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Experimental
CHICK
Cell Research 102 (1976) 9-13
ERYTHROCYTE
Their Expression Haematopoietic
ANTIGENS
in Irradiated Chicks Grafted with Tissues from Different Origins
J.-P. BLANCHET, Dipartement de Biologie Universite’ Claude
MEMBRANE
&hale Bernard,
J. SAMARUT
and V. NIGON
et appliquke, Laboratoire associi au CNRS, Lyon I, F-6%21 Villeurbanne, France
SUMMARY Three erythrocyte populations (E, EA, A) were characterized during normal chick development by presence on cells of the embryonic (E) or adult (A) antigen or both (EA). Embryonic and adult stem cells were grafted into irradiated animals in order to distinguish the respective influence of stem cell origin and physiological conditions in the production of antigens. Adult marrow stem cells produce A erythrocytes. Embryonic stem cells (from 6- or I l-day-old embryo yolk sac) give rise first to E, then to EA populations. These results confirm the existence of adult stem cells with their own properties. It was not possible to decide whether the E and EA populations arise from a unique embryonic stem cell or from the existence of two stem cell populations.
In the preceding paper [l] two antigens detected on chick erythrocytes allowed the characterization of three erythrocyte populations (E, EA and A) after hatching. To explain their succession (E disappears during the first month, EA appears on 3rd day following hatching and disappears 5 months later, A appears 3 weeks after hatching and forms adult blood) two hypotheses were put forward: these populations either arise from separate stem cell populations or result from the influence of changing physiological conditions on the same stem cell population (or on its progeny). Stem cells present in haematopoietic tissues grafted into irradiated chicks restore erythropoiesis [7, 81. The respective influences of environmental factors and stem cell properties on the erythropoiesis could be checked by comparing results obtained
when stem cells from different age donors were grafted into hosts of same age [4]. In order to apply this method to erythrocyte antigen detection, we chose adult bone marrow and 11-day-old embryo vitelline cells whose stem cells produced, respectively, A and E cells in vivo. The results showed that the antigenic constitution of erythrocytes produced in 23-day-old irradiated chicks depended on the origin of grafted stem cells. MATERIAL
AND
METHODS
Random bred white Leghorns were used. 23-day-old chicks received 760 R and 990 R on 2 consecutive days from an X-ray Stabilipan Siemens machine [7]. They were injected immediately after the second irradiation with 4-5x 108 1 l-day-old vitelline or adult bone marrow cells prepared according to Samarut & Nigon [7]. Adults received 700 R on the first day and 900 R on the second day (62 R/min flow, source-target distance=75 cm, 0.2 mm Al filter, 190 kV, 20 mA) and Exp
Cell
Res 102 (1976)
10
Blanchet,
Samarut and Nigon 7
30
20
IO
Fig. 1. Abscissa: time following graft (days); ordinate: no. of cells x IOr’. l - - -0, Vitelline cells from 1 l-dayold embryos; *---*, adult marrow cells; t--+, irradiated, non-grafted chicks. E, EA and A cell production in blood of irradiated 23-day-old chicks, non-grafted (controls) or grafted with various haematopoietic tissues. For control values, 50 chicks were irradiated and 4 were studied. Each time one of the four died, a new chick from the initial irradiated flock was introduced. On the 10th day, only two chicks were examined and from 12th day onward only one.
were immediately injected with 3~ 109 vitelline cells from 6- or II-day-old embryos. The embryonic and adult antigen detection, allowing the evaluation of E, EA and A populations, was described in the preceding paper [ 11. The erythrocyte count was determined with a Malassez haematimeter. The blood volume which remains unmodified by irradiation [7] was estimated from the body weight [6].
10 days onward was due to regeneration of the normal erythropoiesis occurring in the single surviving chick (see caption to fig. 1). In irradiated chicks grafted either with vitelline or bone marrow cells, the following results were observed: E cells: They disappear in the same way whether chicks are irradiated only or receive adult marrow cells. The E population level was maintained in vitelline cell-injected chicks. EA cells: Their number rapidly decreases and the EA restoration (observed in the sole irradiated, non-grafted surviving chick) is not clearly apparent in adult marrow-injected chicks. The EA population was less diminished in vitelline cell-grafted chicks. A cells: Their total number increases rapidly from the 5th day onward in marrow cell-grafted chicks. By contrast, no A cells were produced during the first 10 days, in vitelline cell-injected chicks, Then regeneration of erythropoiesis occurred.
