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NEW OFFICIAL PUBLICATION! Scientific basis for the HemaXellerate I™:

( DD taken from http://regenbiopharma.com/news/2013.02.05.html as well as http://www.translational-medicine.com/content/pdf/1479-5876-10-231.pdf )

"HemaXellerate I™ is a patient-specific composition of cells that have previously been demonstrated to repair damaged bone marrow and stimulate production of blood cells based on previous animal studies. The company, together with an internationally-renowned group of stem cell researchers, recently published the scientific basis for the HemaXellerate I™ product which may be found at www.translational-medicine.com/content/pdf/1479-5876-10-231.pdf"

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REVIEW Open Access
Exogenous endothelial cells as accelerators of
hematopoietic reconstitution
J Christopher Mizer
1
, Thomas E Ichim
1,2*
, Doru T Alexandrescu
1
, Constantin A Dasanu
3
, Famela Ramos
1
,
Andrew Turner
4
, Erik J Woods
5
, Vladimir Bogin
1,2
, Michael P Murphy
6
, David Koos
1
and Amit N Patel
7
Abstract
Despite the successes of recombinant hematopoietic-stimulatory factors at accelerating bone marrow reconstitution
and shortening the neutropenic period post-transplantation, significant challenges remain such as cost, inability to
reconstitute thrombocytic lineages, and lack of efficacy in conditions such as aplastic anemia. A possible means of
accelerating hematopoietic reconstitution would be administration of cells capable of secreting hematopoietic
growth factors. Advantages of this approach would include: a) ability to regulate secretion of cytokines based on
biological need; b) long term, localized production of growth factors, alleviating need for systemic administration of
factors that possess unintended adverse effects; and c) potential to actively repair the hematopoietic stem cell
niche. Here we overview the field of hematopoietic growth factors, discuss previous experiences with mesenchymal
stem cells (MSC) in accelerating hematopoiesis, and conclude by putting forth the rationale of utilizing exogenous
endothelial cells as a novel cellular therapy for acceleration of hematopoietic recovery.
Background
During hematopoietic stem cell (HSC) transplantation,
the recipient is exposed to combinations of chemother-
apy and/or radiotherapy that result in destruction of en-
dogenous HSC thereby creating space in the bone
marrow niche for donor cells to engraft [1,2]. Unfortu-
nately, as a result of existing disease and due to the con-
ditioning regimen, there is a delayed time that elapses
while the donor cells are re-establishing hematopoiesis
in the recipient bone marrow microenvironment. This
period is associated with pancytopenia and increased
risk of bacterial, fungal, and viral infection. During bone
marrow or mobilized peripheral blood stem transplant-
ation, this “ risk period ” is approximately three to four
weeks after myeloablative transplantation [3].
One of the drawbacks of HSC transplantation is the lack
of donors. Approximately 30% of patients have a related
donor that can meet the stringent requirement of a 6/6 or
5/6 HLA match when HSC transplants are performed.
When unrelated donors are required, it takes approximately
four months to find a match using registries and minorities
are almost impossible to match [4-6]. In contrast to HSC
transplants, cord blood transplantation does not require
stringent matching and can be performed using 4/6 or even
3/6 matching, in part due to the immature nature of the
cells [7,8]. This obviously increases the donor pool avail-
able. As a result, there is an increasing use of cord blood as
a source of stem cells for transplants. Cord blood is super-
ior to bone marrow and peripheral blood HSC in terms of
reduced graft versus host disease and matching ability [9-
11]. The time to engraftment for cord blood, however, is
approximately four to seven weeks [12]. Studies have
shown that adults receiving cord blood transplants have a
relatively high rate of adverse events. For example, in one
study, of 68 patients with hematological malignancies, 60
patients survived 28 days or more after transplantation. Of
these, 55 had neutrophil engraftment at a median of 27
days. At 22 month follow-up, 19 of the 68 patients were
alive and only 18 of these were disease-free 40 months after
transplantation [13]. In another study, myeloablative ther-
apy followed by infusion of unrelated umbilical cord blood
cells was performed in 57 adult patients with high-risk,
hematological disease. All patients received granulocyte
colony-stimulating factor (G-CSF) after transplantation
until neutrophil recovery. Neutrophil recovery (neutrophil
count of 500/microL) occurred at 26 days and platelet re-
covery (>2,0000/microL) at 84 days. The median survival of
* Correspondence: [e-mail]
1
Regen BioPharma Inc, San Diego, CA, USA
2
Medistem Inc, San Diego, CA, USA
Full list of author information is available at the end of the article
© 2012 Mizer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Mizer et al. Journal of Translational Medicine 2012, 10:231
http://www.translational-medicine.com/content/10/1/231
the entire group was 91 days. Of the 57 patients, 11 were
alive at a median follow-up of 1,670 days with infection
being the primary cause of death [14].
Thus one of the limiting factors to HSC transplant-
ation is acceleration of engraftment of the donor cells,
particularly in cord blood transplantation. In order to
provide a framework for discussing means of acceler-
ation HSC reconstitution, we will describe various
hematopoietic growth factors that have entered clinical
use. This will position us to make the case for utilization
of various cells as therapeutics for hematopoietic recon-
stitution, with the concept that cells may function as
“ homeostatic producers ” of growth factors according to
the body ’ s needs.
