Parathyroid Hormone Stimulates Circulating

Osteogenic Cells in Hypoparathyroidism

M. R. Rubin, J. S. Manavalan, D. W. Dempster, J. Shah, S. Cremers, S. Kousteni,
H. Zhou, D. J. McMahon, A. Kode, J. Sliney, E. Shane, S. J. Silverberg,
and J. P. Bilezikian
Department of Medicine, Division of Endocrinology, Metabolic Bone Diseases Unit, College of Physicians
and Surgeons, Columbia University, New York, New York 10032
Context: The osteoanabolic properties of PTHmay be due to increases in the number and maturity
of circulating osteogenic cells. Hypoparathyroidism is a useful clinical model because this hypoth-
esis can be tested by administering PTH.
Objective: The objective of the study was to characterize circulating osteogenic cells in hypopar-
athyroid subjects during 12 months of PTH (1-84) administration.
Design: Osteogenic cells were characterized using flow cytometry and antibodies against osteo-
calcin, an osteoblast-specific protein product, and stem cell markers CD34 and CD146. Changes in
bone formation frombiochemical markers and quadruple-labeled transiliac crest bone biopsies (0
and 3 month time points) were correlated with measurements of circulating osteogenic cells.
Setting: The study was conducted at a clinical research center.
Patients: Nineteen control and 19 hypoparathyroid patients were included in the study.
Intervention: Intervention included the administration of PTH (1-84).
Results: Osteocalcin-positive cells were lower in hypoparathyroid subjects than controls (0.7 0.1 vs.
2.0 0.1%; P 0.0001), with greater coexpression of the early cell markers CD34 and CD146 among
theosteocalcin-positivecells inthehypoparathyroidsubjects (11.01.0vs. 5.60.7%; P0.001). With
PTH (1-84) administration, the number of osteogenic cells increased 3-fold (P 0.0001), whereas the
coexpression of the early cell markers CD34 and CD146 decreased. Increases in osteogenic cells corre-
latedwithcirculatingandhistomorphometric indices of osteoblast function: N-terminal propeptideof
type I procollagen (R
2
0.4, P 0.001), bone-specific alkaline phosphatase (R
2
0.3, P 0.001),
osteocalcin (R
2
0.4, P 0.001), mineralized perimeter (R
2
0.5, P 0.001), mineral apposition rate
(R
2
0.4, P 0.003), and bone formation rate (R
2
0.5, P 0.001).
Conclusions: It is likely that PTHstimulates bone formationby stimulatingosteoblast development
and maturation. Correlations between circulating osteogenic cells and histomorphometric indices
of bone formation establish that osteoblast activity is being identified by this methodology. (J Clin
Endocrinol Metab 96: 176186, 2011)
T
he mechanisms by which PTH increases bone forma-
tion are not well understood. When PTH acts on
bone, communication between several different special-
ized cell types occurs, including osteoblasts, bone marrow
andstromal cells, hematopoietic precursors of osteoclasts,
and mature osteoclasts and osteocytes (1). PTH increases
the number of osteoblasts by decreasing apoptosis of
preosteoblast cells and osteoblasts (2, 3) and increasing
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright 2011 by The Endocrine Society
doi: 10.1210/jc.2009-2682 Received December 15, 2009. Accepted August 30, 2010.
First Published Online September 29, 2010
Abbreviations: ALP, Alkaline phosphatase; BAP, bone-specific alkaline phosphatase activ-
ity; BFR, bone formation rate; FBS, fetal bovine serum; MAR, mineral apposition rate;
Md.Pm, mineralizing perimeter; OCN, osteocalcin; PBMC, peripheral blood mononuclear
cell; PE, phycoerythrin; P1NP, N-terminal propeptide of type I procollagen; Runx2, Runt-
related transcription factor 2.
O R I G I N A L A R T I C L E
E n d o c r i n e R e s e a r c h
176 jcem.endojournals.org J Clin Endocrinol Metab, January 2011, 96(1):176186
osteoblast proliferation (4). Increasing the supply of os-
teoblast precursor cells inthe bone marrowis another way
that PTH might stimulate bone formation (5).
Osteoblast precursor cells were previously identifiedby
specific qualities, namely their ability to adhere to plastic,
tostimulate the expressionof osteoblast-relatedgenes and
to formmineralized nodules (6, 7). In the circulation, such
plastic-adherent osteoblastic cells have been demon-
strated (8, 9), but they are present in exceedingly lowcon-
centrations (9). Thus, until recently, osteoblast lineage
cells were difficult to study in the peripheral blood. Long
et al. (10, 11) demonstrated that in addition to the adher-
ent bone marrow-derived osteoblastic cells, there is an-
other population of nonadherent cells with osteogenic po-
tential in the circulation. Using flow cytometry with
antibodies to the osteoblast-specific protein products os-
teocalcin and bone-specific alkaline phosphatase to iden-
tify osteogenic cells in the circulation, the investigators
demonstrated that these osteogenic cells do, in fact, cir-
culate in physiologically significant numbers (12). This
concept has also been supported by studies in mice, which
have similarly shown that osteogenic cells can be readily
found in nonadherent circulating stromal cells (13, 14).
Potential markers of osteoblast progenitor cells include
other cell surface markers, besides osteocalcin. CD34, for
example, a marker of hematopoietic stem cells, is also a
marker for cells that give rise tofunctional osteoblasts that
are capable of forming mineralized nodules and can heal
fractures (15). Cells that are positive for both osteocalcin
and CD34 (OCN

