Analysis and characterization of hematopoietic progenitor cells from fetal bone marrow, adult bone marrow, peripheral blood, and cord blood

ABSTRACT Hematopoietic stem cell transplantation has been increasingly used to replace a defective hematopoietic system and to treat various genetic defects as well as malignant diseases.

However, the limitations of conventional bone marrow transplantation have stimulated an intense interest in exploring the use of alternative sources of hematopoietic stem cells, including

peripheral blood mononuclear cells (PBMC) and cord blood (CB). A major investigative effort of our laboratory has been focused on evaluating fetal bone marrow (FBM) for transplantation. The

current study compares and characterizes the functional and phenotypic characteristics of FBM, CB, adult bone marrow (ABM), and PBMC by clonogenicity assays, immunogenicity, and the

quantification of progenitor cells. There was a striking difference in the proportion of CD34+ cells in FBM, ABM, PBMC, and CB (24.6%, 2.1%, 0.5%, and 2.0%, respectively). The clonogenic

potential, as measured by colony forming unit in culture (CFU-C) assay, was significantly higher in FBM when compared with ABM, PBMC, and CB (202.5, 73.5, 40.8, and 65.5 colonies/105 cells,

respectively). There was a significant decrease in proliferative responsiveness in mixed lymphocyte reaction (MLR) assay of FBM and CB compared with ABM and PBMC. These observations indicate

that each source of hematopoietic stem cells has different intrinsic properties closely correlated with ontogenetic age that is a vital determinant for phenotypic characteristics, lineage

commitments, immunogenicity, and proliferative potentials. SIMILAR CONTENT BEING VIEWED BY OTHERS IN VITRO EXPANSION OF FETAL LIVER HEMATOPOIETIC STEM CELLS Article Open access 04 June 2021

LINEAGE TRACKING TO REVEAL THE FATE OF HEMATOPOIETIC STEM CELLS INFLUENCED BY FLK2− MULTIPOTENT PROGENITORS AFTER TRANSPLANTATION Article Open access 13 January 2023 NON-CONDITIONED BONE

MARROW CHIMERIC MOUSE GENERATION USING CULTURE-BASED ENRICHMENT OF HEMATOPOIETIC STEM AND PROGENITOR CELLS Article Open access 11 June 2021 MAIN The reduced immunogenicity of fetal tissue

makes it a useful donor source for transplantation. In addition, fetal tissues produce abundant trophic substances and growth factors, which promote cell growth and regeneration of damaged

tissues and cells. The ability for rapid cellular growth, proliferation, differentiation, revascularization, and tissue repair has resulted in the successful use of fetal tissue

transplantation in the treatment of various diseases, including Parkinson's disease, neural tissue injury, diabetes, and myocardial infarction as well as various genetic defects(1–8).

Hematopoietic stem cell transplantation has not only been increasingly used for correction of abnormal hematopoiesis in patients with malignant and nonmalignant diseases but also has made

high dose chemotherapy possible for the treatment of many solid malignancies. Historically, the adult bone marrow has been the major source of hematopoietic stem cells. However, the shortage

of availability of donors for allogeneic bone marrow transplantation and the risk of malignant contamination in autologous bone marrow have stimulated a search for alternative sources of

stem cells. One of the prime sources has been PBMC, which offers several advantages including rapid hematopoietic and immunologic reconstitution and frequently lower risks of contamination

with tumor cells compared with bone marrow grafts(9,10). Similarly, CB is being used as an alternative source and offers the advantages of easy accessibility and a good hematopoietic

cellular yield, which is equivalent to that of ABM(11–14). Although there are variable levels of maternal cell contamination observed in CB that may be associated with GVHD, this has not

posed any major clinical problem(15,16). However, the relatively small number of primitive stem cells and large number of committed progenitors per donor in CB may affect its therapeutic

efficacy. On the other hand, transplantation of fetal tissue in animal models and in human experimental treatments with fetal liver, thymus, spleen, and bone marrow have shown distinct

advantages over adult tissues including lowered immunogenicity and higher percent of primitive cells. Fetus-derived stem cells potentially remove the problems of tissue typing and also have

a high capacity to differentiate into the complete repertoire of erythroid, myeloid, and lymphoid lineages. The present studies were performed to compare the phenotypic characteristics of