Use of hematopoietic growth factors for acceleration of
engraftment/hematopoiesis
Currently, hematopoietic growth factors are used to re-
duce the period of cytopenia in the context of trans-
plantation. The most widely used hematopoietic growth
factor is Neupogen
TM
(Filgastrim), which is an E coli
produced form of granulocyte colony stimulating factor
(G-CSF) [15]. Early, open label studies demonstrated this
biological drug was capable of augmenting absolute neu-
trophil counts dose-dependently 1.8 – 12.0 fold in can-
cer patients undergoing chemotherapy [16], shortening
time until neutrophil recovery post myeloablation, and
shortening median febrile days and days in hospital [17].
Potency of Neupogen in non-transplant associated neu-
tropenia was demonstrated in a double blind, placebo
controlled trial, 123 patients with recurrent infections
(absolute neutrophil count < 0.5 x 10
9
/L) were rando-
mized to either receive Neupogen or enter a four month
observation period followed by Neupogen administra-
tion. Blood neutrophil counts, bone marrow histology,
and infection-related events were evaluated. Neupogen
administration was associated with significant decreases
in infection related events of approximately 50% as well
as an almost 70% reduction in duration of antibiotic use
[18]. Another double blind study examined 218 patients
with cancer who had fever (temperature > 38.2 degrees
C) and neutropenia (neutrophil count < 1.0 x 10
9
/L)
after chemotherapy. Patients were randomly assigned to
receive Neupogen (12 micrograms/kg of body weight
per day) (n = 109) or placebo (n = 107). Patients
received treatment and remained in the study until the
neutrophil count was greater than 0.5 x 10
9
/L and until
four days without fever (temperature < 37.5 degrees C)
occurred. Compared with the placebo, Neupogen
reduced the median number of days of neutropenia and
the time to resolution of febrile stage but not days of
fever. The frequency of the use of alternative antibiotics
was similar in the two groups. The median number of
days patients were hospitalized while in the study was
the same (8.0 days; P = 0.09). Neupogen did decrease
the risk for prolonged hospitalization by half [19].
Based on the above and numerous other studies
[15,18,20-28], Neupogen was approved in the USA by
the FDA for decreasing the incidence of infection as
manifested by febrile neutropenia in patients with non-
myeloid malignancies receiving myelosuppressive
chemotherapy drugs associated with a significant inci-
dence of severe neutropenia with fever. It is indicated
for reducing the time to neutrophil recovery and the
duration of fever following induction or consolidation
chemotherapy in patients with acute myeloid leukemia.
In patients with bone marrow transplant, is indicated to
reduce the duration of neutropenia and neutropenia-
related clinical sequelae (e.g. ‚ febrile neutropenia in
patients with nonmyeloid malignancies undergoing mye-
loablative chemotherapy followed by marrow transplant-
ation). Additionally, it is indicated for treatment of
chronic neutropenia [29,30]. A delayed-release form of
Neupogen, termed Neulasta (pegfilgrastin), is now also
approved for similar indications by the FDA [31].
Another biological drug belonging to the HSC-
stimulating family is recombinant granulocyte monocyte
colony stimulating factor (GM-CSF; Sargramostim, Leu-
kine) which was approved by the FDA for treatment of
post-chemotherapy hematopoietic recovery in older
adult patients with acute myelogenous leukemia (AML)
to shorten time to neutrophil recovery and acceleration
of myeloid reconstitution after autologous or allogeneic
bone marrow transplantation (BMT). Additionally, Leu-
kine was approved for use in bone marrow transplant-
ation failure or engraftment delay [32]. Clinical trials
with Leukine that supported its claim include a 125 pa-
tient double blind trial of post-chemotherapy AML
patients in which the growth factor was given 11 days
post therapy until neutrophil recovery. Time to neutro-
phil recovery, infectious toxicity, and treatment-related
toxicity was significantly reduced in the Leukine-treated
arm compared to placebo. Additionally, the median sur-
vival for patients was 10.6 months in the treated group
and 4.8 months in the placebo arm [33]. Clinical trials
with Leukine also included a 128 patient, multicenter,
double blind trial examining neutrophil recovery after
autologous bone marrow transplant. In this study,
patients in the treated group had a recovery of the neu-
trophil count to 500 × 10
6
per liter seven days earlier
than the patients who received placebo (19 vs. 26 days),
suffered from fewer infections, required 3 fewer days of
antibiotic administration (24 vs. 27 days), and required
six fewer days of initial hospitalization (median, 27 vs.
33 days). Unfortunately, there was no difference in sur-
vival rate at 100 days [34]. Another clinical trial with 134
patients in a double blind trial of cancer (hematological
and solid) patients suffering febrile neutropenia in which
Mizer et al. Journal of Translational Medicine 2012, 10:231 Page 2 of 12
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Leukine enhanced neutrophil recovery [35]. The current
practice is to utilize to some extent interchangeably
Neuopogen and Leukine based on the patient character-
istics and the familiarity of the institute.
Platelet recovery takes a longer time as compared to
granulocyte recovery, during HSC reconstitution.
Thrombopoietin (TPO) is a cytokine involved in stimu-
lating megakaryocyte production from the bone marrow.
