Increased graft contentof vascular progenitor cells is associated with reduced

toxicity following autologous hematopoietic transplantation

Tariq Iqbala,b, Thivisha Rajagopald,e,Michael Halpennye, Lin Yange, Lisa Martine,

Antonio Giulivic,e, Sheryl McDiarmida, Lothar Huebscha,b, and David S. Allana,b,d

aBlood and Marrow Transplant Program,

The Ottawa Hospital; bDepartments of Medicine and cPathology, University of Ottawa; dMolecular Medicine Program,

Ottawa Health Research Institute; eStem Cell Processing Laboratory, Canadian Blood Services, Ottawa, Ontario, Canada

(Received 22 April 2007; revised 13 November 2007; accepted 6 December 2007)

Objective. Endothelial-like vascular progenitor cells (VPCs) can be collected in peripheralblood stem cell (PBSC) products that are used in hematopoietic stem cell transplantation(HSCT). The association between VPCs in PBSC products and transplant-related toxicitycaused by high-dose chemo/radiotherapy was assessed to identify potential mediators ofvascular repair.

Materials and Methods. PBSC grafts in 29 patients (mean age: 48 years; range, 20–67 years)undergoing autologous HSCT were analyzed using a cell culture assay for VPC cluster forma-tion in fibronectin-coated dishes in serum-rich angiogenic conditions. Transplant toxicity wasestimated using total length of hospital stay (LOS) following HSCT and the Seattle criteria fortransplant-related organ toxicity for 8 organ systems (grade 0–4).

Results. LOS following graft reinfusion was lower (14.7 vs 20.0 days, p [ 0.002) and the meannumber of organs with any toxicity (1.0 vs 2.4, p [ 0.016) or with toxicity grade $ 2 was re-duced (0.2 vs 1.6 organs, p [ 0.007) in patients with high graft VPC content (n [ 10, O2.0 3103 VPCs/kg) compared with reduced VPC content (n [ 19, #2.0 3 103 VPCs/kg). An asso-ciation between graft CD34+ levels and LOS or organ toxicity was not observed. In addition,graft VPC levels were independent of graft CD34 counts, peripheral blood monocytesand hemoglobin levels, age, and disease (p [ NS).

Conclusion. PBSC products enriched for VPCs are associated with reduced toxicity followingHSCT. Identifying specific factors that contribute to high graft VPC levels is needed. � 2008ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Experimental Hematology 36 (2008) 506–512

Endothelial-like vascular progenitor cells (VPCs) are impli-cated in the repair of ischemic tissue damage through devel-opment of new capillary networks in areas of ischemictissue damage [1]. VPCs are defined by their vascular func-tion and include marrow-derived progeny from ‘‘angio-blast’’ precursors [2], vessel wall progenitor cells, andmonocyte-derived vascular cells [3,4]. VPCs can be effi-ciently collected in peripheral blood, bone marrow, umbil-ical cord blood [5], and cytokine-stimulated leukapheresisgrafts [6–8] used in hematopoietic stem cell transplantation

Offprint requests to: David Allan, M.D., F.R.C.P.C., Blood and Marrow

Transplant Program, The Ottawa Hospital, General Campus, 501 Smyth

Road, Box 704, Ottawa, ON K1H 8L6 Canada; E-mail: daallan@ohri.ca

(HSCT), although the frequency and biological propertiesmay vary. Importantly, several animal models of ischemictissue damage have been instrumental in demonstratingthe potential therapeutic role of VPCs and include mousemodels of myocardial ischemia, hind limb ischemia, andstroke [9,10]. In addition, clinical ischemic events, suchas myocardial infarction [11] and stroke [12], are morelikely to occur in patients with low levels of circulatingVPCs and acute ischemic damage, such as trauma or severeburns [13], are associated with repair responses that includeincreased levels of circulating VPCs. Full characterizationof this response and the individual cell populations most re-sponsible for repairing tissue injury remains hampered bya lack of definitive cell markers for their efficient isolation.

0301-472X/08 $–see front matter. Copyright � 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

doi: 10.1016/j.exphem.2007.12.005

507T. Iqbal et al. / Experimental Hematology 36 (2008) 506–512

Cell therapy using VPCs, however, is emerging as a promis-ing therapeutic approach in several types of acute tissueinjury.