/CD34

) are probably more immature

than cells that are positive only for osteocalcin (OCN

/
CD34

). The cells that are positive for OCN and CD34

diminish when osteoblasts develop (16). Bianco and col-
leagues (17) suggested that CD 146

stromal cells also

function as self-renewing, clonogenic skeletal progenitors
and that CD146 may be a marker of early osteoblasts.
Hypoparathyroidism is a useful model to study the ac-
tions of PTH on circulating osteogenic cells. Because en-
dogenous PTHis absent, this disorder can be used to eval-
uate the effects of PTH deficiency and its administration
on these cells. To this end, we studied 19 untreated hypo-
parathyroid subjects before and after 1, 2, 3, 6, and 12
months of PTH(1-84) administration. At each time point,
we measured the number of circulating osteogenic cells as
well as early cell markers, CD34 and CD146. We hypoth-
esized that the number of circulating osteogenic cells
would be lower in hypoparathyroid subjects, compared
with controls, with an altered profile of early cell markers
that would change with PTH exposure. To validate our
methodology, we correlated changes in osteoblast lineage
populations in the hypoparathyroid subjects after PTH
(1-84) administration with changes in circulating bone
formation markers and data frompercutaneous iliac crest
bone biopsies. For histomorphometric assessment, we
used the quadruple-labeled iliac crest bone biopsy, a
method that permits time-dependent assessment of
changes after PTH (1-84) administration with only a sin-
gle biopsy. Our data support the hypothesis that PTH
regulates osteoblast development and may facilitate the
maturation of osteogenic cells.
Subjects and Methods
Subjects
Nineteensubjects withdocumentedhypoparathyroidismand
19controls participatedinthe study. Eachhypoparathyroidsub-
ject was matched with an age- (within 3 yr), sex-, and meno-
pause-matched control. The diagnosis of hypoparathyroidism
was established by the simultaneous presence of serum calcium
and PTH concentrations below the lower limits of normal on at
least twoprior occasions, separatedbyaninterval of at least 30d.
Hypoparathyroidismhad to have been present for at least 3 yr to
establish a chronic state of PTH deprivation. All subjects had to
be on stable regimens of supplemental calcium and vitamin D
intake for 6 months before enrollment. Patients and controls
were excluded if they had been on a bisphosphonate within 5 yr
before study entry or for more than 6 months duration at any
time or if they were women within 5 yr of menopause. Patients
and controls were also excluded if they used any of the following
medications: estrogens, progestins, raloxifene, calcitonin, sys-
temic corticosteroids, fluoride, lithium, statins, loopdiuretics, or
methotrexate. Potentially confounding disorders were also ex-
clusionary criteria, if present: Pagets disease of bone, diabetes
mellitus, chronic liver or renal disease, acromegaly, Cushings
syndrome, rheumatoid arthritis, or multiple myeloma.
Patients were recruited from the Metabolic Bone Diseases
Unit of Columbia University Medical Center and from the Hy-
poparathyroidism Association. The control subjects were re-
cruited by local posted announcements. The study was approved
by the Institutional ReviewBoard of Columbia University Med-
ical Center. All subjects and controls gave written informed
consent.
Protocol
Time course of PTH administration
The hypoparathyroid subjects were given PTH (1-84), pro-
vided by NPS Pharmaceuticals (Bedminster, NJ), for 12 months
at a sc dose of 100 g every other day. This dose was selected
because we showed previously that this regimen restores sup-
pressed bone turnover markers in hypoparathyroidism to levels
that are in the normal range (18). Circulating osteogenic cells
were measured at baseline (T 0) and after 1, 2, 3, 6, and 12
months of PTH. To validate osteogenic cell measurements, bio-
chemical and histomorphometric indices were determined. Bio-
chemical markers of bone formation[aminoterminal propeptide
of type I procollagen (19), bone specific alkaline phosphatase
(20), and osteocalcin (16)] were measured at the same time
points. Percutaneous iliac crest bone biopsies were performed in
12 of the 19 hypoparathyroid subjects with a quadruple tetra-
cycline-labeling protocol (21). In contrast to conventional label-
J Clin Endocrinol Metab, January 2011, 96(1):176186 jcem.endojournals.org 177
ing with one set of tetracycline labels, two sets of tetracycline
labels were sequentially administered, at baseline and 3 months
after PTH(1-84), withthe single biopsy obtainedafter 3months.
This method permits the determination of bone formation indi-
ces at baseline and after PTH(1-84) by measuring characteristics
of each set of double labels separately and then comparing them
with each other (21).
Biochemical markers of bone formation
Intact N-terminal propeptide of type I procollagen (P1NP)
was measured by RIA (22). The interassay and intraassay vari-
abilities are 6.010.2% (normal range: 1983 and 1696 g/
liter for pre- and postmenopausal women, respectively); osteo-
calcin was measured by ELISA (23) (N-mid osteocalcin; IDS
Ltd., Fountain Hills, AZ) (mean SD: 17.9 6.5 and 28.4 9.5
ng/ml for pre- and postmenopausal women, respectively). The
intraassay and interassay variability is 1.8 and 2.7%, respec-
tively. Bone-specific alkaline phosphatase activity (BAP) was
measured by immunoassay (20) (Metra BAP; Quidel Corp, San
Diego, CA). This assay has low cross-reactivity with the liver
form of alkaline phosphatase (38%). Interassay variability is
8.6%at 13.7 U/liter and 6.0%(normal range: 11.629.6 U/liter
and 14.242.7 U/liter in pre- and postmenopausal women,
respectively).
Histomorphometric assessment of bone formation
with quadruple-labeled protocol
Two tetracycline labels were administered (Sumycin 250 mg
four times daily) using a standard format of 3 d on, 12 d off, 3 d
on immediately before initiation of PTH(21). After 3 months of
PTH administration, the tetracycline labeling protocol was re-
peated using the same schedule but with a different tetracycline
(Declomycin 150 mg four times daily). Percutaneous iliac crest
biopsies were performed 1 wk after the second double-label pro-
tocol. The method yields two sets of fluorescent labels repre-
senting bone formation before (the first set) and after PTH (the
second set) administration (21). Each set of double labels was
easily distinguishable by color under fluorescent light. Biopsy
specimens were processedandanalyzedby histomorphometry as
previously described in detail from our laboratory (24). Histo-
morphometry was performed using an OsteoMeasure digitizing
image-analysis system (OsteoMetrics, Inc., Atlanta, GA). Bone
formation was evaluated on cancellous, endocortical, or intra-
cortical bone surfaces and expressed by the variables of miner-
alizing perimeter (Md.Pm), mineral apposition rate (MAR), and
bone formation rate (BFR). All indices are expressed according
to the recommendations of the American Society for Bone and
Mineral Research Nomenclature Committee (25).
Flow cytometry and cell sorting
Peripheral blood mononuclear cells (PBMCs) were isolated
by density gradient centrifugation using Ficoll-Hypaque and
were counted with Tryptan blue for viability using a hemacy-
tometer. PBMCs were resuspended in flow-staining buffer [PBS
plus 2% fetal bovine serum (FBS)] and the primary antibodies
were added. After 30 min incubation at 4 C, the cells were
washed twice and fluorochrome-conjugated primary and sec-
ondary antibodies were added. The cells were then incubated for
an additional 30 min at 4C and washed twice before flow cy-
tometryanalysis. The primaryunconjugatedantibodywas agoat
polyclonal antihuman osteocalcin (Santa Cruz Biotechnology,
Santa Cruz, CA) antibody (a control isotype antibody was used at
the same concentrations); secondary conjugated antibodies in-
cluded fluorescein isothiocyanate-conjugated AffinityPure IgG
f(ab)
2
fragment donkeyantigoat (JacksonImmunoResearch, West
Grove, PA) antibodies. Primary conjugated antibodies were allo-
phycocyanin-conjugated anti-CD15, phycoerythrin (PE)-conju-
gated anti-CD146, and anti-PE-Cy7-conjugated CD34 (all from
Becton Dickinson, San Diego, CA). Five-color flow cytometry ac-
quisition was performed using a LSR II flow cytometer (Becton
Dickinson) and analysis using FLO-JO software (Treestar, Inc.,
Ashland, OR). Cells were gated for size, shape, and granularity
using forward- and side-scatter parameters. The positive popula-
tions were identified as cells that expressed specific levels of fluo-
rescence activity above the nonspecific auto fluorescence of the iso-
typecontrol. Theregionwas set toencompass boththelymphocyte/
monocyte-enriched area and the granulocyte-enriched area and to
exclude dead cells. All CD15

