Large-scale ex vivo generation of human neutrophils from cord blood CD34+ cells
Large-scale ex vivo generation of human neutrophils from cord blood CD34+ cells
Zhenwang Jie 1 2
Yu Zhang 1 2
Chen Wang 0 2
Bin Shen 1 2
Xin Guan 1 2
Zhihua Ren 0 1 2
Xinxin Ding 1 2
Wei Dai 1 2
Yongping Jiang 0 1 2
0 Biopharmagen corp. , Suzhou , China , 3 College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, New York City, United States of America, 4 Environmental Medicine, NYU Langone Medical Center , Tuxedo, New York City , United States of America
1 Biopharmaceutical R&D Center, Peking Union Medical College of Tsinghua University , Suzhou , China
2 Editor: Suresh kumar Subbiah , Universiti Putra Malaysia , MALAYSIA
Conventional high-dose chemotherapy frequently leads to severe neutropenia, during which patients experience a high risk of infection. Although support care with donor's neutrophils is possible this choice is largely hampered by the limited availability of matched donors. To overcome this problem, we explored a large-scale ex vivo production of neutrophils from hematopoietic stem cells (HSCs) using a four-stage culture approach in a roller-bottle production platform. We expanded CD34+ HSCs isolated from umbilical cord blood (UCB) using our in-house special medium supplemented with cytokine cocktails and achieved about 49000-fold expansion of cells, among which about 61% were differentiated mature neutrophils. Ex vivo differentiated neutrophils exhibited a chemotactic activity similar to those from healthy donors and were capable of killing E. coli in vitro. The expansion yield as reported herein was at least 5 times higher than any other methods reported in the literature. Moreover, the cost of our modified medium was only a small fraction (<1/60) of the StemSpan™ SFEM. Therefore, our ex vivo expansion platform, coupled with a low cost of stem cell culture due to the use of a modified medium, makes large-scale manufacturing neutrophils possible, which should be able to greatly ameliorate neutrophil shortage for transfusion in the clinic.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was funded by the State
Scientific Key Projects for New Drug Research and
Development (2014ZX09101042-004 and
2011ZX09401-027) and the International
Cooperation and Exchange Program
(2013DFA30830), China. Biopharmagen Corp
provided support in the form of salaries for authors
[C.W. and Z.R.], but did not have any additional
role in the study design, data collection and
analysis, decision to publish, or preparation of the
Neutrophils are special phagocytes that are found in the bloodstream. During the beginning
or acute phase of inflammation, particularly as a result of bacterial infection, environmental
], and tumorigenesis [
], neutrophils are among the first-responders of
inflammatory cells that migrate towards the site of inflammation. In the clinic, patients who undergo
extensive chemotherapy often experience frequent and prolonged periods of neutropenia, a
major risk factor for severe bacterial and fungal infection  [
]. Despite the use of modern
antibiotics and/or hematopoietic growth factors to shorten the period of treatment-induced
neutropenia, infection remains the major cause of morbidity and mortality in these patients
]. For typical leukemic patients who receive chemotherapy and subsequent bone marrow
manuscript. The specific roles of these authors are
articulated in the ªauthor contributionsº section.
transplantation, there is a gap about 8 to 12 days of severe neutropenia before their neutrophil
counts return to the normal (0.5 x 109 neutrophils/L) [
]. G-CSF is often ineffective for some
patients with a loss of bone marrow function. Moreover, there are fungal or bacterial infections
that are unresponsive to antimicrobial treatments as demonstrated by visible spreading lesions
on skin, mucosa or radiological examination [
To date, neutrophil transfusion is the only logical approach to the treatment of infections
in neutropenic patients. Haylock and colleagues [
] have proposed that administration of ex
vivo-expanded neutrophil precursors can shorten the neutropenic period, thus reducing the
risk of infection. Methods for collecting cells from normal donors have been available for
more than 25 years. However, due to a lack of an effective method for obtaining HSC-derived
neutrophils with high purity and quantity, successful clinical transfusion of ex vivo expanded
neutrophils has not been achieved. In the past, a few research groups have obtained
immortalized neutrophil cell lines from induced pluripotent stem (iPS) cells [10±12]. However, due to
safety concern, iPS cell-derived neutrophils have not been used for clinical applications. It has
been proposed that ex vivo expanded neutrophils from CD34+ hematopoietic stem cells can
be used as an autologous source of cells for transplantation because of their ease of collection
and less stringent HLA matching, as well as a high rate of cell proliferation [
]. In fact, several
groups have obtained ex vivo-expanded CD34+ which are granulocyte-specific [13±16].
Moreover, small clinical studies were performed using human cord blood-derived CD34+ cells in
patients receiving chemotherapy, which markedly reduced the recovery period of granulocyte
The major obstacle of generating neutrophils ex vivo is that CD34+ cells are not efficiently
expanded before inducing them to mature neutrophils. Several research groups have tried to
produce neutrophils from CD34+ cells (from a single UCB collection which yields about 5x106
CD34+ cells) that were not sufficiently expanded [
]. Currently known methods for a
large scale ex vivo expansion are at best capable of generating two doses of clinical neutrophils,
which are roughly equivalent to 10,000-fold expansion. Although increasing the amount of
starting cells from mobilized peripheral blood can potentially generate up to ten doses of
neutrophils  the total neutrophils generated through this approach are only sufficient for a
single treatment per donation.