FBM, harvested from second trimester of lost pregnancies, with ABM, PBMC, and CB. In addition, the clonogenic potential and immunogenicity of each of these cell sources were evaluated to

determine their suitability for transplantation. METHODS _CELL SOURCES AND PROCESSING._ FBM (_n_ = 8) was individually harvested from fetuses of lost pregnancies in the second trimester (18

to 20 wk gestation). FBM cells were collected from femurs, tibias, vertebral columns, pelvic bones, and scapulae, which were harvested en bloc, dissected aseptically, and cut into small

pieces. After removing the cortex, the marrow-rich cancellous portion was minced with a homogenizer. In all cases, the tissues were collected after informed consent was obtained. Normal ABM

(_n_ = 8) was obtained from normal allogeneic bone marrow donors according to standard techniques developed earlier(17). The volume of each aspiration was limited to < 5 mL to minimize

the contamination of peripheral blood with the harvested marrow(18,19). ABM cells were procured by flushing marrow collection bags and filter screens used in harvesting marrow from normal

allogeneic donors. Recombinant human granulocyte-colony stimulating factor (rhG-CSF) mobilized PBMC (_n_ = 8) were collected from normal volunteer donors. CB (_n_ = 12) was obtained

immediately after delivery by draining samples from the umbilical cord vein into sterile collection tubes containing preservative-free heparin. PBMC and CB were diluted to 1:3 with PBS. The

low-density mononuclear cells were separated by Ficoll-Hypaque (Atlanta Biologicals, GA) density centrifugation and washed twice with PBS. Cell yield and viability were determined by trypan

blue dye exclusion assay. The logistics of human donors were handled according to Department of Health and Human Services/National Institutes of Health guidelines on protection of human

subjects, with the authorization of the Georgetown University Medical School IRB Committee. _PHENOTYPIC ANALYSIS._ Two-color immunofluorescent staining was used to analyze the phenotypic

characteristics of mononuclear cells. Mononuclear cells were resuspended in PBS containing 0.3% BSA, 0.25% human immune globulin, and 0.3% sodium azide at a concentration of 106/100 µL and

incubated for 15 min at 25°C. An aliquot of 5 × 105 cells was removed and stained with monoclonal antibodies directed against various human lymphocyte and/or myeloid antigens. Monoclonal

antibody preparations directed against the following surface markers were used: CD34, CD38, CD3, CD16, CD19, CD56, and HLA-DR (Becton Dickinson, San Jose, CA). Cell analysis was performed on

FACStarplus (Becton Dickinson). The data were analyzed with Reproman and Lysys software. _CLONOGENIC CULTURES._ Mononuclear cells from all sources were added to semisolid media containing

0.8% of methylcellulose (HCC 4230; StemCell Technologies, Inc., Vancouver, Canada) supplemented with erythropoietin (Epo, 2 u/mL) and granulocyte/macrophage colony stimulating factor

(GM-CSF, 20 ng/mL). The cells were plated at a concentration of 1 × 105/mL and for each group, two 1-mL cultures were set up in 35-mm Petri dishes. Plates were incubated at 37°C in a

humidified atmosphere of 5% CO2 for 2 weeks. The number of CFU-C was scored on d 14 with a colony consisting of 40 or more cells. _MIXED LYMPHOCYTE REACTIONS (MLR)._ MLRs were conducted by

culturing 1 × 105 mononuclear cells with equal numbers of irradiated autologous or allogeneic bone marrow or blood cells in 96 well round-bottomed microtiter plates. The stimulator cells

were irradiated with 1500 rads with a JL Shepard Cs-137 Irradiator. All cultures were performed in triplicate in 0.2 mL of RPMI 1640 supplemented with 10% human AB serum. Cultures were

maintained in a 37°C and 5% CO2 humidified incubator for 5 d. DNA synthesis was measured by pulsing with 1 µCi of 3H-thymidine during the final 18 h of the culture. Cultures were harvested

onto glass fiber filter paper using a PHD™ cell harvester (Cambridge Technology Inc., MA) and 3H-thymidine incorporation was determined by liquid scintillation counting. _STATISTICAL