granulocytes were excluded before

gatingfor specific populations toexclude contaminationof isolated
mononuclear cells with granulocytes.
For flow sorting, PBMCs were resuspended in flow staining
buffer at 1 10
6
/ml and labeled with polyclonal antihuman
osteocalcin. After 30 min incubation at 4 C, the cells were
washed twice and the following fluorochrome-conjugated pri-
mary and secondary antibodies were added: fluorescein isothio-
cyanate-conjugated AffinityPure IgG f(ab)
2
fragment donkey
antigoat antibody, allophycocyanin-conjugated anti-CD15, and
PE-Cy7-conjugated anti-CD34. After 30 min incubation, cells
were washed twice using flow buffer. Flow sorting was per-
formed using FACSAria (BD). Cells were sorted into the follow-
ing populations; OCN

/CD34

and OCN

/CD34

. Sorted
populations were thenstoredinRNeasylysis tissue buffer at 80
C for extraction of RNA.
Expression analysis for osteoblast molecular
markers
Total RNA was isolated from tissues using TRIZOL reagent
(Invitrogen, Carlsbad, CA) followed by a clean-up step using the
RNeasy minipurification kit (Qiagen, Valencia, CA). Only the
RNAsamples that gave A260/A280 about 1.81.9 in Nanodrop
ND-1000 UV-VIS spectrophotometer (Thermo Scientific, Wil-
mington, DE) were used. One microgramof total RNAwas first
treated with deoxyribonuclease at roomtemperature, and RNA
was then reverse transcribed using Superscript III reverse tran-
scriptase at 42 Cfor 60 min. The resulting cDNAs were used for
real-time PCR analysis of various genes using the Stratagene
qPCR machine (La Jolla, CA). Primers for the assays were ob-
tained from Superarray Biosciences (Frederick, MD). Reactions
were set up in the total volume of 25 l with the BIORAD 2X
qPCRmix in triplicate (Bio-Rad Laboratories, Hercules, CA) for
each sample and were measured against standard curves for re-
spective genes. Using real-time PCR, the expression of osteoblast
gene markers, including OCN, alkaline phosphatase (ALP),
Runt-related transcription factor 2 (Runx2), and TGF, was
evaluated in sorted OCN