In studies on small animals and non-human primates,
administration of TPO resulted in a rapid rise in platelet
counts to levels previously unattainable with other
thrombopoietic cytokines [36]. In myelosuppressed
models, use of TPO following chemotherapy, radiation,
or stem-cell transplantation accelerated megakaryocyte
and platelet recovery [37,38]. These studies prompted a
12 patient trial in sarcoma patients treated with a single
dose of TPO. Treatment was associated with an increase
in platelet counts, peaking at a 213% over baseline. This
increase began by day four in most patients and peaked
on a median of day 12 [39]. Other trials provided results
that were somewhat inconclusive. Nash et al examined
37 patients after bone marrow transplant (both autolo-
gous and allogeneic) who suffered from thrombo-
cytopenia that were treated with increasing doses of
TPO. Ten patients had recovery of platelet counts dur-
ing the 28-day study period; three of these ten had an in-
crease in marrow megakaryocyte content seven days
after completing treatment with TPO. When all baseline
marrows were compared with samples after TPO treat-
ment, there was no difference in marrow megakaryocyte
content [40]. No association between dose and megakar-
yocyte recovery was observed. Due to further studies
that demonstrated development of autoantibodies to
TPO in treated patients, as well as general lack of effi-
cacy, the clinical development of TPO was discontinued
in the USA [41].
In order to overcome these potential issues associated
with administration of proteins (eg sensitization), scien-
tists started to develop agonists of the TPO receptor.
One of these that was successfully developed is the TPO
mimetic peptide Romiplostim (originally named
AMG531) developed by Amgen. Early studies demon-
strated that in vitro administration of Romiplostim binds
the TPO receptor with similar affinity as TPO, induces
activation of the same molecular pathways (eg Mpl,
JAK2, and STAT5) and stimulates of megakaryocytic col-
ony growth from bone marrow cells [42]. Interestingly,
Amgen chose to focus clinical development of Romi-
plostim on immune mediated thrombocytopenia (ITP)
instead of post-transplant acceleration of thrombopoi-
esis. Two studies demonstrated safety and improvement
in platelet counts in a dose dependent manner in
patients with severe ITP [43]. Another study randomized
234 adult patients with immune thrombocytopenia and
provided the standard of care (77 patients) or weekly
subcutaneous injections of Romiplostim (157 patients).
The rate of a platelet response in the Romiplostim group
was 2.3 times that in the standard-of-care group.
Patients receiving Romiplostim had a significantly
reduced incidence of treatment failure (18 of 157
patients [11%]) than those receiving the standard of care
(23 of 77 patients [30%]). Splenectomy, a last resort
treatment for ITP, also was performed less frequently in
patients receiving Romiplostim (14 of 157 patients [9%])
than in those receiving the standard of care (28 of 77
patients [36%]). The Romiplostim group had a lower rate
of bleeding events, fewer blood transfusions, and greater
improvements in the quality of life than the standard-of-
care group. Serious adverse events occurred in 23% of
patients (35 of 154) receiving Romiplostim and 37% of
patients (28 of 75) receiving the standard of care [44]. In
August 2008, Romiplostim was approved by the FDA for
the treatment of thrombocytopaenia in patients with
chronic ITP who have had an insufficient response to
corticosteroids, immunoglobulins, or splenectomy [45].
Current hematopoietic growth factors have several
limitations: their inability to stimulate all hematopoietic
lineages, their inability to heal damaged bone marrow
microenvironment, and their adverse effects associated
with prolonged usage. From a health economics perspec-
tive, these agents are associated with high costs. For ex-
ample, according to one report, the cost of treating
neutropenic fever with Neupogen is approximately
$40,000 [46]. This included the cost of drug, hospital
stay, and care of the patient.
Cellular approaches for acceleration of hematopoietic
reconstitution: use of mesenchymal stem cells (MSC)
It is well known that chemotherapy and radiation are
damaging to the bone marrow microenvironment
[47,48]. Additionally, administration of human MSC has
been shown to accelerate hematopoietic reconstitution
in animal models [49,50]. Although the in vivo signifi-
cance of MSC is still highly debated, one theory is that
MSC in the bone marrow provide a suitable environ-
ment for hematopoiesis. Accordingly, one of the first
clinical uses of MSC has been to accelerate
hematopoietic recovery. In a 1995 paper, Lazarus et al.
reported the use of autologous, in vitro expanded, “ mes-
enchymal progenitor cells ” to treat 15 patients suffering
from hematological malignancies in remission. The
authors demonstrated feasibility of expanding bone mar-
row derived by MSC in vitro. They showed that a 10
milliliter bone marrow sample was capable of 16,000-
fold growth over a four to seven week in vitro culture
period. Cell administration was performed in total doses
ranging from 1 - 50 × 10
6
cells and was not causative of
Mizer et al. Journal of Translational Medicine 2012, 10:231 Page 3 of 12
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treatment associated adverse effects [51]. In a subse-
quent study from the same group in 2000, the use of
MSC to accelerate hematopoietic reconstitution was per-
formed in a group of 28 breast cancer patients who
received high dose chemotherapy. MSC at concentra-
tions of 1.0 - 2.2 × 10
6
/kg were administered intraven-
ously. No treatment associated adverse effects where
observed, and leukocytic and thrombocytic reconstitu-
tion appeared to undergo “ rapid recovery ” [52]. It is
interesting that these initial uses were actually in
patients with neoplasia and no overt acceleration of can-
cer progression was noted. Besides feasibility, these stud-
ies were important because they established the
technique for ex vivo expansion and readministration.
Despite these initial safety data, the use of mesenchymal
stem cells in the context of cancer has been raised in sev-
eral academic contexts. The recent regulatory approvals
of the Osiris Prochymal product in Canada and New
Zealand for graft versus host disease suggests that there is
some degree of safety, however long term follow up stud-
ies are necessary to convincingly state that MSC grafts do
not accelerate tumor formation/recurrence.