In this report, we describe an optimized four-stage culture approach using our in-house
culture medium and the roller-bottle production platform that can generate neutrophils ex
vivo on a large scale. We believe that our new stem cell expansion and differentiation platform
is capable of providing large amounts of high quality neutrophils for clinical applications.
Materials and methods
All studies that involved the use of animals were conducted according to relevant national
and international guidelines. Both male and female NOD/SCID mice of 6±8 weeks of age were
purchased from the Shanghai Laboratory Animal Co (SLAC, Shanghai, China, http://www.
slaccas.com/). Experiment protocols were approved by the Institutional Animal Care and Use
Committees of Soochow University [IACUC permit number: SYXK(Su) 2013±0018], and
were in accordance with the Guidelines for the Care and Use of Laboratory Animals (National
Research Council, People's Republic of China, 2012). We further attest that all efforts were
made to ensure minimal animal suffering. All fresh UCB samples were provided with a written
consent from volunteer patients at Suzhou Municipal Hospital (Suzhou, China). Consent
forms were signed by participated patients. The overall study and all necessary signed forms
were approved by the Hospital's Ethics Committee and Research Ethics Advisory Committee.
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Cytokines, antibodies, and reagents
Recombinant human stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt-3L),
granulocyte colony-stimulating factor(G-CSF), granulocyte-macrophage colony-stimulating
factor (GM-CSF), interleukin (IL)-3, thrombopoietin (TPO) and insulin were purchased from
Biopharmagen Corp (Suzhou, China, http://www.biopharmagen.com/). IL-1β and IL-8 were
purchased from PeproTech Inc. (Rocky Hill, NJ; http://www.peprotech.com). Fluorescein
isothiocyanate (FITC)-conjugated anti-CD66b antibody, FITC-conjugated anti-38 antibody,
Allophycocyanin (APC) and phycoerythrin (PE) -conjugated anti-CD34 antibody, and
FITCconjugated anti-CD45 antibody were purchased from BD Biosciences (San Diego, CA, http://
www.bdbiosciences.com). Putrescine, selenium, transferrin, zymosan, HEPES, human AB
serum, Hanks' balanced saline solution (HBSS) and formyl-methionyl-leucyl-phenylalanine
(FMLP) were from Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com). B-27
supplements were from Thermo Fisher Scientific Life Sciences (Waltham, MA, http://www.
Collection of CD34+ cells
Fresh UCB samples were obtained within 6±8 hours of delivery from Suzhou Municipal
Hospital (Suzhou, China) with written consents from donors. The protocol was approved by the
Suzhou Municipal Hospital Ethics Committee and Research Ethics Advisory Committee.
Mononuclear cells were isolated by Ficoll-Paque (GE Healthcare, Marlborough, MA, http://
www.gehealthcare.com) density gradient centrifugation [
]. CD34+ cells were enriched from
mononuclear cells by magnetic cell sorting using a MACS Direct CD34 MicroBead Kit
(Miltenyi Biotec, Amsterdam, The Netherlands, http://www.miltenyibiotec.com) per manufacturer's
instructions. Enriched CD34+ cells with purity ranging from 91% to 98% were confirmed by
flow cytometry (BD Biosciences) after staining with a PE-conjugated anti-CD34 antibody.
Isolated CD34+ cells were cultured and expanded in 25-T flasks (Corning, USA). After 6 days'
culture, expanded cells were cultured in a supplement bottle turning device (HERAcell 240i,
Thermo Fisher Scientific, USA). Culture bottles were placed horizontally in an incubator at
37ÊC with 5% CO2 in the air with a rotation rate set at 0.82U per minute(min). Cells were
cultured in a modified medium based on Iscove's modified Dulbecco's medium (IMDM) (Life
Technologies, USA) after the addition of nutrition supplements [
] consisting of
putrescine (100 μM), selenium (5 ng/mL), insulin (25 μg/mL), transferrin (50 μg/mL), and B-27
Supplements (2%, v/v). A staged culture protocol was designed for ex vivo expansion and
differentiation, which included Stage 1 (days 0±6), Stage 2 (days 7 ~9), Stage 3 (days 10~15), and
Stage 4 (days16~18), respectively, fresh cytokines are replaced every three days. To optimize
progenitor cell proliferation and neutrophil differentiation, different combinations of growth
factors and cytokines, including SCF, Flt-3L, G-CSF, GM-CSF, IL-3, TPO, and fetal bovine
serum (FBS) (10%, v/v, Hyclone, USA), were supplemented to modified culture media at
various concentrations (Table 1). Culture optimization was carried out in a 2L-bottle with 200 ml
medium. Once the optimal culture condition was achieved and finalized, Take three
independent cord blood samples as described above for CD34+ hematopoietic stem cell separation
method, part of isolated CD34+ cells(5×105) were cultured and expanded in 25-T flasks.
After 6 days' culture, expanded cells were cultured in the same-size bottle containing 600 ml
medium. Cells were sub-cultured and cryopreserved to maintain an optimal cell density which
ranged from 2×105 to 1×106 cells/ml .
