标题: Isolation of Mesenchymal Stem Cells of Fetal or Maternal Origin from Human Place [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:35 标题: Isolation of Mesenchymal Stem Cells of Fetal or Maternal Origin from Human Place
a Department of Obstetrics, $ C6 Q+ J" E' K1 \) s/ l: f " C' g( j' b! Y# b6 K- pb Department of Immunohematology and Blood Transfusion, and : p6 k* G5 Q7 S$ N3 U6 u v3 S+ Q! Y' k# d6 U$ ?
c Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands$ x+ x$ h1 ~: L, V) G8 b; G
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Key Words. Mesenchymal stem cells ? Placenta ? Amniotic fluid ? Decidua basalis ? Decidua parietalis ? Fetal 1 @+ X4 O; s* V: v. H W( m5 N- N( h. a$ ?! {: i
Correspondence: Pieternella S. in ＆t Anker, M.D., Ph.D., Department of Obstetrics, K6-32, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Telephone: 31-71-5262872; Fax: 31-71-5266741; e-mail: E.in_t_Anker@lumc.nl 4 H8 k4 F5 }) R" L4 a% R a; U7 y9 S! V& K3 n* x, l
ABSTRACT3 r5 G4 Y% V$ k, K- I
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Mesenchymal stem cells (MSCs) are capable of differentiating into different mesenchymal lineages, including adipose and connective tissue, bone, and cartilage . MSCs were initially identified in human postnatal bone marrow (BM) and later in peripheral blood, periosteum, muscle, adipose tissue, and connective tissue of human adults . For clinical use, human adult BM is the most common source of MSCs ; however, the frequency of MSCs in human adult BM is relatively low. Because the frequency and differentiating capacity of MSCs are decreasing with age , different fetal tissues have been studied for the presence of MSCs. Human first-trimester fetal BM, liver, and blood and second-trimester BM, liver, lung, spleen, pancreas, and kidney have been found to be rich sources of MSCs.# a- s0 \. v" ~ W/ P, L
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Because the use of fetal tissues for stem cell therapy has ethical restrictions and is associated with a high rate of bacterial and fungal contamination, other potential sources of fetal MSCs applicable for human therapies have been sought. Umbilical cord blood (UCB) is an attractive source of fetal MSCs; however, it is shown that MSCs are present in UCB in a low frequency or are even undetectable (in ＆t Anker et al., unpublished data). Recently, we reported that second-trimester amniotic fluid (AF) is a novel and rich source of fetal MSCs useful for clinical application . " Q- ^8 d' Y S$ p4 Z" g8 U9 D1 J9 v# `" e, Q$ O! n+ s- k( `
AF contains a heterogeneous population of cells from fetal origin. Potential sites contributing to the presence of cells in the AF are the fetal skin, the fetal membranes of the placenta, and the epithelial and mucosa of the fetal digestive, respiratory, and urinary tract . The origin of the fetal MSCs in AF is yet unknown, and therefore we studied the fetal membranes as a possible source of fetal MSCs. In addition, we analyzed the presence of MSCs in human third-trimester AF, because the presence of those cells in third-trimester AF had not been studied yet.7 M# c5 p' Q0 }5 n2 j
Z! f8 S0 J* j) oIn their recent report, Zhang et al. describe the presence of MSCs in term human placenta. They did not analyze the origin of the placenta villi-derived cells. The human placenta is an organ with both a fetal and a maternal portion (Fig. 1). The amnion and chorion are from fetal origin, and the different regions of the decidua are from maternal origin. Here we studied whether MSCs from fetal origin and maternal origin could be cultured from the fetal versus maternal side of the placenta.% n9 W# N2 b) u) {" G8 Y1 [
" d/ H% ]; q/ W j- \Figure 1. Schematic drawing of the human placenta showing the amnion (fetal part), decidua basalis, and decidua parietalis (maternal part). 7 i+ |6 H+ W' d- D8 O# {5 x' V$ n1 X! C `
MATERIALS AND METHODS) o4 ~7 u0 L/ g4 J6 W" C) {0 P
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Identification of Mesenchymal Stem Cells $ P1 X; N+ A' m: E& p6 p 5 }$ X! ?2 [5 h+ B! TThe mean volume of AF collected from second-trimester pregnancies by transabdominal puncture was 8.7 ml (SD, ± 1.7) and by transcervical punctures was 32.3 ml (SD, ± 13.9). MSCs were cultured from all 10 consecutive samples of both transabdominally and transcervically collected second-trimester AF. In 8 of 10 samples, MSCs were isolated from second-trimester amnion, and in 8 of 10 samples, MSCs were isolated from second-trimester decidua (Table 1). ( ^6 h9 _" y( e 6 I- `# ?3 Y9 ~& g7 I$ |$ h0 }Table 1. Isolation of MSCs from second-trimester and term third-trimester AF, amnion, and decidua; R3 @9 L C& U. D: I
! ~1 h/ m3 S8 `# Z+ Q- [8 l/ y5 WWe successfully isolated MSCs from only 2 of 10 term AF samples (mean volume of AF, 10.7 ml ), from 7 of 10 term amnion samples, from 6 of 10 term decidua parietalis samples, and from 4 of 10 term decidua basalis samples (Table 1). The cell suspension of approximately 10 specimens of amnion (1 cm2), decidua basalis (1 cm2), and decidua parietalis (1 cm3) was necessary to culture MSCs. & u$ ?3 i* K ]; p! E4 j7 H: L- ]# t. \