Studies along these lines continued which reaffirmed
the feasibility of the approach of “ repairing bone marrow
stroma ” with expanded MSC cells. In 2005, Lazarus et al
treated 46 patients suffering from hematological malig-
nancies with HLA-matched allografts comprising bone
marrow and donor-derived expanded MSC. The num-
bers of MSC administered were 1 - 5 million/kg. On
average, the time to neutrophil reconstitution (as defined
by absolute neutrophil count > or = 0.500 × 10
9
/L) and
platelet reconstitution (as defined by platelet count > or
= 20 x 10
9
/L was 14.0 days (range 11.0 - 26.0 days) and
20 days (range 15.0 - 36.0 days). Incidence of acute,
Grade II-IV GVHD was 13/46 and chronic was 22/36
patients that survived for at least 90 days. Relapse of ma-
lignancy occurred in 11 patients with a median time to
progression of 213.5 days (range 14 - 688 days). The
authors concluded that cotransplantation of HLA-
identical sibling culture-expanded MSCs with an HLA-
identical sibling HSC transplant was feasible and safe,
without immediate infusional or late MSC-associated
toxicities [53]. These data were of importance since one
of the concerns regarding MSC treatment is associated
with growth factor production. Leukemic patients have
minimally residual disease which seems to be at least in
part controlled by recipient immune function [54,55].
The demonstration that the recipients did not have an
overtly higher incidence of relapse, based on clinical
experiences of the authors with the specific
hematological malignancies, suggests that MSC do not
endow a preferential advantage to leukemic cells. This is
interesting given that MSC are generally considered im-
mune suppressive cells [56,57].
Other studies also supported the safety aspect and
included several variations. For example, Ball et al
reported on the use of purified, donor-specific MSC (1 -
5 million/kg) injected alongside with isolated CD34 from
HLA-mismatched relatives in 14 pediatric leukemia
patients. They showed that in contrast to traditional
graft failure rates of 15% in 47 historical controls, all
patients given MSCs showed sustained hematopoietic
engraftment without any adverse reaction. Interestingly,
children given MSCs did not experience more infections
compared with controls [58]. Zhang et al [59] reported
that 12 patients cotransplanted with donor MSC (1.77
+/- 0.40) × 10
6
/kg and HSC. No observable, adverse re-
sponse during and after the infusion of MSCs was
reported and hematopoietic reconstitution occurred rap-
idly. Two patients developed grade II-IV acute GVHD
and two extensive chronic GVHD. Four patients suffered
from cytomegalovirus infection but were cured ultim-
ately. Up to the time of publication, seven patients had
been followed as long as 29 - 57 months and five
patients died. It was concluded by the authors that
MSCs can be expanded effectively by culture and are
safe and feasible to cotransplant in patients with allo-
genic, culture-expanded MSCs.
As mentioned above, engraftment of cord blood
occurs over a more protracted time period as compared
to bone marrow. Macmillan et al used parental haploi-
dentical MSC to promote engraftment in 15 pediatric
recipients of unrelated donor umbilical cord blood for
acute leukemias. Eight patients received MSCs on day
zero with three patients having a second dose infused on
day 21. The average dose of the first infusion was 2.1
million/kg (range 0.9 – 5.0/kg). The second infusion was
1 million, 600,000, and 5 million per kg. The reason for
the inconsistency was lack of ability to expand cells
in vitro. No serious adverse events were observed with
any MSC infusion. All eight evaluable patients achieved
neutrophil engraftment at a median of 19 days. Probabil-
ity of platelet engraftment was 75% at a median of 53
days. At the median follow-up of 6.8 years, five patients
were alive and disease free [60]. Meuleman et al used
donor-derived expanded MSC (10
6
/kg) to treat six
patients to accelerate hematopoietic recovery. Two
patients displayed rapid hematopoietic recovery (days 12
and 21) and four patients showed no response. One pa-
tient developed cytomegalovirus (CMV) reactivation 12
days following the MSC infusion and died from CMV dis-
ease, although the authors stated that it was impossible to
discern whether the reactivation was associated with the
MSC therapy or prior immune suppressive regimen [61].
Use of third-party MSC to enhance peripheral blood
stem cell grafts was performed by Baron et al in 20
patients who received non-myeloablative hematopoietic
stem cell transplant. The outcomes were compared to a
Mizer et al. Journal of Translational Medicine 2012, 10:231 Page 4 of 12
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control group of 16 patients receiving a similar trans-
plant protocol without MSC. MSC were administered
one-half hour to two hours before the hematopoietic
graft. Out of the 20 patients, one had primary graft fail-
ure. One year non-relapse mortality was 10% and relapse
occurred in 30%. Overall survival was 80%, progression-
free survival was 60%, and 1-year incidence of death
from GVHD or infection with GVHD was 10%. In the
control group, the one year incidence of non-relapse
mortality was 37% (P = .02); the one year incidence
of relapse was 25% (NS). The one year overall survival
and progression free survival was 44% (P = .02), and
38% (P = .1), respectively. The one year rate of death
from GVHD or infection with GVHD of 31% (P = .04)
[62]. Of particular interest is that the nonmyeloablative
protocol used in this study depends largely on donor
graft versus leukemia effect [63]. Therefore, because the
MSC did not cause a greater increase in leukemic re-
lapse, there is suggestion that these cells may not be
cancer-promoting, at least not from the perspective of
immune suppressive activities. These data suggest that
MSC coinfusion does not accelerate relapse may actually
possess beneficial properties in terms of graft versus
tumor events. There is still some controversy in that
Ning et al showed that out of ten patients who received
MSC coinfusion, six had relapses, whereas only three of
the fifteen who received transplants without MSC had
relapses [64]. There is some debate whether patient se-
lection in the study was appropriately matched between
controls and treated groups [65]. Drawbacks of using
MSC for hematopoietic engraftment also include the po-
tential risk of acceleration of tumor relapse [64]. Specif-
ically, the immune modulatory properties of these cells
may decrease the GVL effect.
