the density. The presence of cells up to 1 X 107 per ml does not significantly influ-ence the refractive index. The fractions are washed with BSS to free the cells from Ficoll priorto injection. The desired cell dilutions are prepared and injected intravenously (i.v.) into groupsof 10-20 irradiated test mice. Eight days after injection, these mice are sacrificed, the spleensfixed in Bouin's solution, and the colonies counted.

Quantitation of stem cells by direct injection into the spleen (local assay): Because of the uncer-tainty that an intravenously injected cell will lodge in the spleen, it is difficult to determine the

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true number of stem cells present in a cell suspension by the number of spleen colonies formed.Much greater sensitivity and accuracy can be obtained by injecting cells directly into the spleen.The spleens of anesthetized irradiated mice are exposed aseptically by lateral laparotomy, andvarious numbers of cells in 1-5 ul ofJBSS are injected 1 mm deep into the spleen with a Hamiltonsyringe mounted on a micromanipulator. A siliconized glass microneedle (25-30 1 i.d. at the tip)is prepared for each experiment. To avoid excessive bleeding only one injection per spleen is done.The laparotomic incision is closed by clamps or suture and covered with adhesive wound spray.Microscopy of cells in various fractions: Microscopical analysis of the cells found in the various

density gradient fractions is performed on smears, stained with May-Griinwald-Giemsa. A totalof 1,000-2,000 cells are classified by two independent observers. The cells are grouped as follows:erythroblasts and nuclei, cells "B," cells "B1" (see Results), granulocytes and precursors, lympho-cytes.' These groups include more than 95% of the cells seen in the unfractionated samples (therest being damaged or broken cells of difficult identification) and more than 99% of the cellsfound in fractions with densities between 1.020-1.040.

Results.-(a) Effect of irradiation on the density distribution of spleen cells: It isdesirable to distinguish between cells of the irradiated host and those derived fromthe irradiated stem cells. Fortunately, it was found that irradiation of mice at960 rads produces a considerable change in the density profile of spleen cells. Thisis shown in Figure 1. The profile for "first recipient" spleen cells (Fig. lc) issimilar to that for unirradiated controls (Fig. lb). In the gradient region betweenp = 1.020 and 1.045, about 7 per cent of the total cell input from these preparationsis recovered, whereas only 0.01 per cent is recovered from the irradiated cells (Fig.lc) in a comparable density range. In all three preparations most of the cells

DENSITY 1.020 1.030 1.040 1.050 1.060 1.080 T UBE

a~~ ~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~Tas 50a 1.. 5 4.0 4.6 5.0 5.5 6.1 1400 500

b 0.1 .05 .05 .025 .02 -.02 7 9 0 1200

C 1.2 4.8 7.7 120 2175 1 0 20 550 59011000

FIG. 1.-Effect of irradiation on the density distribution of total spleen cells. (a) Unirradiatedcells from untreated donor; (b) irradiated spleens, 10 days after irradiation at 960 rads; (c) "firstrecipient" spleens with confluent colonies. Cell count in millions.

band at higher densities. This experiment strongly suggests that the cells from the"first recipient" found between p = 1.015 and 1.045 are mainly derived from theconfluent colonies of the "first recipient."

(b) Distribution of stem cells from "first recipient" spleen cells in the density regionbetween p = 1.015 and 1.040: The cells found in the density region between p =1.015 and 1.040 are of the most interest since they are mainly derived from coloniesproduced by transplanted stem cells in "first recipients." Stem cells were assayedby the colonies produced after i.v. injection of cell aliquots from various fractions(Ap = 0.005). The enrichment of stem cells in each fraction can be expressed bythe ratio of the colonies produced by a given number of injected cells, comparedwith the unfractionated preparation. In 21 experiments the maximal enrichmentalways occurred in fractions p = 1.020-1.025. The enrichment values ranged from2.4 to 28 with an average of 5.4. Values above 20 were obtained in four experi-ments. The data for one of these are presented in Table 1. Essentially no en-

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VOL. 58, 1967 PATHOLOGY: NIEWISCH ET AL. 2263

richment was observed at densities ranging from p = 1.045-1.085. Consequently,our studies on stem cells were concentrated on the density range p = 1.015-1.045.

