Taylor HS. Endometrial Cells Derived From Donor Stem Cells in Bone Marrow Transplant
Recipients. JAMA. 2004;292(1):81-85. doi:10.1001/jama.292.1.81
Author Affiliation: Division of Reproductive Endocrinology, Yale University School of Medicine, New Haven, Conn.
Context Regeneration of the endometrium in each menstrual cycle is required
for reproduction. Endogenous endometrial stem cells reside in the basalis
layer and serve as a source of cells that differentiate to form the endometrium.
Bone marrow–derived cells have been shown to take on functions outside
the hematopoietic system.
Objective To investigate the possibility that cells of extrauterine origin could
repopulate the endometrium.
Design, Setting, and Patients Endometrium from 4 HLA-mismatched bone marrow transplant recipients
(1998-2002) was evaluated for donor HLA expression. Each recipient had a bone
marrow donor with an HLA type that enabled determination of the origin of
any cell. Endometrial biopsies also were obtained from 4 healthy control women.
Main Outcome Measure HLA type was determined by immunohistochemistry and by reverse transcription–polymerase
Results Donor-derived endometrial cells were detected in endometrial biopsy
samples from all bone marrow recipients and accounted for 0.2% to 48% of epithelial
cells and 0.3% to 52% of stromal cells. None of the controls demonstrated
HLA mismatch in endometrial samples.
Conclusion These findings demonstrate that endometrial cells can originate from
donor-derived bone marrow cells and suggest that nonuterine stem cells contribute
to the regeneration of endometrial tissue.
Disorders of the uterine endometrium are common, leading to abnormal
uterine bleeding, infertility, pregnancy complications, miscarriage, endometriosis,
and cancer.1- 3 Although
the generation and growth of the uterine endometrium is important in normal
pregnancy and disease, it is poorly understood. The uterine endometrium is
one of the most dynamic human tissues and consists of a glandular epithelium
and stroma that are completely renewed in each monthly menstrual cycle. Endometrial
stem cells are thought to reside in the basalis layer and serve as a source
of cells that differentiate to form the endometrium. These endogenous stem
cells allow the rapid regeneration of the endometrium necessary to support
Stem cells of bone marrow origin have the ability to transdifferentiate
into multiple nonhematopoietic cell lineages. Adult bone marrow stem cells
differentiate into hepatocytes, skin, muscle, lung, and neuronal cells in
rodents.1- 9 Human
bone marrow–derived circulating stem cells have been reported to differentiate
into normal endothelial cells, hepatocytes,10,11 neurons,12 cardiomyocytes,13,14 skin,
and gastrointestinal epithelium.13,15 To
date, however, there have been no reports that circulating stem cells of extrauterine
origin can differentiate into human endometrial tissue and contribute to endometrial
We studied 4 female allogeneic bone marrow transplant recipients who
received marrow from a single-antigen mismatched related donor and therefore
had an HLA type that allowed determination of the origin of any cell. To avoid
the possibility that men may not harbor female reproductive tract stem cells,
each recipient had received marrow from female donors. All bone marrow transplants
were performed for leukemia treatment. All women were of reproductive age
(28-43 years) and had received total body irradiation and chemotherapy with
cyclophosphamide and in 2 cases also cytarabine or busulfan at least 2 years
Endometrial biopsies were performed for clinical indications; 1 was
performed for the evaluation of infertility after sex steroid treatment and
the other 3 for abnormal vaginal bleeding. All endometrial biopsies obtained
from HLA-mismatched recipients and identified by the investigator in 1998-2002
were included. An equal number of endometrial biopsies were obtained from
healthy nontransplanted control women of reproductive age (32-41 years) as
part of fertility evaluation or for abnormal vaginal bleeding. Endometrial
biopsies were obtained under an approved human investigation committee protocol.
Written informed consent was obtained from each patient at the time of endometrial
biopsy from Yale University School of Medicine, New Haven, Conn.