Thus the use of cells to stimulate hematopoietic re-
constitution appears to be clinically feasible as a possible
adjuvant or replacement for hematopoietic growth factor
administration. Possible advantages of using cellular
based therapies as compared to individual growth factors
include: a) that multiple cytokines and growth factors
are produced, which may have synergistic effects not
observed with single cytokine administration; b) produc-
tion of factors may be regulated based on physiological
and microenvironmental signals; and c) anti-
inflammatory effects of cells may have additional bene-
fits in terms of healing bone marrow microenvironment.
Below we will discuss.
Endothelial cells as a component of the hematopoietic
stem cell niche
Originally thought of as an inert structure, over the past
two decades the endothelium has received significant at-
tention as a dynamic surface cell that acts as an adapt-
able, anticoagulated barrier between the blood stream
and interior of the blood vessel. This allows for selective
transmigration of cells in and out of the blood stream,
regulates blood flow through controlling smooth muscle
contraction, and participates in tissue remodeling and
angiogenesis [66-70]. Embryonically, endothelial cells are
believed to originate from a stem cell, the hemangio-
blast, which is capable of giving rise to both
hematopoietic and endothelial cells [71]. During adult-
hood, the endothelium is continually self-renewed by a
population of bone marrow-derived cells termed endo-
thelial progenitor cells (EPC). This progenitor popula-
tion has previously been characterized as expressing the
CD34 HSC marker as well as VEGF-receptor 2 and
AC133 [72]. These cells have been demonstrated using
in vivo chimeric models to repair damaged blood vessels
in non-diseased [73] as well as in pathological settings
[74,75].
Given the importance of endothelial cells in so many
aspects of biological systems, it would be reasonable to
explore the ways in which endothelial cells provide sup-
port, if not control, for hematopoietic processes. Specif-
ically, the observation that hematopoietic and
endothelial cells originate developmentally from a com-
mon precursor may suggest that in adulthood these cells
are inter-related. The original experiments highlighting
the interaction between hematopoietic cells and endo-
thelial cells were studies which attempted to recapitulate
hematopoiesis in vitro. Early experiments demonstrated
that an endothelial cell layer was essential as part of the
“ stroma ” for in vitro hematopoiesis [76,77]. Interestingly,
soluble factors generated by endothelial cells that sup-
ported in vitro hematopoiesis were not only identified
[78], but it was demonstrated that their production was
inducible by various agents such as lipopolysaccharide
[79]. This suggests that the endothelium was an indu-
cible source of factors stimulating hematopoiesis when
physiologically necessary, such as during infection [80].
Detailed characterization of the role of endothelium in
hematopoiesis was performed initially by anatomical
studies which defined the sinusoidal aspects of the bone
marrow hematopoietic endothelium [81,82]. Morpho-
logical changes of this specialized endothelium have
been identified during times of excessive production of
various blood cells [83], hinting at a possible involve-
ment in the process of hematopoiesis [84]. During times
of inflammation, structural changes occur in the bone
marrow endothelium, in part to support release of gran-
ulocytes [85]. Such changes also occur in response to ad-
ministration of bone marrow stem cell mobilizing
agents, such as Neupogen, which causes stimulation of
localized complement activation through activation of
proteases that expose neoepitopes in the bone marrow
microenvironment [86,87]. Because of the physical prox-
imity of HSC and endothelial cells, as well as the ability
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of endothelial cells in vitro to support hematopoiesis,
investigators have sought to identify molecular means by
which endothelial cells may control hematopoiesis.
Endothelial cells produce hematopoietic growth factors
The factors controlling hematopoiesis were originally
described by biological activity in colony forming assays
before the era of molecular biology unfolded. Knudtzon
and Mortensen in 1975 described experiments in which
bone marrow mononuclear cells were plated on agar
and colony formation was assessed as a means of quanti-
fying stem cell proliferation and differentiation. They
observed that human endothelial cells were capable of
specifically stimulating granulocyte colonies. Specifically,
they co-cultured vein fragments or isolated endothelial
cells separated from the vein of human umbilical cords.
The granulocyte colony stimulating activity was superior
to that obtained by co-culture with leukocytes. They
suggested that in addition to monocytes, that bone mar-
row resident endothelial cells supported proliferation
and granulocytic differentiation of hematopoietic stem
cells [88].
Five years later, Quesenberry and Gimbrone used a
similar assay system and observed not only that endo-
thelial cells were capable of stimulating hematopoiesis
and granulopoiesis, but also that pretreatment of the
endothelial cells with either endotoxin or granulocytes
augmented the ability of endothelial cells to promote
granulopoiesis [79]. Further studies revealed that treat-
ment of endothelial cells with TNF-alpha, a potent medi-
ator of inflammation, actually resulted in augmentation
of hematopoietic stimulatory activity, in part through
production of a protein that was later identified as GM-
CSF [89]. A similar finding was demonstrated with the
inflammatory cytokine IL-1 [90].