Culture-expanded cells derived from AF, amnion, decidua basalis, and decidua parietalis were immunopheno-typically analyzed. The phenotype of the cultured cells was similar to that of MSC derived from adult BM and fetal second-trimester tissues , i.e., CD90, CD105, CD166, CD49e, SH3, SH4, and HLA-ABC positive and CD31, CD34, CD45, CD49d, CD123, and HLA-DR negative. No difference was found among the expression of one of these markers on MSCs from the different sources. 4 N- D3 D9 r; d; u" |5 S8 {4 H, o1 ?. p+ _8 A4 X+ o
Culture-expanded cells from AF, amnion, decidua basalis, and decidua parietalis and adult BM were all able to differentiate into both osteoblasts and adipocytes. / r! {0 S* L. @9 z! V5 G/ O7 s3 j$ F& `1 Z6 }
HLA Typing & ]' A% H; J* H# h2 y9 G n& `% J5 q8 C( e- `! a1 M
HLA analysis of the culture-expanded cells from transabdominally collected second-trimester (n = 10) and term (n = 2) AF showed that all these samples were of fetal origin, i.e., only fetal-specific and no maternal alleles were present (Table 1). On the basis of HLA typing, all second-trimester (n = 8) and term (n = 7) amnion-derived culture-expanded cells were of fetal origin (Fig. 2A). In contrast, only 4 of the 10 transcervically collected AF samples were of fetal origin. Six of the 10 transcervically collected AF samples expressed both fetal- and maternal-specific alleles., b* l+ V0 E" o! b
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Figure 2. HLA typing of culture-expanded MSCs. The Dynal Reli SSO (Dynal Biotech, Hamburg, Germany) reverse-line blot strip assay was used for molecular typing of the HLA-A and HLA-B locus alleles of maternal cells, fetal cells, and culture-expanded MSCs from the same sample. The HLA-A and HLA-B type of the culture-expanded amnion-derived MSCs (A) is identical to the fetal HLA-A and HLA-B type and mismatched with the maternal HLA-A and HLA-B type. The HLA-A and HLA-B type of the culture-expanded deciduas basalis–derived MSCs (B) is identical to the maternal HLA-A and HLA-B type and mismatched with the fetal HLA-A and HLA-B type. Upward arrows indicate maternal-specific HLA antigens, and downward arrows indicate fetal-specific HLA antigens. Abbreviations: C, cultured amnion-derived MSCs; F, fetal cells; M, maternal cells; MSCs, mesenchymal stem cells. 7 J) l) v1 r4 o% F; A: l r1 N ! z0 Z5 W: }6 q( ySix of the eight cell populations expanded from second-trimester decidua were of maternal origin. However, two of the eight culture-expanded cell populations expressed both fetal- and maternal-specific alleles. Culture-expanded cells from term decidua basalis (n = 4) and decidua parietalis (n = 6) were of maternal origin (Fig. 2B).+ M' w' r3 b$ C5 D& j! x$ a* z" M: b
. F* |( z5 N$ lThe growth characteristics of MSCs derived from different fetal (second-trimester transabdominal-collected AF and amnion ), maternal (decidua from second-trimester placenta and decidua basalis and decidua parietalis from term placenta), and adult (BM ) sources were compared during 3 weeks. The growth of second-trimester fetal AF (collected transabdominally) and amnion-derived MSCs was similar (Fig. 3). There was no significant difference in cell numbers and growth kinetics during this culture period. After 11 days, a plateau in the growth was reached. Maternal cells derived from second-trimester decidua tissue and term decidua parietalis tissue had a similar growth pattern (Fig. 4). The number of cells derived from these two sources at the days of counting was not significantly different. From day 11, the number of adherent cells derived from term decidua basalis was significantly higher (p 3 e6 }6 U% Y- ~' u+ Y # c1 a7 i4 k' ]/ U; zFigure 3. Growth curves of amnion and amniotic fluid. At t = 0, 200,000 mesenchymal stem cells were seeded in culture flasks. Duplicate cultures were harvested twice weekly for 3 weeks, and adherent cells were counted. Results are expressed as mean ± standard error of the mean. Growth curves of second-trimester amnion (n = 3, gray line) and second-trimester amniotic fluid (n = 7, black line). " S7 o7 [5 A; z: v6 F, d* S# I( {- [; L& Q3 ~7 c% z' P
Figure 4. Growth curves of decidua basalis, decidua parietalis, and adult BM. At t = 0, 200,000 mesenchymal stem cells were seeded in culture flasks. Duplicate cultures were harvested twice weekly for 3 weeks, and adherent cells were counted. Results are expressed as mean ± standard error of the mean. Growth curves of adult BM (n = 2, ), second-trimester decidua (n = 5, ), term decidua basalis (n = 2,), and term decidua parietalis (n = 5,x).Abbreviation: BM, bone marrow. ! p( u- d# d1 S6 l6 u3 T" `4 L( r6 V, ?
DISCUSSION # W* C1 _5 {# R4 v+ h6 t+ e1 t- J3 X H; l. h2 u* k& ?
We thank the gynecologists from the department of Obstetrics of the Leiden University Medical Center and W. Beekhuizen of the Center of Human Reproduction in Leiden for collecting the amniotic fluid and P.H.C. Eilers, Ph.D., from the Leiden University Medical Center for help with statistics., { Z- w9 E; ~8 ~( j6 G
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