3 / 18
Cell numbers were determined at various time points using the Count Star1 cell counting
system (Ailite, China) and were stained with Wright-Giemsa reagents (NJJCBIO, Nanjing,
China) for morphological analysis. Images of stained cells were taken under a microscope with
a digital camera (Olympus, Tokyo, Japan) at 400x magnification. Neutrophils were identified
and quantified with a FACSVerse flow cytometer (BD Biosciences) after staining with an
antiCD66b antibody according to the procedure described in Saeki et al [
4 / 18
In vitro bacterial killing assay
Escherichia coli BL21 was picked from single colonies grown on LB-agar plates, inoculated into
LB broth and grown for 18h at 37ÊC. Microorganisms were pelleted by centrifugation at 2000g
for 5 min, transferred into a microtube, washed once in 0.9% NaCl solution by centrifugation
at 12,000g for 10s, and suspended in 1.5ml 0.9% NaCl solution. Bacterial concentration was
determined by measurement of turbidity at 500nm. Suspensions were diluted to 1×108 cells/
ml in 1 ml medium [
], HEPES-buffered saline with 10% human AB serum and opsonized
for 30 min at 37ÊC in a shaking water bath. Opsonized bacteria were kept on ice until use.
Neutrophils were suspended in 100μl HEPES-buffered saline with 40% human AB serum at a
concentration of 5×106/ml. The opsonized E coli were added to the suspension of neutrophils at a
neutrophil/bacterium ratio of 2:1. After incubation for 1 h, 50μL of aliquots with and without
neutrophils were diluted in 2.5 ml alkalinized water (pH 11) for neutrophils lysis. All the
samples were then inverted twice. After standing for 5 min at room temperature and then vortex
vigorously for 5 s, 50 μl of the samples were diluted in PBS to achieve a bacterial concentration
of about 2×103/ml. Ten microliter aliquots of the diluted suspensions were spread on LB with
1.5% agar. Bacterial colonies were counted after overnight incubation.
Chemotactic ability was determined using a modified Boyden chamber method as described
]. Chemotaxis was assessed using Transwell (3-μm pore size; Corning Inc., Corning,
NY 14831 USA, http://www.corning.com/cn/zh/products/life-sciences.html), which was
placed in a 24-well dish. The lower chamber, a 24-well culture dish, was filled with 500 μl
Hanks' balanced saline solution (HBSS) per well supplemented with 2.5% FBS whereas the
upper chamber, a chemotaxel cup, was filled with ex-vivo differentiated neutrophils (EDN) or
human peripheral blood-derived neutrophils (PBN) suspended in 500μl of HBSS per well
supplemented with 2.5% FBS (4×105 cells per milliliter). As chemoattractants, 100nM
formylmethionyl-leucyl-phenylalanine (FMLP) and 10ng/ml IL-8 were added to lower chambers.
After incubation at 37ÊC for 4 h, cells from three randomly selected areas of the lower chamber
were counted. In the meantime, cells in the chemotaxel cup were fixed and stained with the
CD66b antibody for flow cytometer analysis.
Transplantation of human UCB-derived neutrophils into mice and airpouch chemotaxis assay
Eight-week old female NOD/SCID mice were made neutropenic by a single intraperitoneal
injection of 5-fluorouracil (5-FU; 150mg/kg). Air pouches were produced by subcutaneous
injection of 10 ml sterile air into the back of female mice [
]. Three days later, 5ml sterile air
was injected into the same cavity. After another 3 days, mice received i.v. transplants of 2× 106
ex-vivo differentiated neutrophils (EDN) or human peripheral blood-derived neutrophils
(PBN) or vehicle (saline). Five hundred microliters of PBS with or without Zymosan (1mg/ml)
and human IL-1β (10 ng/ml) were injected into the air pouch. Sixteen hour post injection,
mice were sacrificed with 1% pentobarbital (50 mg/kg), and the air pouch was washed with 1
ml of ice-cold PBS to obtain cumulated leukocytes, which were subsequently confirmed by
flow cytometric analysis after staining with the anti-CD66b+ cells [
In vivo safety and function study in NOD/SCID mouse
The safety and function study of mice in vivo experiments using the method of Guan and
]. NOD/SCID mice were intravenously administered with 1x107 human
UCB5 / 18
derived, differentiated neutrophils (day 18 culture as above), human peripheral blood
neutrophils (PBN), or saline after sub-lethal irradiation (2.5Gy). Mouse peripheral blood samples
were then collected from the retro-orbital plexus at different times post transplantation and
stained with antibodies against human CD66b and CD45 followed by flow cytometric analysis.
Human neutrophil progenitor cells cultured on the ninth day which contains 3.2×105 CD34+
cells were also injected via tail vein into irradiated mice, after which mouse PB samples were
collected and human neutrophil cells were determined by flow cytometry as above. Mouse
bone marrow was harvested from both femurs 2 months after transplantation. Hematopoietic
stem cells (CD34+), mature neutrophils (CD66b+) and promyelocytes (CD38+) [
] in bone
marrow were determined by flow cytometry [
One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests was
used for comparison among the various treatment groups. Results were considered statistically
significant when the P value was less than 0.05.