ANALYSIS._ Each of the markers was tested independently using an analysis of variance (ANOVA). In order to achieve normality and equal variance assumptions for the ANOVA, the marker

expression data were transformed using natural logs. Differences in the collection types (FBM, CM, PBMC, and ABM) were tested using Tukey's multiple comparison method(20) to control the

overall significance level at 0.05 for each marker. The colony formation data were tested in a similar manner, but the data were transformed using the square root, which is a standard

transformation instrument for assessment of data to achieve normally distributed values. The overall significance level was 0.01 for the multiple comparison method to control for using the

combined data. Where applicable, simple correlations were performed. RESULTS _PROPORTION OF HEMATOPOIETIC PROGENITORS._ The quantitative yield of mononuclear cells from each single fetus was

approximately 1-3 × 108 and was dependent on the age and the available number of fetal bones. The most striking and unique characteristics of FBM were the number of CD34+ cells in the

mononuclear cells after Ficoll separation. The percentage of CD34+ cells in FBM ranged from 14.8 to 30.7 (mean 24.6 ± 3.5) which was significantly higher when compared with the other three

sources, _i.e._ ABM, PBMC, and CB (Fig. 1). In comparison, the percentages of CD34+ cells in ABM and CB were equivalent, ranging from 1.3-3.4% and 1.2-2.9%, respectively. PBMC had the lowest

number of CD34+ cells (0.2-0.8%). Because the majority of the CD34+ cells are committed to either the hematopoietic or stromal cell lineages, we further compared the most common lineage

associated antigens, CD38 and HLA-DR, expression within the fraction of CD34+ cells. As shown in Table 1 and 2, FBM had the highest number of CD34+/38- and CD34+/HLA-DR- cells (2.11 ± 0.21%

and 3.12 ± 0.19%, respectively). The number of CD34+ cells was statistically analyzed by using ANOVA (Table 2). FBM had the highest log expression (3.13), CB and ABM were better than PBMC,

but were not different from each other (mean log expression: 1.09, 1.11, and 0.376, respectively). For the CD34+/DR- and CD34+/CD38- combination, FBM (1.10) remained the highest. PBMC and

ABM were better than CB, but they were not significantly different from each other. _PHENOTYPIC ANALYSIS OF LYMPHOID POPULATIONS._ In addition to the unique finding of CD34+ cells in FBM,

other phenotypic characteristics were also observed. The percentage of each marker expressed in different sources was shown in Figure 1. For each marker, the type of cell source made a

difference in log expression levels (_p_ < 0.001) for each marker except for CD56+ (_p_ = 0.025). Table 2 shows the mean log expression level for each collection type within each marker.

CD3 was significantly expressed in CB (3.46) and PBMC (2.79), compared with ABM (1.76) and FBM (0.12), but there was no statistical difference between CB and PBMC. Although CD19+ had the

highest expression in CB (1.68), a great percentage was also observed in ABM (0.91). Interestingly, the percentage of CD19+ cells in PBMC (-0.54) was lower than other sources. CD56+ cellular

concentrations in two samples from the ABM collection gave outlier values, causing higher standard deviations, thus potentially violating the normality assumption for the ANOVA. PBMC (1.04)

had the highest CD56 expression but was only significantly higher than CB (0.41). The percentages of CD56+ cells in FBM (0.88) and ABM (0.58) were not significantly different from each

other. CD16 was similarly expressed in both CB and PBMC but was significantly lower in ABM and FBM. _HEMATOPOIETIC PRECURSORS DEFINED BY CLONOGENIC ASSAYS._ Based on our initial finding that

the percentages of CD34+ cells in the four sources were quite different, we further compared the cloning efficiencies of bone marrow and blood cells. The cloning efficiency was defined as

the number of colonies per 1 × 105 cells after a 2-wk culture in semisolid media. Two types of colonies were formed from seeding of the collected cells, _i.e._ CFU-GM and BFU-E, in the

presence of GM-CSF and Epo. The number of CFU-C (CFU-GM + BFU-E)/105 cells from FBM was significantly higher than that from other sources (Fig. 2). The number of colonies from CB was less

than ABM but greater than PBMC. The number of CFU-GM was lower than BFU-E in all cell populations except in CB, where CFU-GM was greater than BFU-E. Table 3 shows the statistical analysis of

colony enumeration from different sources. It can be seen that although FBM showed, in general, higher numbers of colonies from various hematopoietic progenitors, individual differences

were seen between CFU-GM and BFU-E. Strong correlation was found between CD34+ cells and the number of colonies BFU-E and CFU-C. The correlation coefficients were 0.92 (_p_ = 0.01) and 0.93