A
6
1
y-7 I
RESULTS Antigenic changes in the blood of irradiated, non-grafted or grafted 23-day-old chicks are shown in fig. 1. In non-grafted chicks, the E cells disappeared rapidly, the EA population was greatly reduced and the appearance of A cells, as observed in control chicks [l], was delayed for about 10 days. However, these results must be viewed with reserve since this experiment inevitably involves the selection of the most resistant animals and thus does not reflect the response of a homogeneous population. In fact, the increase in EA and A cells from Exp Cell Res IO2 (1976)
Fig. 2. Abscissa: time folIo@ng graft (days); ordinate: cell no. X log. 0. . . l , E; *-*, EA; *-*, A population. Estimation of the erythrocytic production of vitelline stem cells from (A) ll-day-old embryos; (II) adult marrow stem cells. Values are the difference between the total number of E, EA and A erythrocytes present in grafted irradiated and non-grafted irradiated 23-dayold chicks. Negative values were plotted as zero.
Antigen expression in irradiated
and grafted chicks
11
bryos (fig. 4B, table 1). These results clearly show that vitelline stem cells give rise to E and EA cells. No megalocyte has ever been observed, even with vitelline cells from 6-day-old embryos. DISCUSSION
3. Immunofluorescent detection of embryonicantigen-bearing cells in the blood of irradiated adult grafted with yolk sac from 6-day-old embryos. (Top) UV illumination; (bottom) the same field as seen with tungsten light.
Fig.
In conclusion, adult marrow stem cells apparently produce A cells, vitelline stem cells E and EA cells. This is obvious when the production of grafted stem cells is evaluated (fig. 2). However, the normal production of EA erythrocytes by 23-day-old chicks leaves some doubt on the origin of EA erythrocytes which crop up in vitelline cell-grafted chicks. To resolve this ambiguity, vitelline cells from 1l-day-old embryos were grafted into irradiated adults. Only A cells are present in adult blood [l] and no modification was observed after irradiation without graft. Thus the appearance of embryonic antigenbearing cells was easily detected (fig. 3). The first cells which appeared belong to the E population (fig. 4A, table 1); their number decreased on the 4th day following graft. Then EA cells appeared. Similar results were obtained in irradiated adults having received vitelline cells from 6-day-old em-
During post-embryonic chick development, three erythrocyte populations (E, EA, A) have been characterized by their antigenic composition. To determine if these populations arise from different stem cell populations or from different environmental factors acting on a unique stem cell population, haematopoietic tissues from embryos or adults were grafted into 23-day-old chicks or into adults. Our results show that stem cells from adult marrow give rise to A erythrocytes only in an environment which still allows production of embryonic antigen-bearing cells. This confirms previous results [l] demonstrating the inability of anaemic adults to produce cells other than A cells. By contrast, vitelline stem cells grafted
A
7t
* I”,
E
r-*/
I\, ‘, \
I
5
\ *\
f \
It,
I \
I
I
\ *
I
I )A ‘O-j:
e
1
J
\
\
\
\
\ 1
/,A /’ ,,,,,.,.,.. *-* 1 3 5
)j *-s-J 10
1
3
5
10
time following graft (days); ordinate: ceil no. x 108. O-e, E; *-*, GA erythrocytes. Time of atmearance of E cells and EA cells in the blood of irradiated adults grafted with vitelline cells from (A) 11-day-old embryos; or (B) bday-old embryos.