(c) Microscopy of the cells in the maximal fraction: Unfractionated preparationsof "first recipient" spleen cells are mainly composed of erythroblasts at variousstages of maturation, isolated nuclei, and polymorphs and their precursors. Lym-phocytes, reticulum cells, and ill-defined cell types appear in minor proportions.However, cells in the region p = 1.020-1.025 show less heterogeneity.

TABLE 1ENRICHMENT OF STEM CELLS IN DENSITY GRADIENT

Colony count for Total number of Enrichment4.8 X 10' i.v. cells in fraction (fractionated vs.

Density injected cells (millions) unfractionated)Unfractionated 22 2,100 1

1.015 33 0.45 1.51.020 518 1.9 241.025 144 8.3 6.51.030 103 9.9 4.71.035 100 25 4.51.040 66 26 3.01.0451.045 27 2,000 1.21.085

"First recipient" spleen cells were fractionated by density gradient. 2 X 104 cells from each fraction wereinjected i.v. into each of 24 mice. The standard deviations in colony count for each batch of 24 mice did not de-part significantly from a Poisson distribution.

Figure 2 shows a view of the cells found in this fraction in the experiment ofTable 1, which gave a particularly high yield of colonies. Easily identifiable are thenaked nuclei which are numerous and appear as intensely stained round bodies of4-6 u in diameter. These nuclei are sometimes surrounded by residual cytoplasmwith traces of hemoglobin, and are probably derived from the erythroblasts whichare constantly seen in this fraction. The next most frequent cell type in this frac-tion, particularly frequent in the above-mentioned experiment (20%), is a largecell with a rather homogeneous over-all appearance. In smears, the cell diameter is14-17 g. The nucleus, 13-16 u in diameter, possesses a loose chromatin structure.The cytoplasm is intensely basophilic. These cells will be denoted as type "B."Another basophilic cell type is similar to "B" but contains scattered granules, about0.5 u in diameter. It is observed in much smaller numbers. Other cells occurringin small amounts are lymphocytes and granulocytes at various stages of develop-ment.

(d) Correlation of cell type with colony-forming ability in various density fractions:Examination of various fractions denser than p = 1.025 showed much smaller pro-portions of type "B." To check for the correlation of different cell types withcolony formation, various density fractions and their respective unfractionatedcells were evaluated by differential cytological counts. The average percent-ages of the various cell types from nine centrifugation runs are shown in Table2. It is evident even from these averages that the only single cell type showing anobvious correlation is cell "B."In order to test this correlation in detail, a correlation matrix was set up between

cell types and colony counts, using the data of the 29 fractionated and unfrac-tionated samples included. This was done for each cell type as well as for somecombinations of cell types. The most pertinent results are given in Table 3. It

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TABLE 2DISTRIBUTION OF CELL TYPES EXPRESSED AS PER CENT OF TOTAL CELLS*

Unfractionated - ---Fractionated CellsCell type cells 1.020-1.025 1.025-1.030 1.030-1.035E-N 74 + 13 89±i 6 87 ± 9 86 + 3G 19 + 14 0.7 ± 0.7 2.0 +E 2.4 5.4 i 3.1L 2.7 + 2.5 2.0 + 3.3 2.1 ± 3.0 2.2 + 1.8B 1.1 + 0.14 7.3 + 6.6 4.6 ± 3.0 3.8 + 2.0

B, 0.25 ± 0.36 1.4 + 1.1 3.4 ± 3.4 1.4 + 1.7Others 3.6 0.1 0.1 1.0Colonies per 2 X 104

i.v. inj. cells 1.2 ± 0.6 8.0 + 8.1 6.1 i 4.(0 3.4 i 2.0

"First recipient" spleen cells were used (pooled data of 9 experiments).* E-N, erythroblasts and their nuclei; G, granulocytes and precursors; L, lymphocytes; B, cells type "B"; Bi

cells "B" with granules; Others, damaged cells of uncertain identification. i standard deviation.

is shown that cell "B" not only has a correlation coefficient closest to 1 (0.909), butalso its regression line is closest to the origin, which is also a necessary criterion ofproportionality. Furthermore, this cell type has the smallest standard error of theslope of the regression line.