Culture optimization for ex vivo generation of neutrophils from UCB
We first designed and optimized a four-stage culture protocol for ex vivo expansion and
differentiation of neutrophils from human UCB CD34+ cells (Table 1). Distinct culture medium
formulas as described were used for each stage.
In Stage 1, isolated CD34+ cells were expanded for 6 days to expand hematopoietic stem
and progenitor cells. There was no significant difference between the MNF+SFGM3T and MN
+SFGM3T group in terms of hematopoietic stem cell proliferation although the total number
of cells in the group MNF+SFGM3T that contained serum was higher than that of group MN
+SFGM3T. We chose the MNF+SFGM3T group as the optimized one mainly based on the
principle of a higher total cell number. Specifically, the MNF+SFGM3T group consisted of
IMDM, nutrition supplements, 10% FBS, SCF at 100 ng/ml, Flt-3L at 100 ng/ml and TPO at
20 ng/ml, IL-3 at 25 ng/mL, GM-CSF at 15 ng/ml, and G-CSF 50 ng/ml. The proliferation
folds of CD34+ cells and total cells were at 30 ± 2.4 and 110 ± 15, respectively. CD34+ cell
percentage was maintained at 32% ± 4.3% throughout the culture (Fig 1A).
Stage 2 intended to evaluate various optimal cytokine combinations for differentiation
toward the myeloblast lineage (Fig 1B). We selected a panel of cytokines to ensure minimal
non-paracrine differentiation. For example, TPO is not necessary for neutrophil progenitor
] and its inhibitory effect on neutrophil maturation is marginal. Thus, we
removed TPO in Stage 2 culture protocol. Total cell counts were higher in the group
supplemented with MNF+SFGM3(GM20) (a combination of SCF, Flt-3L IL-3, G-CSF, and
GMCSF(20 ng/ml)) and MNF+SFGM3(GC75) (a combination of SCF, Flt-3L IL-3, G-CSF(75
ng/ml), and GM-CSF) cocktails than other groups supplemented with various cytokine
cocktails (Fig 1B). Although MNF+SFGM3(GM20) and MNF+SFGM3(GC75) cocktails induced
the highest total cell expansion the percentages of neutrophils were no difference, suggesting
that a high concentration of G-CSF and GM-CSF had little effect on the induction to mature
neutrophils (Fig 1B). On the basis of minimal non-paracrine differentiation and the
percentage of induced neutrophils, the cocktail MNF+SFGM3 was selected for induction of
differentiation and maturation of neutrophils in Stage 2. On average, after 6-day culture with MNF
+SFGM3T and 3-day culture with MNF+SFGM3, the normalized yield of total cells was
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Fig 1. Optimization of culture conditions for ex vivo expansion of human neutrophils from human UCB CD34+ cells. (A)
Absolute cell numbers were calculated on a particular day (e.g., 5×104 CD34+ cells seeded on day 0). In Stage 1, isolated CD34+
cells were cultured for six days in various medium formulas (Table 1), absolute numbers of the total cell [A(i)] and CD34+ cell [A(ii)]
were calculated on day 6. (B) Stage 2 started with the cells derived from group MNF+SFGM3T (IMDM +Nutrition Supplements +
100ng/ml SCF + 100 ng/ml Flt-3L + 50ng/ml G-CSF + 20ng/ml TPO + 25ng/ml IL-3 +15ng/mL GM-CSF) of Stage 1. Absolute
numbers of total cells [B(i)], CD66b+ cell [B(ii)] were calculated on day 9 with G-CSF ranging from 50 to 100 ng/ml and GM-CSF
7 / 18
ranging from 5 to 20 ng/ml in MNF+SFGM3 medium (IMDM + nutrition supplements + FBS + 100 ng/ml SCF + 100 ng/ml Flt-3L + 75
ng/ml G-CSF + 15 ng/ml IL-3 + 10 ng/ml GM-CSF). (C) Stage 3 started with the cells derived from group MNF+SFGM3 of Stage 2.
Absolute numbers of total cell [C(i)], CD66b+ cell [C(ii)] and were calculated on day 15 in different medium formulas with G-CSF
ranging from 100 to 500 ng/ml in MNF+SFG (IMDM + nutrition supplements + FBS + 100 ng/ml SCF + 100 ng/ml Flt-3L + 100 ng/ml
G-CSF) medium. Results are presented as means ± SD of 6 independent experiments. One-way ANOVA followed by Dunnett's
multiple comparison tests was used for comparison among the various treatment groups.* P<0.05; ** P<0.01; *** P<0.001;
**** P<0.0001; ns, No significant difference.
approximately 1.0×103 cells from one CD34+ cell with 22.4%±4.1% of CD66b+ cells (n = 6,
In Stage 3, neutrophil cell expansion was further induced by specific cytokine
combinations. In order to induce the highest neutrophil production, various combinations of G-CSF
(range 100~500 ng/ml), SCF (100 ng/ml) and Flt-3L (100 ng/ml) were added to the culture
medium for optimization as these cytokines were routinely used in neutrophil maturation.