Formalin-fixed paraffin-embedded biopsy specimens were cut into serial
sections 6 µm thick, placed on coated slides, and deparaffinized through
a series of xylene and ethanol washes. Immunohistochemistry was performed
by using monoclonal antibodies against human HLA antigens A11, A3, and B7
(One Lambda; Pel Freez, Rogers, Ark); calcitonin (Neomarkers, Cambridgeshire,
England); CD45 (Neomarkers); and β galactosidase (Vector, Burlingame,
Calif). Slides were incubated with primary antibodies, followed by biotin-conjugated
appropriate secondary antibodies. 3-3'-Diaminobenzidine or Nova red (Vector)
was used as the substrate for the peroxidase reaction with Vectastain (Vector).
Secondary antibodies used for immunofluorescence were conjugated to fluorescein
isothiocyanate or tetrarhodamine isothiocyanate. Controls consisted of tissue
sections obtained from nontransplanted women undergoing endometrial biopsies
and immunostaining of transplanted patients' sections without primary or without
More than 200 high-power fields were counted for each patient. Three
observers assessed each of the samples. The interobserver and intraobserver
reliability were 6% and 4%, respectively. Each observer counted 5 identical
slides twice; error was defined as differences in counts between observers
or repeated counts by each observer.
Total RNA was extracted from endometrial tissue by using TriZol reagent
in accordance with the manufacturer's guidelines (Life Technologies Inc, Gaithersburg,
Md). The reverse transcription (RT) step was done by using the bulk first-strand
reaction mix (Amersham/Pharmacia Biotech, Piscataway, NJ) according to the
manufacturer's direction. Briefly, 1 µg of extracted RNA was diluted
in 20 µL of RNase-free water, heated to 65°C for 10 minutes, and
then chilled on ice. For first-test-strand complementary DNA synthesis, heat-denatured
RNA solution, along with deoxynucleotide triphosphates and 200-mM deoxynucleoside
dithiothreitol, was added to 11 µL of reagent, followed by incubation
at 37°C for 1 hour, and then heated to 90°C for 5 minutes and chilled
Primers used for polymerase chain reaction (PCR) are as described for
clinical use in HLA typing for bone marrow transplantation.16,17 To
amplify the 164–base-pair region of HLA-A11 (Figure 1) , the following primers
were used: A11N, which consists of base pairs 381 through 399 of exon 3; and
AL1, which consists of base pairs 527 through 544 of the same exon.18 Polymerase chain reaction was performed as follows:
2 minutes at 95°C, 35 cycles of 95°C for 1 minute, 52°C for 30
seconds, 72°C for 2 minutes, and extension at 72°C for 8 minutes.
The characteristics of the bone marrow transplant recipients are shown
in Table 1. Donor-derived endometrial
cells were detected in endometrial samples of bone marrow recipients. Endometrial
samples subjected to RT-PCR demonstrated the expression of messenger RNA (mRNA)
for a donor-derived HLA type. Endometrial samples from nontransplanted controls
did not demonstrate expression of mRNA for a discrepant HLA type (Figure 1). Because this mRNA may have derived
from migratory bone marrow–derived leukocytes, immunohistochemistry
was performed to determine cell identity.
Immunohistochemistry revealed endometrial epithelial cells and stromal
cells of donor origin in the uterus of women who were bone marrow transplant
recipients (Figure 2). Endometrial
sections from nontransplanted controls did not demonstrate cells with a discordant
HLA type. The percentage of bone marrow–derived cells was greater in
stroma than glandular epithelium and was localized in focal areas, suggesting
local proliferation of donor-derived stem cells. In one patient, the endometrium
consisted of more than 50% donor-derived cells (Figure 2C). Others consisted of approximately 0.3%, 4%, and 11%
donor-derived cells (Table 1).
Although most glands consisted of entirely host or entirely donor-derived
cells, some glands consisted of a fraction of cells of each origin or contained
only a few cells of donor origin (Figure 2D, E). Large numbers of transient mature leukocytes of donor origin
were observed. Some epithelial cells of donor origin demonstrated cilia characteristic
of functionally differentiated secretory endometrium (Figure 2E).