Fia. 4. Abscissa:
Exp Cell
Res 102 (1976)
12
Blanchet,
Samarut and Nigon
Table 1. E and EA erythrocyte total number in the blood of irradiated &day-old or 11-day-old vitelline cells (IO9 cells/animal)
adults grafted with
Days after grafting 1
3
4
5
Vitelline cells from 11-day-old embryos No. 183 E 2.0 4.3 7.6 6 EA 0.5 0 2.7 7.5 No. 181 E 3.1 2.2 7.4 4.4 EA 0.5 1 0 3.8 No. 381 E 3.5 6.4 15.8 12.3 N:‘?58 0 0 2.6 8.4 li EA
2.2 0
Vitelline cells from No. 744 E 1.8 EA 0.8 No. 729 E 0.9 EA 0
7.2 0
10.9 1.3
4 8.9
6
7
a
9
5.3 5.3
4.8 8.5
-
14.2 7.6
6.8 -
6.2 10.4
2.6 8.4
2 5.4
3 13.4
10
11
12
14
17
21
-
5 10.4
4.7 6.1
2.2 7
-
0.9 10
-
0.7 8
0 4.9
0 6.3
0 4.1
07 318
0 2.6
1
-
1.5 11.4
5.9 5
&day-old embryos 1.2 0.6
1.4 1
1.1 2.9
0.8 5.5
0.8 7.6
0.6 5.3
:::
3.9 0
7.1 0
5.6 2.2
5.6 10.7
11.5 11.5
5.4 14.4
7.2 16.9
6.6 11.1
into chicks or adults give rise to E and EA (2) E and EA populations arise from two stem cell populations, both present in the erythrocytes. There are two possible exyolk sac of 6-day-old embryos, one (EA) planations. (1) E and EA erythrocytes proceed from being inactive. The rapid appearance of E the same stem cell population. EA cell ap- cells could result from immediately active E pearance would then result from the in- precursors. already engaged in erythrofluence of environmental factors either on poietic pathway at the time of grafting by contrast to EA stem cells which would be pluripotent stem cells or on an intermediary inactive in embryo. A reduced (or superythropoietic precursor. In these conditions, the delay observed in the appearance pressed) activity of the corresponding stem of EA cells (fig. 4) could result from the cells would explain the rapid disappearance time necessary for stem cells to express this of E cells. This last possibility agrees with influence. Another explanation could lie in \ recent observations by Samarut & Nigon [8] the late expression of adult antigen dur- Ithat most vitelline stem cells have a more ing maturation of erythrocytes, thus trans- limited proliferative capability than do adult forming E into EA erythrocytes. At any stem cells. rate, a reduced level of adult antigen was During normal development, the EA observed on immature cells [ 11. This would population appears after hatching [ 11, therealso explain the abrupt reduction of E cell fore arising from bone marrow stem cells. number 4 days after graft (fig. 4). The pos- The graft experiments described in this sibility of a rapid destruction of these cells, paper demonstrate the presence of stem however, cannot be ruled out. cells already able to give rise to EA cells in Exp CellRes
102 (1976)
Antigen
expression
the vitelline sac. Presence of similar stem cells in vitelline and bone marrow is not surprising if these haematopoietic tissues are seeded by blood-borne stem cells arising from still unknown intraembryonic sites [2,5]. However, stem cells giving rise to the A population do not appear to be present among vitelline stem cells. In conclusion, this work demonstrates the existence of adult stem cells giving rise to erythrocytes characterized by the presence of the adult antigen and by a reduced synthesis of foetal haemoglobin [3, 41. These properties appear to be possessed by stem cells which still retain a high proliferative capability. In contrast to adult stem cells, vitelline stem cells produce the embryonic antigen and a relatively high proportion of foetal haemoglobin. Whether E and EA Cells arise or not from different
stem from which duced
in irradiated
and grafted
chicks
13
cell populations cannot be deduced our present results. Situations in only E or EA cells would be proare now under investigation.
The authors are indebted to Dr Grantham for reviewing the manuscript. Mrs M. F. Grasset provided excellent technical assistance.
REFERENCES 1. Blanche& J P, Exp cell res 102 (1976) 1. 2. Dieterlen-Lievre, F, J embryo1 exp morph01 33 (1975) 607. 3. Godet, J, Dev biol40 (1974) 199. 4. Godet, J, Samarut, J & Nigon, V, Compt rend acad sci 38 (1975) 624. 5. Le Douarin, N, Houssaint, E & Jotereau, F V, Proc natl acad sci US 72 (1975) 2701. 6. Medway, W & Kare, W R, Poultry sci 38 (1959) 624. 7. Samarut, J & Nigon, V, J embryo1 exp morph01 33 (1975) 259. 8. -Ibid. In press.