The results showed that G-CSF at a concentration of 100ng/ml-500ng/ml had no significant
effect on neutrophil differentiation. Thus, we selected 100ng/ml G-CSF as the final
concentration in the culture media (Fig 1C). We observed that G-CSF alone exhibited a low expansion
potential while it synergized with SCF and Flt-3L during progenitor cell expansion (rather
than only acting on differentiation and maturation of progenitor cells expanded by SCF and
Flt3-L). Furthermore, morphological changes were clearly noticeable after differentiation.
Mature neutrophils were multi-lobulated in shape (Fig 2A). Combined, these results
demonstrate that MNF+SFG medium facilitates differentiation and maturation of human neutrophils
ex vivo after day 9 of culture.
Fig 2. Kinetics, morphology, and characterization of differentiated neutrophils via four-stage culture system. (A) Representative images of
neutrophils on day 18 after Wright-Giemisa staining (magnification ×400). (B) Isolated CD34+ cells were cultured in defined culture conditions. The
expansion rate was calculated as fold-increase in cell counts on each day (day 0, 6, 9, 12, 15, and 18). Static culture (grey line) and turning-bottle
culture (black line) expansion of human UCB CD34+ cells were compared. Data were collected from three independent experiments (C)
Representative FACS plots for CD66b expression of human UCB-derived neutrophils on day 18. (D) Representative histograms of CD66b-positive
populations during neutrophil differentiation.
8 / 18
In Stage 4 of culture, FBS was excluded to eliminate its effect on multi-lineage
differentiation. One percent of human serum albumin (HSA) was used to replace FBS as it is routinely
used as an important nutrient for various cell culture processes [
]. In this Stage, the total
number of differentiated, mature neutrophils was amplified about 1.2 times. G-CSF appeared
to extend the survival time of differentiated neutrophils likely through preventing their
apoptosis . Taken together, the final optimized culture medium for human neutrophil
expansion and differentiation was IMDM supplemented with nutrition supplements and sequential
cytokine combinations (MNF+SFGM3T for Stage 1, MNF+SFGM3 for Stage 2, MNF+SFG for
Stage 3, MNA+SFG for Stage 4) which were summarized in Table 1.
Large-scale generation of human neutrophils
With the selected cytokine combination in each stage, ex vivo large-scale granulocytopoiesis
from human UCB CD34+ cells was performed in a bottle turning device culture system. As
shown in the growth curve (Fig 2B), the expansion ratio of the total cells was slowly increased
during the initial culture period (day 0 to day 6). From day 6 to day 12, the cells entered an
exponential growth phase in which cells maintained high rates of proliferation, leading to a
vigorous growth period. Approximately 8300-folds of initial cell expansion was achieved on
day 12. The expansion rate slowed down from the 12th day, with a combined total cell
expansion folds of 4.9×104 on day18. When the culture continued, cell death was observed from day
18 (data not shown). We also used the same culture conditions (e.g., cytokine cocktails, etc)
and starting cell density using the conventional stationary culture condition as a control, it is
noted that more cells were obtained in the static culture conditions before the ninth day, but
the number of cells in roller bottles significantly exceeded those of stationary culture
conditions after day 12. In addition, the final cell number and the neutrophil percentage in the roller
bottle culture system were significantly higher than those of the stationary culture at day18
The speed of the roller bottle also had a significant impact on cell growth. A higher speed
enables cells to obtain more nutrition whereas too much dissolved oxygen inhibits neutrophil
]. We found that the optimal rotational speed for expansion of human UCB
differentiated neutrophils was 0.82 U.
Isolated human UCB CD34+ cells on day 0 express high levels of HSC markers (CD34+)
and, as expected, low or undetectable levels of neutrophilic markers such as CD66b were
detectable. The CD34+ ratio decreased rapidly on day 9 (3%±1%) and on day 12 (0.8%±0.3%),
respectively, after 18-days of proliferation and differentiation, the percentage of CD34+ and
CD133+ cells was significantly decreased to below 1%. In contrast, the percent of cells
expressing CD66b rapidly increased. For example, on day 18, the neutrophil population reached
61.5% (± 5.3%) as shown in the flow cytometric histogram (Fig 2C). Percentages of CD66b+
cells were determined for the entire course of neutrophils differentiation and maturation (Fig
2D). In addition, differentiated and mature neutrophils were also confirmed by Giemsa
staining. We observed that ex vivo-derived neutrophils contained intra-cellular granules in the
cytoplasm and segmented nuclei (Fig 2A).
Chemotaxis and bactericidal activities of ex vivo differentiated neutrophils (EDN) were similar to human peripheral blood isolated neutrophils (PBN)
In the bacterial killing assay, the negative control group was the culture with no addition of
neutrophils on the coated plates, which typically yielded about 230 bacterial colonies per plate
after overnight incubation. However, after co-incubation with neutrophils, regardless of EDN
9 / 18
or PBN group, only 2±5 colonies grew on each plate, indicating that both EDNs and PBNs
has an excellent bactericidal killing activity (Fig 3A). The chemotactic activity, which is one of
the most critical functions of mature leukocytes including neutrophils, was further evaluated
using bacterial chemotactic peptide FMLP and neutrophil specific chemokine IL-8 as
chemoattractants. As shown in Fig 3B, neutrophils differentiated from human UCB displayed a
chemotactic activity that was similar to neutrophils isolated from human peripheral blood.