(_p_ = 0.004), respectively. However, a weak correlation of 0.57 (_p_ = 0.37) was found between CD34+ cells and CFU-GM. The correlation between CD34+ cells and the number of colonies in the

individual groups [FBM, ABM, PBSC, and CB] were poor. Probably, the small sample size (_n_ = 8) in each group was not suitable for correlation analysis. _MIXED LYMPHOCYTE REACTION ASSAYS._

Mixed lymphocyte reactions in both autologous and allogeneic cell cultures are shown in Figure 3. FBM demonstrated reduced responses to both autologous and allogeneic simulators when

compared with adult cells. Interestingly, the proliferative responses were much lower when irradiated FBM was used as a stimulator to other sources. CB exhibited a profound immunoreactivity

when compared with FBM but it was significantly lower than ABM and PBMC. DISCUSSION Hematopoiesis is the process of formation and development of blood cells that is sustained throughout the

life span of the individual by multi-potential stem cells. The varying sites of human hematopoiesis, which takes place during ontogenesis (yolk sac, spleen, thymus, liver, and bone marrow),

provide new insights for the identification of potential sources of stem cells in both children and adults. Thus, CB, PBMC, and fetal liver have been used in hematopoietic stem cell

transplantation experimentally and clinically(21–26). However, there have been no reports using FBM obtained from spontaneously aborted fetuses as an alternative source of hematopoietic stem

cell for transplantation. The present studies provide additional support for the potential use of this source. Fetal tissue is preimmunocompetent and is characterized by a significantly

reduced capacity to evoke immunogenic responses(27,28). The depressed immunocompetence or immunologic immaturity of the fetus is ontogeny-related. It is a result of the reduced number or

lack of postthymic T-lymphocytes _in utero_, which, when mature, contribute to GVHD and/or from immaturity of antigen-presenting cells. In the course of fetal development, the precursors of

the hematopoietic stem cells arise in the yolk sac, migrate to the liver, and thereafter to the thymus and spleen. Eventually, they locate in bone marrow(2). In the second trimester of

pregnancy (18-22 wk), the bone marrow provides an enriched source of hematopoietic precursors. The number of the progenitor cells at this gestational age increases dramatically because of

the rapid development of the fetus. Recently, there has been an increasing interest in CB as a source of stem cells for transplantation. CB has several distinct advantages: it is easily

accessible and contains large number of cells including immature lymphocytes that are not capable of mediating mature T cell functions associated with GVHD(29). Furthermore, the number of

CD34+ cells in CB is similar or slightly higher than in ABM(30,31). However, because of contamination of CB with maternal blood cells, there is the potential risk of GVHD after CB

transplantation. Recently published registry data(11) from 45 European transplantation centers collected between 1988 and 1996 on CB transplantation (_n_ = 143) revealed that in 78

recipients of CB from related donors the estimated survival at 1 y was 63% in all pediatric patients and in some adults with hematopoietic diseases. GVHD of at least grade II in HLA-matched

recipients occurred at 9%, whereas in HLA-mismatched recipients it was estimated to be 50%, with a 29% survival at 1 y. It was concluded that cytomegalovirus contamination seemed to be the

most important predictor of GVHD. More studies are needed to improve the survival of unrelated recipients(32). The observation that the stem cells exist in the peripheral blood of adults has

led to the exploitation of PBMC as an alternative source for hematopoietic reconstitution. However, because a small number of stem cells circulate in the peripheral blood, the

administration of hematopoietic growth factors and/or chemotherapeutic reagents are often required for mobilizing progenitor cells into the blood stream before harvesting(33,34). The

reported frequencies of CD34+ cells in different sources of stem cells varies between different laboratories and are probably related to differences in methods of detection or analysis

criteria. The number of CD34+ cells in ABM and CB ranged from 0.5-4%(31,35), whereas in PBMC, CD34+ cells comprised < 0.5%. Our results showed a significant increase of CD34+ cells and a

higher clonogenic ability of FBM. Previous studies from other laboratories have shown that CD34+ cells can be divided into at least four subgroups according to CD38 and HLA-DR expression.