/CD34

and OCN

/CD34

cells.
Osteogenic cultures
Flow-sorted OCN

/CD34

cells were suspended in growth

medium (MesenCult basal medium; Stem Cell Technologies,
Vancouver, BritishColumbia, Canada) containing 10%FBS and
1% penicillin-streptomycin mixture and plated in fibronectin-
coated plates (Becton Dickinson) at a plating density of 3.5
178 Rubin et al. Osteogenic Cells in Hypoparathyroidism J Clin Endocrinol Metab, January 2011, 96(1):176186
105 per square centimeter. On d 21 the mediumwas changed to
osteogenic differentiation medium containing MesenCult basal
mediumwith 15%osteogenic stimulatory supplements, 3.5 mM
-glycerophosphate, 10
8
M dexamethasone, and 50 g/ml
ascorbic acid (Stem Cell Technologies). Parallel cultures were
performed with only MesenCult basal medium containing 10%
FBS and no osteogenic differentiation supplements. Throughout
the culture of the cells, the total media with the nonadherent cell
fraction from each well were aspirated once a week and washed
once in the appropriate media, and the nonadherent cells with
fresh media were added back to the respective wells. After 3 wk
of differentiation, the cultured cells underwent either expression
analysis of osteoblast markers or stainingfor calciumdeposition.
The expression of osteoblast gene markers in the cultured cells
was assessed with real-time PCRand included ALP, Runx2, and
Osterix. For calcium staining, the cultured cells were fixed in
10% formaldehyde and then stained for calcium deposits using
2% alizarin red (Millipore Chemicon, Billerica, MA).
Statistical analysis
Data are expressedas meanSEM. For the case-control study,
a matched pair analysis was performed. For the time-course
study, estimates of change in indices frombaseline were assessed
with paired t tests. Linear regression was used to assess the re-
lationship between the change in osteogenic cell populations and
the change in biochemical markers of bone formation and to
assess the relationshipbetweenthe change inosteogenic cell pop-
ulations and the change in dynamic histomorphometric indices
of bone formation. A P 0.05 was considered significant.
Results
Study population
The hypoparathyroid and control subjects were matched
for age (hypoparathyroid: 414yr; controls: 423yr) and
gender (eachgroup: four males, nine premenopausal andsix
postmenopausal females). Hypoparathyroidism was post-
surgical (n10) or idiopathic (n9), witha meanduration
of 9.8 3 yr (range 340 yr). Baseline serum calcium was
9.0 0.2 mg/dl (2.25 0.1 mmol/liter); PTHwas less than
3 pg/ml (3 ng/liter); baseline calciumsupplementation was
2065263mg/d(range 600-5000; median1800mg/d) and
baseline calcitriol supplementation was 0.66 0.1 g/d
(range 02; median 0.50 g/d). Parent vitamin D supple-
mentation was used in nine patients (range 400100,000
IU/d). Nineof thesubjects wereonthyroidreplacement med-
ication. Of thosenine, fivehadsuppressedTSHvalues (mean
0.110.1U/ml; normal range0.344.25U/ml), andone
had a TSH value that was slightly above the normal range
(5.23U/ml). None of these individuals was clinically hypo-
or hyperthyroid. The effect of these abnormal values on the
analyses is described below.
Case control comparisons
The percentage of PBMCs that expressed OCN on the
surface was significantly lower in the hypoparathyroid
FIG. 1. Representative example of flow cytometry dot plots looking at OCN

population. The isotype control shows the nonspecific binding, or

background noise. To measure the specific expression of OCN, the isotype control is subtracted from the total OCN. In the control, the OCN

cells
are 2.90.6% of circulating PBMCs (2.3%), whereas in the matched hypoparathyroid (Hypo) subject, the OCN

cells are 1.20.3% of circulating

PBMCs (0.9%).
J Clin Endocrinol Metab, January 2011, 96(1):176186 jcem.endojournals.org 179
subjects, compared with the matched controls (0.7 0.1
vs. 2.0 0.1%; P 0.0001; Figs. 1 and 2A); a represen-
tative pair is shown in Fig. 1. Although the overall popu-
lationof OCN

cells was lower, the hypoparathyroidsub-

jects tended to showa greater proportion of cells that also
expressed the early markers, CD34 and CD146. Although
the subpopulation of OCN

cells that coexpressed CD34

(OCN

/CD34

) showed a trend to be higher in the hy-

poparathyroid subjects (hypoparathyroid: 35.6 1.5
vs. controls 30.9 2.9%; P 0.08; Fig. 2B), OCN

cells that coexpressed CD146 (OCN

/CD146

) were
twice as high in the hypoparathyroid subjects (hypo-
parathyroid: 31.6 1.8 vs. control: 15.1 1.1%; P
0.0001; Fig. 2C). Similarly, there were significantly
more OCN