Dick et al. [
] reported that although ex vivo-derived neutrophils exhibit a reduced
bactericidal activity in vitro the remaining bactericidal activity is sufficient for protection against
bacterial infection, thus providing therapeutic benefits in patients with chemotherapy-induced
neutropenia. These authors also noted that neutrophils with a reduced activity were likely due
to specific culture conditions or improper in vitro assays employed [
]. Thus, it is of great
interest that ex vivo±derived neutrophils in our distinct culture conditions exhibit the same
chemotactic and bactericidal activities as those of neutrophils freshly isolated from the human
In vivo evaluation of chemotactic activity of neutrophils differentiated from human UCB
The method for evaluating chemotactic activity of neutrophils induced from human UCBs
was as previously described [
]. Because of human IL-1βenhanced zymosan-induced
accumulation of human CD66b-positive neutrophils, we used these two agents as
chemoattractants. Sixteen hours after i.v. transplantation of human UCB-derived neutrophils and injection
of proinflammatory agent(s), neutrophils accumulated in the air pouch were collected and
evaluated for human CD66b-positive cells by flow cytometry. Leukocytes that accumulated in
the zymosan and IL-1β induced air pouch inflammation model in normal mice were shown
to be predominantly neutrophils, along with small numbers of monocytes and lymphocytes.
When NOD mice were transplanted with human UCB-derived neutrophils and injected with
both zymosan and IL-1β, human UCB-derived CD66b-positive neutrophils in the air pouch
were 1.1% of the total accumulated cells [Fig 3C(v)], whereas human CD66b-positive cells
were not significantly detected in the air pouch [
] of control NOD mice that did not receive
transplantation of human UCB-derived neutrophils even when both inflammatory agents
were injected [Fig 3C(iii)]. No significant numbers of CD66b-positive cells were detected in
another negative control mouse group that were not injected with inflammatory agents into
the air pouch but received transplanted human UCB-derived cells [Fig 3C(iv)]. When human
peripheral blood neutrophils were used, the same results as those of human UCB-derived cells
were obtained [Fig 3C(vi)] (i.e., human cord blood cell-derived CD66b-positive neutrophils in
the air pouch of NOD mice were 1.3% of the total accumulated cells). There is no significant
difference between the human UCB-derived neutrophil group and the positive control group.
In vivo maturation of human UCB derived neutrophils in the NOD/SCID mice and long-term safety analysis
The therapeutic potential to ameliorate neutropenia using human UCB-derived neutrophils
remained to be determined. The first approach was to test these cells in preclinical animal
models such as NOD/SCID mice. Our study showed that after ex vivo differentiated
neutrophils (EDNs) and peripheral blood isolated neutrophils (PBNs) were injected, via tail vein, into
NOD/SCID mice, human neutrophils and leucocytes were detected in the peripheral blood of
transplanted mice at a percentage of 2% -5% one day after transplantation (Fig 4A). The
difference between mouse groups receiving either EDNs or PBNs was mostly significant 4 days after
transplantation. No human neutrophils were detected in the peripheral blood of NOD/SCID
10 / 18
Fig 3. Study on bactericidal and chemotactic activities of neutrophils derived from human UCB HSC. (A) Bacterial killing assay. E
coli was opsonized with human AB serum, and incubated with ex-vivo differentiated neutrophils (EDN), human peripheral blood fresh
isolated neutrophils (PBN), or medium alone as a control. Bacterial colonies were significantly reduced to approximately 1% of the control
after overnight incubation. There were no significant differences in the bactericidal activity between EDNs and PBNs, data were collected
from three independent experiments; bars indicate SDs; One-way ANOVA followed by Dunnett's multiple comparison tests was used for
comparison among the various treatment groups. ****(P<0.0001 compared with the control). (B) Chemotactic activity of neutrophils in
response to FMLP and IL-8 was determined using Transwell (3-μm pore; Corning Inc, Corning, NY 14831 USA) as described in Materials
and Methods. The transwell inserts were removed after the culture plates were cultured for 2 h. Cells in the lower chamber were counted
under a phase-contrast microscope. One-way ANOVA followed by Dunnett's multiple comparison tests was used for comparison among
the various treatment groups. ****(P<0.0001 compared with the control). (C) In vivo chemotactic activity of human UCB-derived
neutrophils in the air pouch inflammatory model of NOD/SCID mice. Human UCB-derived neutrophils (v) or vehicle saline (iii) were
transplanted into NOD/SCID mice intravenously. PBS (500μl) with zymosan (1 mg/ml) and IL-1β (10ng/ml) was injected into the air pouch
to induce inflammation. After 16 h, cells accumulated in the pouch were collected and subjected to flow cytometric analysis for expression
of neutrophil-specific marker CD66b. As a positive control, human peripheral blood isolated neutrophils (PBN) were transplanted into NOD/
SCID mice as well. The air pouch inflammatory model was examined as in (vi) using both zymosan and IL-1β as inflammatory agents (Fig
3C(i). Representative dot plots were visualized according to the FSC-log and SSC-log (ii):IgG; Fig 3C (iv) shows the injection of PBS
without zymosan and IL-1β but transplanted with human UCB-derived neutrophils to NOD/SCID mice intravenously followed by detecting
human neutrophils in mouse peripheral blood using the same method.