Received April 2. 1976 Accepted April 27: 1976
Exp Cell Res 102 (1976)
Experimental
CHICK
Cell Research 102 (1976) 9-13
ERYTHROCYTE
Their Expression Haematopoietic
ANTIGENS
in Irradiated Chicks Grafted with Tissues from Different Origins
J.-P. BLANCHET, Dipartement de Biologie Universite’ Claude
MEMBRANE
&hale Bernard,
J. SAMARUT
and V. NIGON
et appliquke, Laboratoire associi au CNRS, Lyon I, F-6%21 Villeurbanne, France
SUMMARY Three erythrocyte populations (E, EA, A) were characterized during normal chick development by presence on cells of the embryonic (E) or adult (A) antigen or both (EA). Embryonic and adult stem cells were grafted into irradiated animals in order to distinguish the respective influence of stem cell origin and physiological conditions in the production of antigens. Adult marrow stem cells produce A erythrocytes. Embryonic stem cells (from 6- or I l-day-old embryo yolk sac) give rise first to E, then to EA populations. These results confirm the existence of adult stem cells with their own properties. It was not possible to decide whether the E and EA populations arise from a unique embryonic stem cell or from the existence of two stem cell populations.
In the preceding paper [l] two antigens detected on chick erythrocytes allowed the characterization of three erythrocyte populations (E, EA and A) after hatching. To explain their succession (E disappears during the first month, EA appears on 3rd day following hatching and disappears 5 months later, A appears 3 weeks after hatching and forms adult blood) two hypotheses were put forward: these populations either arise from separate stem cell populations or result from the influence of changing physiological conditions on the same stem cell population (or on its progeny). Stem cells present in haematopoietic tissues grafted into irradiated chicks restore erythropoiesis [7, 81. The respective influences of environmental factors and stem cell properties on the erythropoiesis could be checked by comparing results obtained
when stem cells from different age donors were grafted into hosts of same age [4]. In order to apply this method to erythrocyte antigen detection, we chose adult bone marrow and 11-day-old embryo vitelline cells whose stem cells produced, respectively, A and E cells in vivo. The results showed that the antigenic constitution of erythrocytes produced in 23-day-old irradiated chicks depended on the origin of grafted stem cells. MATERIAL
AND
METHODS
Random bred white Leghorns were used. 23-day-old chicks received 760 R and 990 R on 2 consecutive days from an X-ray Stabilipan Siemens machine [7]. They were injected immediately after the second irradiation with 4-5x 108 1 l-day-old vitelline or adult bone marrow cells prepared according to Samarut & Nigon [7]. Adults received 700 R on the first day and 900 R on the second day (62 R/min flow, source-target distance=75 cm, 0.2 mm Al filter, 190 kV, 20 mA) and Exp
Cell
Res 102 (1976)
10
Blanchet,
Samarut and Nigon 7
30
20
IO
Fig. 1. Abscissa: time following graft (days); ordinate: no. of cells x IOr’. l - - -0, Vitelline cells from 1 l-dayold embryos; *---*, adult marrow cells; t--+, irradiated, non-grafted chicks. E, EA and A cell production in blood of irradiated 23-day-old chicks, non-grafted (controls) or grafted with various haematopoietic tissues. For control values, 50 chicks were irradiated and 4 were studied. Each time one of the four died, a new chick from the initial irradiated flock was introduced. On the 10th day, only two chicks were examined and from 12th day onward only one.
were immediately injected with 3~ 109 vitelline cells from 6- or II-day-old embryos. The embryonic and adult antigen detection, allowing the evaluation of E, EA and A populations, was described in the preceding paper [ 11. The erythrocyte count was determined with a Malassez haematimeter. The blood volume which remains unmodified by irradiation [7] was estimated from the body weight [6].
10 days onward was due to regeneration of the normal erythropoiesis occurring in the single surviving chick (see caption to fig. 1). In irradiated chicks grafted either with vitelline or bone marrow cells, the following results were observed: E cells: They disappear in the same way whether chicks are irradiated only or receive adult marrow cells. The E population level was maintained in vitelline cell-injected chicks. EA cells: Their number rapidly decreases and the EA restoration (observed in the sole irradiated, non-grafted surviving chick) is not clearly apparent in adult marrow-injected chicks. The EA population was less diminished in vitelline cell-grafted chicks. A cells: Their total number increases rapidly from the 5th day onward in marrow cell-grafted chicks. By contrast, no A cells were produced during the first 10 days, in vitelline cell-injected chicks, Then regeneration of erythropoiesis occurred.