Ascensao et al sought to identify other mechanisms re-
sponsible for endothelial cell stimulation of bone mar-
row hematopoiesis. They identified not only the
stimulation of granulopoiesis but also erythropoiesis.
Specifically, they found that endothelial cells produce
proteins of approximately 30,000 Daltons with isoelectric
focusing points of 4.5 and 7.2 that stimulated the growth
of human BFU-E and CFU-mix. They also found heat-
labile proteins of 30,000 and 15,000 Daltons inducing
the proliferation and differentiation of granulocyte-
macrophage (CFU-GM) colonies. Interestingly, no
erythropoietin was found in the culture [91]. More
detailed examination revealed that GM-CSF was one of
these proteins [92]. Several other studies have confirmed
GM-CSF production from endothelial cells. Malone et al
used adipose derived endothelial cells and demonstrated
GM-CSF production in an unstimulated state [93]. A
table describing some of the hematopoietic growth
factors produced by endothelial cells both under stimu-
lated and unstimulated conditions is provided below
(Table 1).
Endothelial cells stimulate hematopoiesis
One of the major goals of hematology research has been
the development of methods of expanding hematopoietic
stem cells outside of the body. This would hypothetically
allow for various approaches to purging leukemic cells
out of autologous grafts while expanding non-malignant
hematopoietic cells, as well as expanding cord blood
stem cells in order to accelerate engraftment after trans-
plantation [103-105]. As previously discussed, early stud-
ies have used bone marrow stromal cells for expanding
hematopoietic stem cells [106]. Components of the bone
marrow stroma include monocytes, adipocytes, and mes-
enchymal stem cells [107]. An interesting finding was
that endothelial cells, whether originating from bone
marrow, brain, or fat, all possessed the ability to stimu-
late hematopoietic cell expansion. Specifically, Davis et
al [108] examined the ability of porcine microvascular
endothelial cells (PMVECs) together with combinations
of cytokines (GM-CSF, IL-3, SCF, IL-6) to support the
expansion and development of purified human CD34+
bone marrow cells. In seven day cultures, the greatest
HSC expansion was observed when the HSC were in dir-
ect contact with PMVEC monolayers, followed by
PMVEC noncontact and liquid suspension cultures.
Maximal expansion of nonadherent cells (42-fold) and
total CD34+ cells (12.6-fold) occurred in PMVEC con-
tact cultures treated with GM-CSF + IL-3 + SCF + IL-6,
with similar increases in the number of granulocyte-
macrophage colony-forming units (CFU-GM), CFU-mix,
erythroid burst-forming units (BFU-E), CFU-blast and
CFU-megakaryocyte (CFU-Mk) progenitor cells. In long-
term PMVEC contact cultures, CD34+ cells seeded onto
PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6
showed a total calculated expansion of over 5,000,000-
fold of nonadherent cells over 35 days in culture. These
experiments not only demonstrated efficacy of endothe-
lial cells at stimulating HSC proliferation, but also that
such factors are not species-specific, thus enabling ani-
mal experiments with human cells. These studies were
also confirmed in another paper [109].
Other studies have associated endothelial cell produc-
tion of IL-6, SCF, G-CSF and GM-CSF ex vivo HSC ex-
pansion [110]. The expansion of HSC by endothelial
cells has been shown to accelerate bone marrow engraft-
ment in vivo [111]. Acceleration of hematopoietic recov-
ery has been demonstrated not only with endothelial
cells but also with conditioned media of these cells, sug-
gesting both contact dependent and contact independent
effects [112]. Ex vivo expansion of HSC using endothe-
lial cells was demonstrated to generate HSC that were
Mizer et al. Journal of Translational Medicine 2012, 10:231 Page 6 of 12
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functional in a large animal model [113], specifically, a
control baboon received no transplant and two animals
that received a suboptimal number of marrow mono-
nuclear cells died 37, 43, and 59 days post-irradiation.
Immunomagnetically selected CD34(+) marrow cells
from two baboons were placed in porcine microvascular
endothelial cells (PMVEC) co-culture with exogenous
human cytokines. After 10 days of expansion, the grafts
represented a 14-fold to 22-fold increase in cell number,
a 4-fold to 5-fold expansion of CD34(+) cells, a 3-fold
to 4-fold increase of colony-forming unit-granulocyte-
macrophage (CFU-GM), and a 12-fold to 17-fold
increase of cobblestone area-forming cells (CAFC) over
input. Both baboons receiving the treatment became
transfusion independent by day 23 post-transplant and
achieved absolute neutrophil count ANC >500/microL
by day 25 ± 1 and platelets > 20,000/microL by day 29 ± 2.
In addition to expanding functional HSC ex vivo,
the direct injection of exogenous endothelial cells has
demonstrated hematopoietic stimulatory effects. Salter
et al treated BALB/c mice with total body irradiation
(TBI) followed by infusion with C57Bl6-derived endo-
thelial progenitor cells (EPCs). TBI caused pronounced
disruption of the BM vasculature, BM hypocellularity,
ablation of HSCs, and pancytopenia in control mice.