11 / 18
Fig 4. Safety and functional study of human UCB-derived neutrophils. (A) Human UCB-derived neutrophils (1x107) and fresh human peripheral
neutrophils (1x107) (or saline as control), were transplanted into sublethally-irradiated NOD/SCID mice. Human neutrophils were detected in mouse
peripheral blood 2 days after transplantation. Representative dot plots and flow cytometric profiles were shown. CD66b (top) and CD45 (bottom)
profiles were presented for IgG (i), saline (ii), human CB-derived neutrophils (iii), and human peripheral blood neutrophils (iv) 2 days after
transplantation. (B) Human UCB-derived neutrophils and peripheral blood neutrophils were detected in the mouse peripheral blood at the indicated
times post-transplantation. Data are summarized as mean±SD (n = 5) at each time point. (C) Ex-vivo-expanded human neutrophil progenitors
engrafted in the mouse bone marrow two months after transplantation. IgG (i), human UCB-derived neutrophils (CD66b+), promyelocytes (CD38+),
and hematopoietic stem cells (CD34+) (ii). Abbreviations: FSC, forward scatter; SSC, side scatter.
mice of the PBN group whereas 1% to 2% of human neutrophils were detected in the EDN
group. The average survival period of human neutrophils was 1±2 days [
]. Thus, it was likely
that human UCB-derived neutrophils continued to mature in recipient mice. CD66b+ cells
derived from UCB survived for at least 4 more days in mouse peripheral blood (PB) after
18-day culture. On the other hand, fresh PB neutrophils lasted for only 2 days
post-transplantation. These results strongly suggest that ex vivo-derived neutrophils/progenitors can further
12 / 18
mature in vivo (Fig 4B). In fact, we continuously detected the presence of human neutrophils
in mouse peripheral blood and mouse bone marrow cells contained human hematopoietic
stem cells, neutrophil progenitor cells, and mature neutrophils during two months post
transplantation. Percentages of human hematopoietic stem cells (CD34+), neutrophil progenitor
cells (CD38+), and neutrophils (CD66b+) in the mouse bone marrow were 4.72%, 50.5%, and
10.6%, respectively (Fig 4C), indicating that cells (on the 9th day of culture) transplanted into
the recipient mice have been successfully engrafted into the bone marrow of immunodeficient
mice, leading to sustained production of neutrophil progenitors and mature cells.
Transplantation of UCB-derived neutrophils elicited no adverse reactions in mice. No signs of fever,
emesis, or diarrhea were observed. Moreover, 4 months after transplantation, all recipient mice
survived with no apparent abnormalities.
It is not a routine clinical practice to collect donor neutrophils despite their potential use for
supporting neutropenic patients through transfusion [
]. Major limitations for utilizing
donor neutrophils for transfusion medicine including complicated processes to identify and
screen donors, the necessity to mobilize HPCs with hematopoietic cytokines, and the difficulty
to obtaining sufficient numbers of neutrophils even after mobilization [
]. In the current
study, we have designed a four-stage culture protocol to expand UCB stem cells and
differentiate them toward the neutrophilic lineage cells using the bottle turning device system with the
cost-efficient culture media. The large scale ex vivo cell expansion system involves no gene
manipulation, which is compatible with good manufacturing practice to produce neutrophils
for clinical applications. Using this protocol, we can obtain 2.4x1011 transplant-ready
neutrophils from one input human UCB CD34+ cells. Our yield is approximately 5-fold higher than
the best one reported previously [20±22]. Theoretically speaking, neutrophils from one UCB
unit (~5x106 CD34+ cells) can be used for treating 12 or more neutropenic patients (mean
weight 70 kg). Given that a large number of cells is required for transfusion, neutrophil
expansion and production in standard tissue culture flasks are not feasible. Therefore, we
implemented a roller-bottle culture system, which can hold up to 10 L culture medium in 16 roller
bottles packed in one incubator. In fact, a sufficient number of neutrophils for one dose of
clinical use can be obtained by this primary culture approach. To our knowledge, this is the first
report that a bottle-turning device platform is used to scale up the production of granulocytes/
It has been also an issue that ex vivo production of a sufficient number of neutrophils for
clinical applications is costly. To address this problem, we have modified the basal culture
medium by the addition of nutrition supplements to IMDM so that HSCs can be better
supported for expansion and differentiation. The cost of modified medium is estimated at only 1/
60 (or even lower) of the StemSpan™ SFEM, which has been a gold-standard for a large-scale
expansion or production of hematopoietic cells ex vivo.