Among these subgroups, the CD34+/CD38- immunophenotype is considered to be a highly primitive subpopulation of CD34+ cells(36). It has been previously reported that CD34+ cells from ABM with

low or absent expression of HLA-DR antigen are enriched for primitive blast-cell-containing colonies, high proliferative potential-colony-forming cells, and long term culture initiating

cells(37). However, data derived from CB clearly showed that the HLA-DR+ subpopulation of CD34+ cells is more primitive than the CD34+/HLA-DR- population(38,39). Our studies showed that the

percentage of CD34+/HLA-DR- cells in the FBM was significantly higher than other sources (Table 1) but it was not related to the clonogenic potential. Although the percentage of single CD34+

cells in CB was lower than other sources, its general clonogenic ability (CFU-C) was still comparable to ABM. The only significant difference was that the total colonies from CB were

dominated by CFU-GM. This result indicates that CB demonstrates a trend toward a higher proportion of primitive committed progenitor cells. Moreover, most primitive cells in ABM were found

in the CD34+, CD38-, and HLA-DR- cell populations suggesting that ABM and FBM differ in the HLA-DR and CD38 expression levels. This further suggests that during human development, the

primitive progenitor cells differ in their behavior with respect to the ontogeny of hemopoietic system(40,41). MLR is induced by culturing mononuclear cells (which include T cells, B cells,

NK cells, dendritic cells, and monocytes) from one individual as a responder with mononuclear cells derived from another individual as a stimulator. Cells used as stimulators need to be

treated with irradiation or mitomycin C. Two populations of alloreactive T cells are stimulated during allogeneic MLR. One type of T cells is CD8+, which is major histocompatibility complex

(MHC) class I restricted, the other one is CD4+, which is MHC class II restricted. Meanwhile, B cells, dendritic cells, and monocytes also participate in this reaction by presenting antigens

to T cells and/or producing cytokines. It is difficult to say which type of cells dominates over the other because of the collective function of the responder cells in the reaction. Our

data showed a poor correlation between CD3+ cells and the MLR when the same cells were used as stimulators for different responders. A similar level of MLR was observed in PBSC and ABM,

although CD3+ cells in PBSC were three-fold higher than ABM. On the other hand, CB had the highest percentage of CD3+ cells, but its MLR was much lower than PBSC and ABM. The same phenomenon

was also observed when HLA-DR expression was used as a reference marker. It indicates that a single marker cannot predict the level of MLR. The ontogenetic influence to MLR that includes

functional maturity of the responder cells must be considered. In our comparative studies of MLR assays, the FBM revealed a significantly decreased responsiveness after stimulation with

autologous and allogeneic hematopoietic cells. We assume that this immunoincompetence was related to either the reduced number of CD3+ cells or immaturity of antigen presenting cells in FBM.

Further, FBM as a stimulator caused a decreased proliferation in all sources of hematopoietic cells. Thus, these data support the hypothesis that FBM may not be recognized as foreign to the

same extent as ABM. The reduced immunologic responses may reduce the incidence and severity of GVHD and potentially obviate the problem of tissue type matching. Further, the increased

HLA-DR+ cells in FBM might suggest greater propensity for GVHD. However, it is known that both HLA class I and II are involved in graft _versus_ host reactions but not class II alone(32).

Together with the decreased MLR reactivity of fetal cells, FBM cells might express reduced HLA class I and/or immature class II antigens. CB is also composed of phenotypically and

functionally immature lymphocytes that are not capable of mediating mature T cell functions associated with GVHD(24). Preliminary functional studies using CB lymphocytes confirm that they

are alloreactive but respond less vigorously in mixed lymphocytes culture than corresponding adult lymphocytes(29,41). As seen in this study, CB revealed relatively reduced immunoreactivity

and immunogenicity when compared with adult hematopoietic cells, but both of these properties were much higher in CB than in FBM. The high frequency of CD3+ cells in CB conflicts with its

low immunogenicity, probably because the naive T lymphocytes contribute to this population. The results of the present study are in agreement with the current knowledge on the ontogenetic