A
6
1
y-7 I
RESULTS Antigenic changes in the blood of irradiated, non-grafted or grafted 23-day-old chicks are shown in fig. 1. In non-grafted chicks, the E cells disappeared rapidly, the EA population was greatly reduced and the appearance of A cells, as observed in control chicks [l], was delayed for about 10 days. However, these results must be viewed with reserve since this experiment inevitably involves the selection of the most resistant animals and thus does not reflect the response of a homogeneous population. In fact, the increase in EA and A cells from Exp Cell Res IO2 (1976)
Fig. 2. Abscissa: time folIo@ng graft (days); ordinate: cell no. X log. 0. . . l , E; *-*, EA; *-*, A population. Estimation of the erythrocytic production of vitelline stem cells from (A) ll-day-old embryos; (II) adult marrow stem cells. Values are the difference between the total number of E, EA and A erythrocytes present in grafted irradiated and non-grafted irradiated 23-dayold chicks. Negative values were plotted as zero.
Antigen expression in irradiated
and grafted chicks
11
bryos (fig. 4B, table 1). These results clearly show that vitelline stem cells give rise to E and EA cells. No megalocyte has ever been observed, even with vitelline cells from 6-day-old embryos. DISCUSSION
3. Immunofluorescent detection of embryonicantigen-bearing cells in the blood of irradiated adult grafted with yolk sac from 6-day-old embryos. (Top) UV illumination; (bottom) the same field as seen with tungsten light.
Fig.
In conclusion, adult marrow stem cells apparently produce A cells, vitelline stem cells E and EA cells. This is obvious when the production of grafted stem cells is evaluated (fig. 2). However, the normal production of EA erythrocytes by 23-day-old chicks leaves some doubt on the origin of EA erythrocytes which crop up in vitelline cell-grafted chicks. To resolve this ambiguity, vitelline cells from 1l-day-old embryos were grafted into irradiated adults. Only A cells are present in adult blood [l] and no modification was observed after irradiation without graft. Thus the appearance of embryonic antigenbearing cells was easily detected (fig. 3). The first cells which appeared belong to the E population (fig. 4A, table 1); their number decreased on the 4th day following graft. Then EA cells appeared. Similar results were obtained in irradiated adults having received vitelline cells from 6-day-old em-
During post-embryonic chick development, three erythrocyte populations (E, EA, A) have been characterized by their antigenic composition. To determine if these populations arise from different stem cell populations or from different environmental factors acting on a unique stem cell population, haematopoietic tissues from embryos or adults were grafted into 23-day-old chicks or into adults. Our results show that stem cells from adult marrow give rise to A erythrocytes only in an environment which still allows production of embryonic antigen-bearing cells. This confirms previous results [l] demonstrating the inability of anaemic adults to produce cells other than A cells. By contrast, vitelline stem cells grafted
A
7t
* I”,
E
r-*/
I\, ‘, \
I
5
\ *\
f \
It,
I \
I
I
\ *
I
I )A ‘O-j:
e
1
J
\
\
\
\
\ 1
/,A /’ ,,,,,.,.,.. *-* 1 3 5
)j *-s-J 10
1
3
5
10
time following graft (days); ordinate: ceil no. x 108. O-e, E; *-*, GA erythrocytes. Time of atmearance of E cells and EA cells in the blood of irradiated adults grafted with vitelline cells from (A) 11-day-old embryos; or (B) bday-old embryos.