We have continued to optimize culture conditions for supporting proliferation and
differentiation capacity of HSCs. We have made special efforts on investigating and screening
hematopoietic stem cell growth and expansion factors [46±48], as well as factors that promote
differentiation toward platelets [
], red blood cells, endothelial cells [
], and neutrophils. No
any gene manipulation process was involved in the culture system and we simply just used the
humanized cytokines, so that the entire culture process would not affect the cell genetic
stability and safety[
]. The other results from our labs show that the expression of oncogene and
genes that related to the telomere is stable after efficient expansion of HSCs (33). It takes 12
days to generate mature neutrophils from myeloblasts. It has been estimated that the transition
13 / 18
time from myeloblasts to myelocytes is about 6 days [
]. The four-stage culture protocol
optimizes cytokine combinations at each stage of proliferation and/or differentiation along a
particular hematopoietic lineage. We have allowed the expansion of hematopoietic stem cells in
the first 6 days and differentiation of myeloid progenitors into neutrophils for the subsequent
12 days. Both SCF and Flt3-L (each at 100 ng/ml) yield a similar expansion rate when they are
used alone. However, these two cytokines appear to have a synergizing effect when they used
in combination [
]. G-CSF is crucial for neutrophil growth, survival, and release from the
bone marrow [
We have systematically investigated a number of cytokine combinations for in vitro
expansion of CD34+ cells from human UCB. It is established that the neutrophilic lineage is
established through the combined action of IL-3, GM-CSF, and G-CSF [
]. Thus, we have
used these cytokines as a Stage-2 cocktail to expand and differentiate neutrophil progenitor
cells. The transit time of the lineage-committed progenitors to the mature neutrophils is
approximately 4 to 6 days [
]. Therefore, SCF, Flt-3L and optimized G-CSF
concentrations have been used as a Stage-3 cocktail to stimulate the expansion of myeloblast
progenitors, as well as promoting differentiation of these cells along the neutrophilic
]. With optimized culture conditions, human UCB CD34+ cells are efficiently
driven toward the neutrophil lineage during 18-day culture. Neutrophils undergo
maturation during the last 3 days of culture. We have used 1% HSA to replace FBS in addition to
the presence of a cytokine combination of SCF, Flt-3L, and G-CSF. Our culture conditions
ensure continued survival of mature neutrophils and in the meantime remove
immunogenic components of FBS so that neutrophils obtained are compatible for clinical
Neutrophils derived from UCB CD34+ cells ex vivo are fully functional as they exhibit
chemotactic and bacterial killing effects similar to those of fresh peripheral blood
neutrophils from heathy donors. In vivo chemotactic activity of UCB-derived neutrophils is
demonstrated using an air pouch inflammatory model in NOD mice transplanted. Besides,
CD66b+ UCB neutrophils are presented in the peripheral blood of recipient mice much
longer than peripheral blood neutrophils, strongly suggesting that the ex vivo generated
neutrophils (and progenitors) can further mature in vivo. To date, a few research groups
have investigated ex vivo expansion of neutrophil progenitors from HSCs [58±60], and in
two such studies, expanded cells have been used clinically for providing autologous HSCs.
In the current study, we have injected cryopreserved neutrophil progenitor cells (day 9
culture) into neutropenia NOD/SCID mice via the tail vein. Human mature neutrophils in the
mouse peripheral blood are capable of sustaining for a period of time as we have detected
the presence of human neutrophil progenitor cells and mature cells in the mouse bone
marrow two months after injection, which provides new insights into clinical applications. Ex
vivo expanded neutrophil progenitors can provide not only a temporary relief for
neutropenic patients but also a long-term support for myeloid progenitor deficiency in the bone
marrow. Furthermore, the neutrophil progenitor cells can be cryopreserved, thus making their
storage and transportation easy. Of note, mice transplanted with human UCB-derived
neutrophils survive well for a long time and no apparent abnormalities including tumor
development are observed.
Taken together, we have established a pilot-scale culture system to produce functional
human neutrophils ex vivo. Considering that one neutrophil transfusion unit (100 ml) contains
2×1010 cells, the CD34+ cells from one UCB unit (80 ml) will generate 2.4×1011 neutrophils,
which are equivalent to 12 units/doses of neutrophils for clinical transfusion. Given excellent
features associated with the neutrophils derived from UCB, we believe that these cells can be
used as an alternative source to conventional neutrophil transfusion in the clinic.
14 / 18
S1 Fig. Kinetics of CD34+ hematopoietic stem/progenitor cell on stages 1 and 2 of culture.
Isolated CD34+ cells were cultured with selected culture conditions, representative dot plots of
CD34 cell-surface markers on uncultured cells(day0) and expanded cells (day6 and day 9).
We thank our co-workers in the laboratory for their valuable discussions and suggestions. We
would also like to extend our thanks to the Ethics Committees of the Institutional Animal
Care and Use Committees of Soochow University to prove the animal studies.
Conceptualization: ZJ XD WD YJ.
Data curation: ZJ ZR BS YZ XG YJ.
Formal analysis: ZJ BS.
Funding acquisition: YJ.
Investigation: ZJ CW BS.
Resources: WD XD YJ.
Writing ± original draft: ZJ.
Writing ± review & editing: XD WD YJ.
15 / 18
progenitor cell transplantation result in a modest reduction of febrile days and intravenous antibiotic
usage. Transfusion. 2006; 46(1):14±23. https://doi.org/10.1111/j.1537-2995.2005.00665.x PMID:
16 / 18
17 / 18
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