Irradiated, EPC-treated mice displayed accelerated re-
covery of BM sinusoidal vessels, BM cellularity, periph-
eral blood white blood cells (WBCs), neutrophils,
platelets, and a 4.4-fold increase in BM HSCs. Systemic
administration of anti-VE-cadherin antibody significantly
delayed hematologic recovery in both EPC-treated mice
and irradiated, non-EPC-treated mice compared with
irradiated controls [114]. Such hematopoietic stimula-
tory effects were observed with endothelial cells from a
variety of sources. For example, it has been shown that
both brain and fetal blood derived endothelial cells have
ability to endow radioprotection, as well as accelerate re-
constitution in C57Bl6 mice treated with 1050 cGy of ra-
diation [115]. In another series of experiments, Fleming ’ s
group demonstrated that transplantation of segments of
adult thoracic aorta or inferior vena cava under the kid-
ney capsule of lethally irradiated recipients (1100 cGy)
resulted in significant radioprotection. Specifically, 10
mg of transplanted vascular tissue would protect 80% of
recipients from lethality. Furthermore, this procedure
gave rise to similar numbers of colony forming units as
rescue using 10
5
bone marrow cells and prevented the
development of severe anemia. Labeling of proliferating
cells using BRDU revealed that the cells within the in-
tima of donor vascular tissue would begin proliferation
within 48 hours of transplantation. It was also demon-
strated in cell tracking studies that donor-derived vascu-
lar cells migrated to the recipient spleen; hematopoietic
colony forming units were of host origin. Although
donor-derived cells were readily detected in the periph-
eral blood two to three weeks after transplant, they rap-
idly declined in frequency to approximately 1.0% by four
weeks and persisted at these levels for more than one
Table 1 Hematopoietic factors produced by endothelial cells
Hematopoietic factors produced by endothelial cells
Source of endothelium Stimulant Cytokine/CSA Reference
Umbilical vein and HUVEC None Granulocyte stimulatory activity [88]
HUVEC Endotoxin and
granulocyte contact
Granulocyte-monocyte colony stimulating activity [79]
HUVEC None Granulocyte-monocyte, and erythroid colony stimulating activity [91]
HUVEC Muramyl peptides Granulocyte-monocyte colony stimulating activity [94]
HUVEC TNF-alpha Granulocyte-monocyte colony stimulating activity [89]
Primary and immortalized
HUVEC
IL-1 Granulocyte-monocyte colony stimulating activity [90]
Immortalized HUVEC None Granulocyte-monocyte colony stimulating activity [92]
Fat capillary endothelial cells None GM-CSF [93]
HUVEC IL-1 Erythrocyte colony stimulating activity [95]
HUVEC Poly (IC) Monocyte, Granulocyte, Granulocyte-Monocyte stimulatory activity [96]
Brain perivascular endothelial
cells
None M-CSF [97]
HUVEC None Stem Cell Factor [98]
HUVEC versus bone marrow
microvessels
None High expression of heparan sulfate proteoglycans only on bone marrow
microvessels, these present SDF-1
[99]
HUVEC None IL-3, IL-6, Stem Cell Factor [100]
HUVEC IL-1 GM-CSF, G-CSF [101]
HUVEC None IL-11 [102]
Mizer et al. Journal of Translational Medicine 2012, 10:231 Page 7 of 12
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year. Bone marrow from rescued primary recipients pro-
vided radioprotection after transplantation into second-
ary recipients, but only CD3(+) donor-derived cells were
detected [116]. The question remained from these ex-
periment about whether similar hematopoietic effects
could be observed by endothelial cells alone or if they
require other vascular cells that mediate therapeutic
effects. This was addressed in another paper by the same
group which assessed vascular endothelial cells adminis-
tered in suspension. The investigators found that as little
as 10
4
endothelial cells either isolated from the murine
lung or brain were capable of radioprotecting mice
[117]. Furthermore, endothelial cell administration was
associated with reconstitution of host hematopoiesis,
with the expanded cells being able to transfer
hematopoiesis to secondary recipients.
These data suggest: a) allogeneic endothelial cells, ap-
parently regardless of source, may conceptually be useful
for acceleration of hematopoietic reconstitution; b)
endothelial cells actively contributed to repair of the
bone marrow microenvironment; and c) cell to cell
interactions mediated by specific adhesion molecules are
essential for therapeutic effects. Such hematopoietic
stimulatory effects were observed with endothelial cells
from a variety of sources.
Practical use of endothelial cells for stimulation of
hematopoietic recovery: hemaxellerate autologous
adipose derived cells as a source of endothelial cells
The most practical embodiment of the concept that
endothelial cells stimulate hematopoiesis in vivo would
be the utilization of autologous, adipose derived, endo-
thelial cells. In this system, it may be useful to utilize the
whole stromal vascular fraction (SVF) as a heterogenous
cellular product. SVF is comprised of the mononuclear
cells derived from adipose tissue and are known to con-
tain not only endothelial cells, but also T regulatory
cells, monocytes, and hematopoietic stem cells. This
term is more than four decades old and is used to de-
scribe the mitotically active source of adipocyte precur-
sors [118,119]. SVF as a source of stem cells was first
described by Zuk et al who identified mesenchymal-like
stem cells cells in SVF that could be induced to differen-
tiate into adipogenic, chondrogenic, myogenic, and
osteogenic lineages [120]. Subsequent to the initial de-
scription, the same group reported after in vitro expan-
sion the SVF derived cells had surface marker
expression similar to bone marrow derived MSC, com-
prising of positive for CD29, CD44, CD71, CD90,
CD105/SH2, and SH3 and lacking CD31, CD34, and
CD45 expression [121,122].