HSCT involves toxic chemotherapy and/or radiationtreatment that causes widespread tissue damage and signif-icant morbidity. The Seattle Criteria for assessing toxicityfollowing hematopoietic transplantation [14] and the Na-tional Cancer Institute toxicity scales for determination oftoxicity following chemotherapy for cancer [15] providevalidated tools for the objective assessments of treatment-related organ damage. Oral mucositis and gastrointestinaltoxicity are the most common toxicities associated with au-tologous hematopoietic transplantation and occur in mostpatients [16]. Intense resource utilization is required to ad-dress organ damage following HSCT, including prolongedhospital stays, complex administration of narcotic analge-sics and total parenteral nutrition, rising costs related tonewer antimicrobials and the involvement of many medicalspecialists and health-care professionals, and the need forcritical care in O10% of patients [17]. HSCT for patientswith hematologic disorders typically remains restricted toyounger patients without significant coexisting organ dys-function, precluding many patients from potentially cura-tive therapy.

In this study, we address the role of graft VPCs in reduc-ing tissue toxicity associated with high-dose chemo/radio-therapy in hematopoietic transplantation. We hypothesizethat increased graft VPC content facilitates more rapidand complete recovery of ischemic tissue injury followingtransplantation.

Materials and methods

PatientsPatients undergoing autologous peripheral blood progenitor cell(PBPC) transplantation in the Blood and Marrow Transplant Pro-gram of The Ottawa Hospital were eligible for inclusion. All pa-tients signed informed consent prior to transplantation allowingthe use of clinical and laboratory data for research purposes.This study was approved by the Ottawa Hospital Research EthicsBoard at the University of Ottawa. Clinical data were retrievedfrom The Ottawa Hospital bone marrow transplantation clinicaldatabase and laboratory values were retrieved from the hospitalcomputer record of each patient and anonymized for analysis.

VPC cell culture assayVPC were evaluated by cell culture assay using an adaptation ofthe method of Hill et al. [18]. Briefly, 5 � 106 mononuclear cellsfrom fresh leukapheresis products were plated in a 9.6-cm2 well offibronectin-coated plastic dishes (BioCoat, BD Biosciences, Bed-ford, MA, USA) in Endocult media (Stem Cell Technologies, Van-couver, Canada) and incubated at 37�C in 5% CO2 and O95%humidity. After 48 hours, nonadherent cells were removed, washed,and replated at 1.0 � 106 cells per 2.0-cm2 well in fibronectin-coated dishes. VPCs were enumerated on day 5 and defined bythe presence of cell clusters with at least 30 cells associated

with a minimum of 3 attached projecting cells using an invertedmicroscope. Immunohistochemistry was performed on a subset ofVPC clusters using primary mouse anti-human antibodies againstCD45, CD14, and VE-cadherin (BD Pharminogen, San Diego,CA, USA) Secondary antibodies against mouse IgG were conju-gated with fluorescein isothiocyanate and cell clusters were fixedand stained according to manufacturer instructions. Fluorescencewas detected using a Zeiss inverted microscope. Nuclei werevisualized using DAPI staining according to methods reportedpreviously.

Clinical parameters of transplant-related toxicityTransplant toxicity was evaluated using the Seattle criteria de-scribed by Bearman et al. [14] and by length of hospital admission(combined admitted and ambulatory days until all acute transplantissues were resolved). Toxicity was assessed prospectively asgrade 0 to 4 (see Table 1) in eight organ systems, including lungs,heart, kidneys, bladder, liver, gut, oral mucosa, and central ner-vous system. Nonrelapse mortality was assessed at day þ100 aftertransplantation.

Statistical analysisMeans are reported 6 1 standard deviation. Comparison of meanswas performed using two-tailed Student’s t–test, assuming un-equal variance. Standard deviation is reported for all mean values.Proportions were compared using c2 analysis.

Results

PatientsGraft VPC analysis was performed on 29 patients undergo-ing autologous hematopoietic transplantation in the OttawaHospital Blood and Marrow Transplant Program betweenSeptember 2005 and August 2007. Subjects had a meanage of 48 6 14 years (range, 20–67 years) and receivedhigh-dose chemotherapy and/or total body irradiation fornon-Hodgkin’s lymphoma (n 5 16), relapsed or refractoryHodgkin’s disease (n 5 3), multiple myeloma (n 5 8),germ cell tumor (n 5 1), and multiple sclerosis (n 5 1).Five patients received total body irradiation as part of themyeloablative regimen (four patients received 500 cGy ina single fraction for follicular lymphoma and one patientreceived 1200 cGy in six fractions for non-Hodgkin’slymphoma). General characteristics of patients analyzedin this study are presented in Table 2.