cells that coexpressed both CD34 and

CD146 (OCN

/CD34

/CD146

) in the hypoparathy-
roid subjects (hypoparathyroid: 11.0 1.0 vs. control:
5.6 0.7%; P 0.0003; Fig. 2D).
Time course after PTH (1-84) administration
With PTH (1-84) administration, the percentage of
OCN

cells in hypoparathyroid subjects increased signif-

icantly, peaking at 2 months. By 6 and 12 months, the
percentage of cells decreased, although they were still
higher than at baseline (Fig. 3A). The profile of OCN

cells also changed after PTH (1-84) administration. The

OCN

cells that did not show the early CD34 marker

(OCN

/CD34

) increased, peaking at
3 months and decreasing by 6 and 12
months, although not to baseline levels
(Fig. 3B). The OCN

cells that were

CD146

(OCN

/CD146

) did not
change. OCN

cells that did not show

either of the early markers (OCN

/
CD34

/CD146

) also increased with

PTH (1-84) but at 12 months had re-
turned to baseline (Fig. 3C). The hema-
topoietic cell populations, which in-
cluded total CD34

cells and OCN

/
CD34

cells, did not change with PTH

administration. Exclusion of the six sub-
jects withabnormal TSHvalues fromthe
analysis did not alter these findings.
Associations with changes in
biochemical markers of bone
formation
The increase in percentage of OCN

cells frombaseline to 3 months of PTH

(1-84) administration was associated
with increases in biochemical markers
of bone formation: P1NP, BAP, and os-
teocalcin. The increase in the subpopu-
lation of OCN

cells lacking the early CD34 marker

(OCN

/CD34

cells) was also associated with in-

creases in biochemical indices of bone formation (Table
1). Increases in the numbers of subpopulations lacking
early cell markers (OCN

/CD146

and OCN

/
CD34

/CD146

) were associated with increases in

P1NP, the most quickly responsive bone formation
marker after PTH administration. The increases in os-
teoblast lineage populations preceded the increases in
biochemical markers of bone formation (Fig. 3D).
Moreover, the osteogenic cells declined after 6 months
of PTH (1-84), whereas the increase in biochemical
markers persisted.
Associations with changes in dynamic
histomorphometric indices of bone formation
To establish further that the increase in the OCN

cells
after PTHadministration reflects changes in dynamic histo-
morphometric indices, correlations were performed with di-
rectly measured mineralized perimeter, mineral apposition
rate, and bone formation rate. Histomorphometric analysis
showed marked increases in indices of bone formation by
both qualitative (Fig. 4) and quantitative (Table 2) assess-
ment. The increase in the percentage of OCN

cells from
baseline to 3 months of PTH (1-84) was associated with
increases in all these histomorphometric indices. All three
bone envelopes, namely cancellous, endocortical, and in-
FIG. 2. Comparison of cell populations in untreated hypoparathyroid subjects and matched
controls. Data are mean SEM. A, Comparison of OCN

cells. The percentage of PBMCs that

was positive for OCN was lower in the 19 hypoparathyroid subjects compared with the 19
matched controls. B, Comparison of OCN

/CD34

cells. The percentage of OCN

cells that
was also positive for CD34 showed a trend to be higher in the hypoparathyroid subjects. C,
Comparison of OCN

/CD146

cells. The percentage of OCN

cells that was also positive for

CD146 was higher in the hypoparathyroid subjects. D, Comparison of OCN

/CD34

/CD146

cells. The percentage of OCN

cells that was also positive for both CD34 and CD146 was
higher in the hypoparathyroid subjects.
180 Rubin et al. Osteogenic Cells in Hypoparathyroidism J Clin Endocrinol Metab, January 2011, 96(1):176186
tracortical (Table 3), showed similar changes. Increases in
lineage cells that had lost the early CD34 or CD146 cell
markers (alone or together) also were associated with in-
creases in dynamic histomorphometric indices (Table 3).
Expression analysis for osteoblast
molecular markers in sorted cells
Toprovide evidence that the cells are
osteogenic, expression profiling of
sorted OCN

/CD34

and OCN

/
CD34

cell populations was per-

formed. The expression profiles of
OCN, ALP, Runx2, and TGF were
evaluated using real-time PCR. In ad-
dition to expressing osteocalcin (Fig. 5,
Aand B), Runx2 was expressed in both
populations, whereas the OCN

/CD34

population also expressed ALP and

TGF.
Expression analysis for osteoblast
molecular markers in cultured cells
To provide further evidence that the
cells are osteogenic, OCN

/CD34

cells were isolated and cultured in

growth medium for 3 wk and then in
osteogenic differentiation medium for
3 wk. The expression profiles of
Runx2, ALP, and Osterix were evalu-
ated at the end of the 6 wk using real-
time PCR. The osteoblast markers were
expressedinthe culturedcells (Fig. 5C).
As a control, OCN

/CD34

cells were
also cultured without osteogenic differ-
entiation media; these cells had significantly less expres-
sion of the osteoblast markers.
Formation of mineralized nodules in cultured cells
In addition to the expression analysis, the cultured
OCN