Fia. 4. Abscissa:
Exp Cell
Res 102 (1976)
12
Blanchet,
Samarut and Nigon
Table 1. E and EA erythrocyte total number in the blood of irradiated &day-old or 11-day-old vitelline cells (IO9 cells/animal)
adults grafted with
Days after grafting 1
3
4
5
Vitelline cells from 11-day-old embryos No. 183 E 2.0 4.3 7.6 6 EA 0.5 0 2.7 7.5 No. 181 E 3.1 2.2 7.4 4.4 EA 0.5 1 0 3.8 No. 381 E 3.5 6.4 15.8 12.3 N:‘?58 0 0 2.6 8.4 li EA
2.2 0
Vitelline cells from No. 744 E 1.8 EA 0.8 No. 729 E 0.9 EA 0
7.2 0
10.9 1.3
4 8.9
6
7
a
9
5.3 5.3
4.8 8.5
-
14.2 7.6
6.8 -
6.2 10.4
2.6 8.4
2 5.4
3 13.4
10
11
12
14
17
21
-
5 10.4
4.7 6.1
2.2 7
-
0.9 10
-
0.7 8
0 4.9
0 6.3
0 4.1
07 318
0 2.6
1
-
1.5 11.4
5.9 5
&day-old embryos 1.2 0.6
1.4 1
1.1 2.9
0.8 5.5
0.8 7.6
0.6 5.3
:::
3.9 0
7.1 0
5.6 2.2
5.6 10.7
11.5 11.5
5.4 14.4
7.2 16.9
6.6 11.1
into chicks or adults give rise to E and EA (2) E and EA populations arise from two stem cell populations, both present in the erythrocytes. There are two possible exyolk sac of 6-day-old embryos, one (EA) planations. (1) E and EA erythrocytes proceed from being inactive. The rapid appearance of E the same stem cell population. EA cell ap- cells could result from immediately active E pearance would then result from the in- precursors. already engaged in erythrofluence of environmental factors either on poietic pathway at the time of grafting by contrast to EA stem cells which would be pluripotent stem cells or on an intermediary inactive in embryo. A reduced (or superythropoietic precursor. In these conditions, the delay observed in the appearance pressed) activity of the corresponding stem of EA cells (fig. 4) could result from the cells would explain the rapid disappearance time necessary for stem cells to express this of E cells. This last possibility agrees with influence. Another explanation could lie in \ recent observations by Samarut & Nigon [8] the late expression of adult antigen dur- Ithat most vitelline stem cells have a more ing maturation of erythrocytes, thus trans- limited proliferative capability than do adult forming E into EA erythrocytes. At any stem cells. rate, a reduced level of adult antigen was During normal development, the EA observed on immature cells [ 11. This would population appears after hatching [ 11, therealso explain the abrupt reduction of E cell fore arising from bone marrow stem cells. number 4 days after graft (fig. 4). The pos- The graft experiments described in this sibility of a rapid destruction of these cells, paper demonstrate the presence of stem however, cannot be ruled out. cells already able to give rise to EA cells in Exp CellRes
102 (1976)
Antigen
expression
the vitelline sac. Presence of similar stem cells in vitelline and bone marrow is not surprising if these haematopoietic tissues are seeded by blood-borne stem cells arising from still unknown intraembryonic sites [2,5]. However, stem cells giving rise to the A population do not appear to be present among vitelline stem cells. In conclusion, this work demonstrates the existence of adult stem cells giving rise to erythrocytes characterized by the presence of the adult antigen and by a reduced synthesis of foetal haemoglobin [3, 41. These properties appear to be possessed by stem cells which still retain a high proliferative capability. In contrast to adult stem cells, vitelline stem cells produce the embryonic antigen and a relatively high proportion of foetal haemoglobin. Whether E and EA Cells arise or not from different
stem from which duced
in irradiated
and grafted
chicks
13
cell populations cannot be deduced our present results. Situations in only E or EA cells would be proare now under investigation.
The authors are indebted to Dr Grantham for reviewing the manuscript. Mrs M. F. Grasset provided excellent technical assistance.
REFERENCES 1. Blanche& J P, Exp cell res 102 (1976) 1. 2. Dieterlen-Lievre, F, J embryo1 exp morph01 33 (1975) 607. 3. Godet, J, Dev biol40 (1974) 199. 4. Godet, J, Samarut, J & Nigon, V, Compt rend acad sci 38 (1975) 624. 5. Le Douarin, N, Houssaint, E & Jotereau, F V, Proc natl acad sci US 72 (1975) 2701. 6. Medway, W & Kare, W R, Poultry sci 38 (1959) 624. 7. Samarut, J & Nigon, V, J embryo1 exp morph01 33 (1975) 259. 8. -Ibid. In press.
Received April 2. 1976 Accepted April 27: 1976
Exp Cell Res 102 (1976)