To distinguish CD45-positive leukocytes from endometrial cells, serial
immunohistochemistry was performed on each slide. CD45 is present on all human
cells of hematopoietic origin, except erythroid cells and platelets. Identification
of leukocytes with CD45 antibody demonstrated the existence of endometrial
cells that were not CD45 positive (Figure
3), which confirmed that the immunohistochemical staining was identifying
endometrial cells of donor origin rather than migratory mature bone marrow–derived
Immunofluorescence was used to colocalize the discrepant HLA type with
calcitonin, which identifies endometrial epithelial cells in the secretory
phase of the menstrual cycle.19 Colocalization
of the HLA-discrepant epithelial cells and calcitonin expression demonstrated
that cells of donor origin expressed this endometrial cell-type specific marker
and were capable of expressing a marker of functional differentiation (Figure 4). Staining with 4,6-diaminidino-2-phenylindole,
which forms fluorescent complexes with natural double-stranded DNA, did not
reveal any evidence of double signal intensity (data not shown), indicating
a low likelihood of cell fusion.
In human or murine bone marrow transplant recipients, donor-derived
cells can differentiate into numerous cell types of mesodermal, ectodermal,
and endodermal origin. This preliminary study suggests that bone marrow–derived
cells can differentiate into human uterine endometrium.
Transient infiltration of the endometrium by bone marrow–derived
cells (primarily macrophages, neutrophils, and eosinophils) occurs predictably
in each cycle; however, these are thought to have a role in inflammation and
immunomodulation rather than to serve as a source of stem cells.20 Transient
infiltration could be distinguished from endometrial cells by CD45 immunoperoxidase
staining. In this study, transient CD45-positive cells were distinct from
glandular epithelium or stroma. Local clusters of donor-derived cells suggest
that single progenitor cells underwent clonal expansion and differentiation.
However, although some endometrial glands appeared to be completely composed
of donor-derived cells, others contained fractions of donor-derived cells
or only a small number of individual cells of donor origin, which implies
that not all glands are clonal in origin. The greater number of donor-derived
stromal cells compared with glandular epithelial cells suggests differential
cell-type–specific rates of transdifferentiation.
The considerable variation in the percentage of cells of donor origin
likely depends on the length of time from treatment to biopsy, as well as
multiple factors that reflect tissue damage, loss of endogenous stem cells,
and tissue repair by recruitment of bone marrow–derived cells; here,
these factors include chemotherapy regimen, radiation dosage, graft-vs-host
disease, and other endometrial conditions unrelated to the transplant. The
woman whose endometrial sample demonstrated the most bone marrow–derived
cells had received dual-agent chemotherapy and total body irradiation and
showed evidence of moderate graft-vs-host disease.
Endometrial ablative techniques in women are fraught with a high long-term
failure rate and rarely result in complete long-term absence of endometrial
tissue or abnormal endometrial bleeding; this treatment failure occurs despite
apparent initial destruction or removal of all endometrial tissue. Conversely,
repair of a damaged uterine cavity with little remaining endometrium (Asherman
syndrome) has a high success rate.21 Both of
these clinical observations suggest that bone marrow–derived regeneration
of endometrium has clinical significance.
Ectopic location of the endometrium is characteristic of endometriosis.
Endometriosis is a common disease, occurring in approximately 15% of women,
causing infertility and pelvic pain.22 The
predominant theory for the origin of endometriosis is retrograde menstruation
through the fallopian tubes, with ectopic implantation; however, this theory
cannot explain foci of endometriosis outside the peritoneal cavity.23,24 A nonendometrial circulating source
of stem cells that can result in endometrial cells suggests an alternative
origin of some endometriosis. In some instances, endometriosis may arise by
differentiation of bone marrow–derived cells into endometrium in ectopic
locations. There is an association between endometriosis and immune disorders,
perhaps indicating that a single disorder may be common to ectopic bone marrow–derived
transdifferentiation of endometrium and other immune phenomena.25- 27 This
hypothesis warrants further investigation.
These preliminary findings suggest that bone marrow–derived cells
can generate endometrium, which may have clinical implications for establishing
and maintaining pregnancy, treating uterine disorders, and therapeutically
augmenting stem cell transdifferentiation into endometrium.