(e) Assay by direct injection into the spleen: Looking at the richest fraction inTable 1, one finds one colony per 1,000 i.v. injected cells. The average for 21 suchruns is one colony per 3,000 cells. The question now arises what proportion ofthese cells could be stem cells, since in the process of the assay there are reasons why

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)X 1FI. :1'~FI.2.-Microscopic view of "first recipient" spleen cells from the density fraction p =

1.020-1.025. Arrow points to a cell of type "B." Several other cells of this kind can beseen. The small dark spots are erythroblasts or their nuclei. There are two lymphocytesin the upper right part of the field (X300). Inset: enlarged view of a type "B" cell(X 1300)).

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TABLE 3RESULTS OF REGRESSION ANALYSIS ON CORRELATION OF CELL TYPE WITH

COLONY FORMATION'Cell type r b a/maxE+N 0.045 21± 91 0.882G -0.346 -154 i 80 0.232L 0.206 25 i 23 0.164B 0.909 175 i 15 0.019Bi 0.113 18.5 i 31 0.061

B + Bi 0.698 194 i 38 0.075B + By + L 0.611 291 i 54 0.086

r, correlation coefficient of cell count X with colony count C; b, slope of regression line X(C)of cell count X ± standard error; a/max, relative distance of regression line from origin, wherea is the intercept of regression line with x-axis, and max is the maximum observed value. Equalweight is given to each point.

an intravenously injected stem cell may fail to produce a colony. Firstly, becauseof random distribution in the body, it might never reach the spleen," and secondly,it might become extinct due to differentiation. The first of these reasons for lossof stem cells can be minimized by injecting the cells directly into the spleen (localassay). Figure 4 shows the colony yield from various preparations produced by thelocal assay. The colony number is proportional to the number of injected cells upto one colony, which is formed after injection of about 70 fractionated (p = 1.020-1.025) and about 200 unfractionated cells from a "first recipient." For highernumbers of injected cells (up to at least 104) the colony number is constant (aboutone). A detailed discussion of this behavior will be given elsewhere.8

In contrast to the local assay, the i.v. injection shows proportionality up to atleast 6' 104 injected cells, as shown in Figure 3. A comparison of the slopes ofFigures 3 and 4 demonstrates that direct injection is much more sensitive than thei.v. injection. Table 4 shows, for several preparations, an average sensitivity gainof 56 ± 16.Discussion.-The results of the regression analysis implicate cell type "B" as a

stem cell. The i.v. assay does not determine the true number of stem cells but only

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number of injected cells (i0 FIG. 4.-Colony yield vs. numberof cells injected directly into the

FIG. 3.-Colony yield vs. number of intravenously in- spleen. (b) "First recipient" cells,jected cells. (a) Normal cells, density p = 1.020-1.025 unfractionated (V). (c) "First re-(i). (c) "First recipient" cells, density p = 1.020-1.025 cipient" cells, density p = 1.020-(e). 1.025 (v).

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TABLE 4COLONY YIELD PER 105 INJECTED CELLS FROM VARIOUS PREPARATIONS

Fractionated.------ Unfractionated p = 1.020-1.025

Normal 1st recipient 1st recipientI.v. assay 2.8 4t 1.9 6.0 i 2.8 32.2 i 5.8

(6 exp) (19 exp) (21 exp)Local assay 130 i 20 470 ±t 32 1330 i 58

(3 exp) (7 exp) (3 exp)Sensitivity gain 47 78 42(local/i.v.)

Average values from the number of experiments indicated in parentheses.4 standard deviation.In the i.v. assay, the number of injected cells was 2 X 104, in the local assay between 25 and 100.