/CD34

cells were stained at the end of the 6 wk

period with alizarin red to assess calcium deposition. Al-
izarin red staining and formation of calciumnodules were
observed (Fig. 5D). No staining was observed in the con-
trol OCN

/CD34 cells that were cultured without the os-

teogenic differentiation media (Fig. 5E).
Discussion
The clinical model of hypoparathyroidism, in which PTH
is chronically absent, has permitted us to gain insights into
the effects of PTH on osteoblast number, development,
and function. We have shown that circulating osteogenic
cells are reduced in hypoparathyroid subjects compared
with matched controls. The results also demonstrate that
agreater proportionof these cells have anearlycell marker
and thus may be immature. With PTH(1-84) administra-
tion, the number of circulating osteogenic cells increases
FIG. 3. Changes in cell populations in hypoparathyroid subjects with PTH (1-84) treatment over
12 months. Data are mean SEM. *, P 0.05 from baseline; **, P 0.001 from baseline. A,
Change in OCN

cells. The percentage of PBMCs that was OCN

cells increased with PTH (1-84)

treatment. B, Change in OCN

/CD34

cells. The percentage of OCN

cells that was lacking

CD34 increased with PTH (1-84) treatment. C, Change in OCN

/CD34

/CD146

cells. The
percentage of OCN

cells that was lacking both CD34 and CD146 increased with PTH (1-84)
treatment. D, Time course of increase in the OCN

cell population compared with time course of

increase in biochemical markers of bone formation with PTH (1-84) treatment.
TABLE 1. Relationship between increases in circulating
osteogenic cells from 0 to 3 months of PTH treatment
and increases in biochemical indices of bone formation
in hypoparathyroid subjects
Increase in cell
population
Increase in
biochemical
marker R
2
Slope ()
P
value
Total OCN

cells (%)
P1NP 0.36 12.63 3.2 0.0004
BAP 0.33 1.85 0.5 0.0007
Osteocalcin 0.35 2.12 0.5 0.0005
Subpopulation
OCN

/CD34

cells (%)
P1NP 0.20 1.20 0.4 0.009
BAP 0.10 0.15 0.07 0.04
Osteocalcin 0.20 0.19 0.07 0.01
Subpopulation
OCN

/CD146

cells (%)
P1NP 0.10 0.92 0.4 0.05
BAP 0.02 0.05 0.07 NS
Osteocalcin 0.04 0.08 0.08 NS
Subpopulation
OCN

/CD34

/
CD146

cells (%)
P1NP 0.20 0.78 0.3 0.007
BAP 0.10 0.08 0.04 NS
Osteocalcin 0.10 0.09 0.05 NS
NS, Not significant.
J Clin Endocrinol Metab, January 2011, 96(1):176186 jcem.endojournals.org 181
significantly, peaking at 23 months, and returning closer
to baseline levels by 12 months. The maturity stage of the
osteogenic cells may also increase, with a marked reduc-
tion in OCN

cells that contain markers associated with

earlier stages of osteoblast development. Measurements of
the circulating osteogenic cell populations correlated with
well-established biochemical, histomorphometric, and
molecular parameters of osteoblast function. The positive
correlations betweenthe measurements of circulating cells
with the osteoblast phenotype and well-established indi-
ces of osteoblast function in the circulation as well as his-
tomorphometric correlations help to confirm that PTH
stimulates bone formation by actions on osteoblasts.
Moreover, our observations that PTH is associated with
an apparent increase in the proportion of cells that have
lost early markers suggest that it may also help to regulate
osteoblast development.
Bone marrowCD34

cells, whichhave beenthought of

as hematopoietic stem cells, are also regarded as early os-
teoblastic cells (15, 16). CD34

cells are able to form

mineralized nodules (26) . Khosla and colleagues (16)
found that CD34

cells constitute 35% of OCN

cells in
human peripheral blood. Similarly, CD34

cells have
been shown to localize to the site of a fracture in rats and
to increase the differentiation of local stem cells into os-
teoblasts, leading to fracture repair (15). We thus used the
presence of CD34 as a marker to assess the maturity of the
OCN

cells. Molecular expression profiling suggested

that osteoblastic genes became detectable in cells that lost
the CD34 marker, suggesting that they are more mature.
We also found the marker CD146 to be useful because
bone marrow stromal cells that have this surface marker,
also known as MUC18, have been shown by Bianco and
colleagues (17) to be osteogenic. In human subjects,
CD146