Currently liposuction is performed routinely and nu-
merous protocols exist for autologous collection and ad-
ministration of SVF which contains all of the cellular
elements mentioned. In fact, safety of this procedure has
been previously published for rheumatoid arthritis in a
13 patient study in which patients were monitored for
over one year [123]. Several other studies have demon-
strated a high content of EPC in adipose tissue
[124,125]. Functional demonstration of adipose EPC was
performed in experiments in which SVF was purified for
CD34 positive cells. This fraction was demonstrated to
induce angiogenesis in immune compromised mice that
were subjected to hindlimb ischemia. Mechanistically,
the cells were identified as EPC based on ability to form
endothelial colonies when cultured in vitro [126]. Nu-
merous groups have reported SVF contains cellular ac-
tivity stimulatory of angiogenesis. For example, Sumi et
al showed that administration of SVF but not adipocytes
led to revascularization in the hindlimb ischemia model
[127]. Other studies have shown that EPC-like activities
are found in SVF [128], and also that conditioned media
from SVF is capable of stimulating host angiogenesis
[129,130]. It is reported that EPC in the SVF capable of
stimulating angiogenesis directly or through release of
growth factors such as IGF-1, HGF-1 and VEGF
[128,130-132].
Use of non-expanded SVF is commonplace in veterin-
ary medicine and has been shown to be safe and effect-
ive. Pioneered by the Harman group at Vet-Stem,
Double blind studies of SVF for canine osteoarthritis
have shown statistically significant improvements in
lameness, range of motion, and overall quality of life
after treatment with autologous SVF [133,134]. To date
Vet-Stem has treated over 5,000 canines using local,
intra-articular administration as well as systemic intra-
venous administration of these cells. Additionally, it is
reported that over 3,000 horses with various cartilage
and bone injuries have been treated with autologous
lipoaspirate fractions without cellular expansion [135].
These studies have not only demonstrated safety and in
some cases efficacy, but also have established the prac-
tical foundation of commercialized stem cell therapeu-
tics, at least in the area of veterinary medicine [136]. For
clinical use closed system point-of-care devices have
been developed by the companies Cytori and Tissue
Genesis to allow for rapid processing of adipose tissue,
without need for a Good Manufacturing Practices com-
pliant laboratory [137,138].
In the published literature, the clinical use of systemic-
ally administered SVF cells has been reported in three
studies. The first study was a description of 3 patients
suffering from multiple sclerosis who received intraven-
ous administration of autologous adipose SVF. All 3
patients reported significant improvement neurologically
and demonstrated a good safety profile [139]. In another
paper, a single patient case report described a remission
of rheumatoid arthritis [140]. More recently, a paper
Mizer et al. Journal of Translational Medicine 2012, 10:231 Page 8 of 12
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demonstrating one year safety of rheumatoid arthritis
patients treated with autologous SVF was published
[123]. The general safety of fat grafting is widely
accepted in that this is a common procedure in cosmetic
surgery [141,142].
The HemXellerate product is an optimized autologous
SVF preparation for stimulation of hematopoiesis that
is covered by one issued patent and two patent applica-
tions. In practice the physician is shipped a kit, which is
used to collect adipose tissue, tissue is sent to a central
processing facility, and a standardized cellular product is
delivered in a ready-to-use manner. Currently the com-
pany is developing various uses for the HemXellerate pro-
duction in conditions involving suppressed hematopoiesis.
Conclusion
While significant advances have been made in the use of
growth factors for stimulation of hematopoietic reconsti-
tution, overall efficacy of this approach is limited. Cell
therapy offers the possibility of administering an “ intelli-
gent ” therapeutic which addresses the hematopoietic
needs of the host in real-time. Autologous SVF therapy,
such as the HemXellerate product, offers the possibility
of delivering a heterogenous population of endothelial
cells, mesenchymal stem cells, and other potential cells
of interest. Given the established safety and ease of
autologous SVF administration, it is anticipated that
the proposed procedure will be rapidly adopted by
the market.
Competing interests
TEI and VB are shareholders of the company Medistem (MEDS) which is
developing universal donor stem cell technologies. TEI, JCM, DK and VB are
shareholders of the company Regen BioPharma (subsidiary of Bio-Matrix
Scientific Corp (BMSN), which is developing the HemaXellerate product.
Authors ’ contributions
JCM, TEI, DTA, CAD, FR, AT, EJW, VB, MPM, DK, and ANP reviewed literature,
wrote the paper, and all read the paper before submission. All authors read
and approved the final manuscript.
Author details
1
Regen BioPharma Inc, San Diego, CA, USA.
2
Medistem Inc, San Diego, CA,
USA.
3
University of Connecticut School of Medicine, Hartford, CT, USA.
4
University of Washington, Seattle, WA, USA.
5
Cook General Biotechnology
LLC, Indianapolis, IN, USA.
6
Indiana University, Indianapolis, IN, USA.
7
University of Utah, Salt Lake City, UT, USA.
Received: 21 August 2012 Accepted: 4 October 2012
Published: 21 November 2012
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doi:10.1186/1479-5876-10-231
Cite this article as: Mizer et al.: Exogenous endothelial cells as
accelerators of hematopoietic reconstitution. Journal of Translational
Medicine 2012 10:231.
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