The colony yield was then computed on the basis of 105 injected cells. Since the standard deviationsdid not depart significantly from the value expected for a Poisson distribution, they were multipliedby the factor (106/number of inj. cells)l".

gives a lower limit (0.03% for the richest fraction, p = 1.020-1.025). The directinjection technique shows that at least 1.3 per cent of the cells (Fig. 3) in the richestfraction of "first recipient" cells are stem cells. Direct injection accounts for onlyone of the reasons for loss of stem cells, namely, dilution in the body of the host.The other main reason, which is the extinction in the stochastic differentiationprocess,7 can be shown to affect about two thirds of all stem cells reaching thespleen.9 Consequently, the estimated true number of stem cells in the richest frac-tion would be at least 4 per cent. This excludes any minority cell type as a stemcell candidate and fits reasonably well with the microscopically observed number ofcells "B" (Table 2).These arguments make a rather strong circumstantial case for cell "B" as a stem

cell. To decide this beyond reasonable doubt, it would be desirable to obtain apurer preparation of cells "B," or grow the cells in tissue culture. Work on bothtechniques, as well as a kinetic study of the development of stem cells and theirdescendents, is in progress.

It is essential to note (Table 1) that the stem cells falling in the density fractionp = 1.020-1.025 represent only a minuscule fraction (about 1%) of the total stemcells in the unfractionated preparation. The great mass of stem cells (about 90%)bands at densities higher than 1.045.Work from other laboratories'0 suggests that at 11 days the spleen colonies are

mainly engaged in the production of granulocytes, while at an early age the coloniesare almost exclusively erythroblastic. The stem cells used in the present work wereobtained from 11-day-old colonies. The pluripotency of the stem cell has beendemonstrated by retransplantation of a single colony containing erythroblasts orgranulocytes.II Both granulocytic and erythroblastic colonies were found in thespleen of the recipient mice. It seems reasonable to propose that the hemato-logically competent stem cell may exist in several metastable states with differentmorphological characteristics, one of which is represented by cell "B."Summary.-By transplantation of spleen cells into irradiated hosts and subse-

quent fractionation by density gradient centrifugation an enrichment of stem cellscapable of forming spleen colonies on injection was obtained. Microscopy re-vealed a cell type with a distribution correlating to the colony-forming stem cells incertain density fractions. By direct injection into the spleen the efficiency of colonyformation was increased 50 times over the usual intravenous technique. In favor-

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able preparations the abundance of the special cell type was consistent with thenumber of colonies produced.

We thank the Department of Nuclear Medicine at UCLA for the use of the Colo irradiationfacilities.

* This research was supported by NIH grant HE 10568, ASCIG, and ACS.t On leave from the Department of Physics, Technische Hochschule, Mfinchen, West Germany.1 Till, J. E., and E. A. McCulloqh, Rad. Re8., 14, 213, 1961.2 McCulloch, E. A., and J. E. Till, Rad. Re8., 22, 383, 1964.3 Becker, A. J., E. A. McCulloch, and J. E. Till, Nature, 197, 453, 1963.4 Wu, A. M., J. E. Till, L. Siminovitch, and E. A. McCulloch, J. Cell. Physiol., 69, 177, 1967.6 Turner, R. W. A., L. Siminovitch, E. A. McCulloch, and J. E. Till, J. Cell. Physiol., 69, 73,

1967.Wintrobe, M. M., Clinical Hematology (Philadelphia: Lea & Febiger, 1961).

7Till, J. E., E. A. McCulloch, and L. Siminovitch, these PROCEEDINGS, 51, 29, 1964.8Matioli, G., H. Vogel, and H. Niewisch, in preparation.9Vogel, H., H. Niewisch, and G. Matioli, in preparation.10 Curry, J. L., J. J. Trentin, Developmental Biol., 15, 395, 1967."Wolf, N. S., and J. J. Trentin, Federation Proc., 25, 296, 1966.12 Correlation analysis was done with a Honeywell 800 computer. The program was formu-

lated according to "Socomp manual, Comp. Sci. Lab," ed. University of Southern California,1967.

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