subendothelial cells have been reported to serve

as skeletal progenitors capable of generating cells that or-
ganize a hematopoietic microenvironment on transplan-
tation (17). Whether the presence of the CD146 marker
reflects an earlier or later stage in the osteoblastic lineage
than the presence of the CD34 marker is unclear. Both
markers appear to be characteristic of progenitor cells.
Further expression studies would be necessary to deter-
mine the precise lineage sequence.
With PTH, the increases in osteoblast numbers that
showed characteristics of more mature cells antedated in-
creases in histomorphometric indices of bone formation,
the gold standard by which bone formation is directly
ascertained. The quadruple-label protocol allowed histo-
logical confirmation on bone formation dynamics both
before and after 3 months of PTH administration. The
observation that cancellous, endocortical, and intracorti-
cal envelopes of bone were similarly affected by PTHsug-
gests that the changes in circulating osteogenic cells are
reflected in all three bone envelopes. A conceptual con-
struct is suggested by these data, namely that new circu-
lating osteogenic cells are recruited and differentiated by
PTH exposure. The time course indicates an early stimu-
lation of number and differentiation followed by reduc-
tion to levels more typical of euparathyroid subjects.
The correlations between osteogenic cells and the se-
rum indices of bone formation were not as strong as the
correlations between osteogenic cells and histomorpho-
metric indices of bone formation. This discrepancy could
be explained by the timing of the samples. The osteogenic
cells peaked by 3 months of PTH treatment, which was
also when the bone biopsies were performed. On the other
hand, biochemical markers of bone turnover, which were
drawn monthly, rose later than the osteogenic cells, peak-
ing at 6 months and remaining elevated thereafter. This
time-course discordance between the osteogenic cells and
FIG. 4. A representative quadruple-label biopsy from a 25-yr-old
hypoparathyroid woman before and after PTH treatment. The first set
of double labels (bottom arrow) was acquired before PTH treatment,
and the second set of double labels (top arrow) was acquired after 3
months of PTH treatment.
TABLE 2. Absolute values of histomorphometric indices
of bone formation in quadruple-labeled bone biopsies in
hypoparathyroid subjects at 0 and 3 months of PTH
treatment (n 12)
Histomorphometric
parameter 0 months 3 months P value
Cancellous
Md.Pm (%) 0.41 0.2 6.55 1.9 0.01
MAR (m/d) 0.32 0.1 0.67 0.1 0.001
BFR (m
3
/m
2
d) 0.003 0.01 0.051 0.02 0.01
Endocortical
Md.Pm (%) 0.82 0.4 10.13 2.5 0.01
MAR m/d) 0.13 0.1 0.61 0.1 0.001
BFR (m
3
/m
2
d) 0.004 0.01 0.083 0.02 0.01
Intracortical
Md.Pm (%) 4.04 1.2 8.53 1.7 0.01
MAR (m/d) 0.70 0.2 0.81 0.1 NS
BFR (m
3
/m
2
d) 0.045 0.02 0.093 0.02 0.01
Data are mean SEM. NS, Not significant.
182 Rubin et al. Osteogenic Cells in Hypoparathyroidism J Clin Endocrinol Metab, January 2011, 96(1):176186
the biochemical markers likely explains the weaker tem-
poral correlations between them.
The effect of PTH on hematopoietic stem cell niche
expansion requires further investigation. We found that
circulating hematopoietic stemcells, as represented by to-
tal CD34

and OCN

/CD34

cells did not change with

PTHadministration. Our expectation was that these pop-
ulations would have increased; that in addition to mobi-
lizing osteoblast lineage cells, which support hematopoi-
esis, PTH would similarly mobilize hematopoietic stem
cells. Such a finding would have been consistent with re-
cent observations in mice illustrating that mobilization of
hematopoietic stem cells occurs within 14 d of PTH ad-
ministration(27). It is infact possible that suchanincrease
did occur but that it occurred before the 1-month time
point that was measured. Another possibility is that PTH
TABLE 3. Relationship between increases in circulating osteogenic cells from 0 to 3 months of PTH treatment and
increases in histomorphometric indices of bone formation in quadruple-labeled bone biopsies in hypoparathyroid
subjects (n 12)
Increase in cell population
Increase in
histomorphometric parameter R
2
Slope () P value
Total OCN

cells (%) Cancellous

Md.Pm (%) 0.5 3.041 0.69 0.0003
MAR (m/d) 0.4 0.107 0.03 0.003
BFR (m
3
/m
2
d) 0.5 0.024 0.01 0.0005
Endocortical
Md.Pm (%) 0.6 4.205 0.86 0.0001
MAR (m/d) 0.7 0.171 0.03 0.0001
BFR (m
3
/m
2
d) 0.6 0.036 0.01 0.0001
Intracortical
Md.Pm (%) 0.6 1.748 0.36 0.0001
MAR (m/d) 0.2 0.067 0.03 0.04
BFR (m
3
/m
2
d) 0.6 0.020 0.01 0.0003
Subpopulation OCN

/CD34

cells (%) Cancellous

Md.Pm (%) 0.3 0.265 0.10 0.01
MAR (m/d) 0.4 0.012 0.01 0.006
BFR (m
3
/m
2
d) 0.3 0.002 0.01 0.01
Endocortical
Md.Pm (%) 0.5 0.431 0.11 0.001
MAR (m/d) 0.6 0.017 0.01 0.0003
BFR (m
3
/m
2
d) 0.5 0.004 0.01 0.0007
Intracortical
Md.Pm (%) 0.5 0.192 0.05 0.0005
MAR (m/d) 0.2 0.007 0.01 NS
BFR (m
3
/m
2
d) 0.5 0.002 0.01 0.0005
Subpopulation OCN

/CD146

cells (%) Cancellous

Md.Pm (%) 0.3 0.286 0.12 0.02
MAR (m/d) 0.1 0.007 0.01 NS
BFR (m
3
/m
2
d) 0.2 0.002 0.01 0.03
Endocortical
Md.Pm (%) 0.3 0.386 0.15 0.02
MAR (m/d) 0.3 0.016 0.01 0.008
BFR (m
3
/m
2
d) 0.3 0.004 0.01 0.01
Intracortical
Md.Pm (%) 0.3 0.155 0.06 0.03
MAR (m/d) 0.2 0.009 0.01 0.05
BFR (m
3
/m
2
d) 0.3 0.002 0.01 0.01
Subpopulation OCN