differences between various sources of hematopoietic stem cells. Namely, each source of hematopoietic stem cells has different intrinsic properties that are closely correlated with

ontogenetic age; and thus, the phenotypic characteristics of human stem cells, the lineage commitment, the level of reconstitution, and engraftment are a function of ontogenetic

age(24,42,43). Our earlier _in vivo_(44–46) as well as _in vitro_(47) studies of FBM transplantation in allogeneic and xenogeneic systems revealed the high efficacy of fetal tissue derived

from miscarriage for transplantation, which is in agreement with data reported by others(48–51). The results of the present study not only support the potential utility of FBM, derived from

spontaneous abortion, for hematopoietic stem cell transplantation, but also suggest that fetal stem cells may provide an important source of cells for gene therapy. A question may be raised

whether a given fetal donor provides a sufficient number of stem cells for effective transplantation. The low immunoreactivity of the fetal tissue suggests that stem cells from more than one

donor could be pooled together. The feasibility of this approach was suggested by our earlier studies in which mixtures of stem cells were transplanted _in utero_, into nonhuman primate

fetuses(44–46). In conclusion, the data in the present study reveal significant ontogenetic differences between sources of stem cells and suggest that fetal bone marrow derived from viable

miscarriages may be a useful source of transplantable cells. More _in vivo_ studies on the engrafting and immune responses in relevant animal model systems are needed to provide predictive

parameters, such as optimal number of stem cells for successful transplantation in the human. ABBREVIATIONS * BFU-E: burst-forming unit - erythrocyte * CFU-C: colony forming unit in culture

* CFU-GM: colony-forming unit - granulocyte macrophage * FBM: fetal bone marrow * ABM: adult bone marrow * CB: cord blood * GVHD: graft _versus_ host disease * PBSC: peripheral blood stem

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1012–1013 CAS  PubMed  Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * International Center for Interdisciplinary Studies of Immunology, Georgetown

University School of Medicine, Washington, 20007, D.C. Ai Guo Wu, Maria Michejda & Joseph A Bellanti * Department of Pediatrics and Microbiology/Immunology, Georgetown University School

of Medicine, Washington, 20007, D.C. Ai Guo Wu, Maria Michejda & Joseph A Bellanti * Bone Marrow Transplantation Program, Georgetown University School of Medicine, Washington, 20007,

D.C. Ai Guo Wu, Amitabha Mazumder & Kenneth R Meehan * Departments of Laboratory Medicine, Georgetown University School of Medicine, Washington, 20007, D.C. Frederick A Menendez *

Obstetrics/Gynecology, Georgetown University School of Medicine, Washington, 20007, D.C. Jean-Gilles Tchabo * Biostatistics Unit, Lombardi Cancer Center, Georgetown University School of

Medicine, Washington, 20007, D.C. Rebecca Slack * Department of Obstetrics/Gynecology, Hutzel Hospital, Wayne State University, Detroit, 48202, Michigan Mark P Johnson Authors * Ai Guo Wu

View author publications You can also search for this author inPubMed Google Scholar * Maria Michejda View author publications You can also search for this author inPubMed Google Scholar *

Amitabha Mazumder View author publications You can also search for this author inPubMed Google Scholar * Kenneth R Meehan View author publications You can also search for this author

inPubMed Google Scholar * Frederick A Menendez View author publications You can also search for this author inPubMed Google Scholar * Jean-Gilles Tchabo View author publications You can also

search for this author inPubMed Google Scholar * Rebecca Slack View author publications You can also search for this author inPubMed Google Scholar * Mark P Johnson View author publications

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Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Wu, A., Michejda, M., Mazumder, A. _et al._ Analysis and Characterization of Hematopoietic Progenitor Cells from Fetal Bone

Marrow, Adult Bone Marrow, Peripheral Blood, and Cord Blood. _Pediatr Res_ 46, 163–169 (1999). https://doi.org/10.1203/00006450-199908000-00006 Download citation * Received: 27 July 1998 *

Accepted: 26 March 1999 * Issue Date: 01 August 1999 * DOI: https://doi.org/10.1203/00006450-199908000-00006 SHARE THIS ARTICLE Anyone you share the following link with will be able to read

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