/CD34

/CD146

cells (%) Cancellous

Md.Pm (%) 0.3 0.166 0.07 0.02
MAR (m/d) 0.2 0.006 0.01 0.04
BFR (m
3
/m
2
d) 0.3 0.001 0.01 0.02
Endocortical
Md.Pm (%) 0.3 0.233 0.09 0.02
MAR (m/d) 0.5 0.011 0.01 0.001
BFR (m
3
/m
2
d) 0.4 0.002 0.01 0.007
Intracortical
Md.Pm (%) 0.4 0.117 0.03 0.002
MAR (m/d) 0.3 0.006 0.01 0.02
BFR (m
3
/m
2
d) 0.5 0.001 0.01 0.001
Data are mean SEM. NS, Not significant.
J Clin Endocrinol Metab, January 2011, 96(1):176186 jcem.endojournals.org 183
is altering hematopoietic stem cell engraftment (29, 30).
Bianco and colleagues (17) have shown that CD45

/
CD34

/CD146

marrowcells containasubpopulationof
cells that can reconstitute a complete bone that is hospi-
table to bone marrow. These observations have recently
been extended to include an even more specific subset of
cell markers essential for bone regeneration (31). Further
investigations delineating the effects of PTH on these cell
types will help to address this point.
Alimitation to this study is that the characterization of
the circulating osteogenic cells, including their relation-
ship to mature osteoblasts on the bone surface, is still pre-
FIG. 5. A and B, Expression of osteoblast differentiation markers in sorted osteogenic cells. OCN

/CD34

cells and OCN

/CD34

cell populations
were isolated. The expression of osteoblast differentiation marker genes was assessed by real-time PCR. Bars indicate means of duplicate
determinations. C, Expression of osteoblast differentiation markers in cultured OCN

/CD34

cells. Sorted OCN

/CD34

cells were cultured in

growth medium for 3 wk followed by either osteogenic differentiation media (black bars) or vehicle (gray bars) for an additional 3 wk. The
expression of osteoblast differentiation marker genes was assessed by real-time PCR. *, P 0.05. D and E, Alizarin red staining in cultured OCN

/
CD34

cells. Sorted OCN

/CD34

cells were cultured in growth medium for 3 wk followed by either osteogenic differentiation media (D) or
vehicle (E) for an additional 3 wk. Panel D shows positive staining as well as the presence of nodules.
184 Rubin et al. Osteogenic Cells in Hypoparathyroidism J Clin Endocrinol Metab, January 2011, 96(1):176186
liminary. It is possible that the OCN

cells might be pro-

viding information about other cells of the mesenchymal
lineage, such as adipocytes and chodrocytes, instead of
osteoblasts. However, the evidence for the osteogenic
identity of these cells includes our observed correlations
with established serum and histomorphometric markers
of osteoblast functionas well as the pertubationinthe cells
coincident with PTH administration. Further supportive
evidence comes fromthe molecular expression data in the
sorted cells. Although the expression of osteocalcin
mRNA is to be expected because the cells were sorted
using an antibody to osteocalcin, the expression of other
key osteoblastic genes in cell cultures supports the osteo-
genic nature of these cells. In addition, cultures of the
sorted OCN

cells showed that they also expressed os-

teoblastic genes and had the potential to formmineralized
nodules in vitro. Our data thus suggest that selection for
these peripheral cells enriches for an osteogenic popula-
tion capable of mineralization. Measurement of osteo-
blast numbers and how they correlate with circulating
osteogenic precursor cells could be an additional way to
validate the measurement of these cells. Furthermore, re-
cent data suggest that the mesenchymal marker CD73
might identify early osteogenic cells (28). Human embry-
onic stem cells that are initially double positive for both
CD34 and CD73 (CD34

/CD73

) subsequently lose
CD34expression. The cells that remain, namely those that
express only CD73, are capable of multilineage mesen-
chymal differentiation, including into osteoblasts (28).
Use of this marker, as well as investigations toconfirmthat
circulating osteogenic cells ultimately return to the mar-
rowas osteoblast precursors, are important future areas of
investigation.
In conclusion, these results provide evidence that the
number and possible maturity of circulating osteogenic
cells are reduced in hypoparathyroidism, a clinical model
in which PTH is absent. The actions of PTH administra-
tion to increase the number and possibly the maturity of
these cells, in the context of this clinical model, help to
support the hypothesis that PTH stimulates bone forma-
tion by actions on the number and maturation of osteo-
genic cells.
Acknowledgments
Address all correspondence and requests for reprints to:
Mishaela R. Rubin, M.D., Department of Medicine, College of
Physicians and Surgeons, 630 West 168th Street, New York,
New York 10032. E-mail: mrr6@columbia.edu.
This work was supported by Grant DK077696 and Florence
Irving Research Award FD-R-02525.
Disclosure Summary: The authors have nothing to declare.
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186 Rubin et al. Osteogenic Cells in Hypoparathyroidism J Clin Endocrinol Metab, January 2011, 96(1):176186