Graft content of VPCsVPC levels enumerated by the cell culture assay varied sig-nificantly in autologous peripheral blood progenitor cellgrafts with a median of 23 clusters per 105 mononuclearcells (range, 0–714). In immunohistochemistry, VPC clus-ters were composed of heterogenous cells with the majoritystaining positively for CD45, CD14, and only a minority ofcells (!10%) was positive for VE-cadherin. Peripheralblood mononuclear cells were CD45þ, CD14þ, and VE-cadherin–negative, while human umbilical vein endothelial

508 T. Iqbal et al./ Experimental Hematology 36 (2008) 506–512

Table 1. Seattle Criteria for organ toxicity following bone marrow transplantation

Organ Grade 1 Grade 2 Grade 3

Bladder Asymptomatic macroscopic hematuria

after 2 days from chemotherapy

Macroscopic hematuria after 7 days from

chemotherapy or symptomatic

Hemorrhagic cystitis requiring intervention

Renal Increased Cr !2� baseline Increased Cr O2� baseline Requires dialysis

Gastrointestinal Noninfectious watery diarrhea

500–2000 mL/d

Noninfectious watery diarrhea O2000 mL/d

or hemorrhagic stools or sub ileus

Ileus requiring NG suction and/or surgery or

hemorrhagic stools requiring transfusion

Hepatic Bilirubin O34 and !102 g/L or

weight gain 2.5–5.0% or AST O2�and !5� baseline

Bilirubin O102 and !340 g/L or weight

gain O5.0% or ascites or AST O5� baseline

Bilirubin O340 g/L or encephalopathy or

ascites causing resp compromise

Oral mucositis Pain and/or ulceration not requiring

continuous systemic narcotic

Pain and/or ulceration requiring continuous

systemic narcotic

Pain and/or ulceration requiring preventive

intubation or aspiration pneumonia

Cardiovascular Asymptomatic heart enlargement of

CXR or asymptomatic ECG changes

Heart failure responsive to diuretics or ECG

changes requiring medical intervention

Heart failure unresponsive to medical

intervention or severe ECG changes

unresponsive to medical intervention

Nervous system Somnolence but easily rousable Somnolence with confusion, or other new CNS

symptoms

Seizures or coma not otherwise explained

Pulmonary Dyspnea without CXR changes or

isolated infiltrate or mild interstitial

changes

Moderate infiltrates or interstitial changes or

10% decrease in PaO2

Requires O50% O2 or mechanical intubation

AST 5 aminotransferase; CNS 5 central nervous system; Cr 5 creatinine; CXR 5 chest x-ray; ECG 5 electrocardiogram; NG 5 nasogastric.

cells were CD45-, CD14-, and VE-cadherin–positive. Interms of total VPC dose reinfused, a median of 1.1 � 103

clusters per kg (range, 0–43) were enumerated in the grafts.VPC levels did not correlate with graft CD34 numbers,graft monocyte levels, diagnosis, or pretransplantation pe-ripheral blood parameters, including hemoglobin, leuko-cyte, or monoocyte levels (p 5 NS). Patients weredivided into ‘‘low’’ graft VPC content and ‘‘high’’ graftVPC content based on whether graft VPC levels were !or O2.0 � 103 per kg. The cutoff of 2.0 � 103 per kgwas selected arbitrarily to provide the best stratificationfor the toxicity outcomes analyzed.

Graft VPC levels andbone marrow transplantation toxicityHigher graft content of VPCs was significantly associatedwith decreased length of hospital stay (Fig. 1). Patientswith high graft VPC content were reviewed daily in the am-bulatory BMT Unit or admitted to the transplant service for14 6 3.0 days compared to 20 6 5.0 days (p 5 0.0017) inpatients with low graft VPC content. Two patients requiredtransfer to critical care and both were in the group withreduced VPC graft content (p 5 NS).

Transplant-related organ injury was assessed using theSeattle criteria for grading organ toxicity in eight organsystems. Higher graft VPC levels were associated with sig-nificantly fewer organ systems manifesting any toxicity(grade O 0) and with a reduction in the number of organswith severe toxicity (grade $ 2). Mean number of organsystems with any toxicity in the high graft VPC groupwas 1.0 6 0.9 compared with 2.4 6 1.9 in the groupwith low graft VPCs (p 5 0.016). In addition, mean numberof organ systems with severe toxicity in the high graft VPCgroup was only 0.2 6 0.4 compared with 1.6 6 1.9 in the

low VPC group (p 5 0.007). If patients who received totalbody irradiation as part of the conditioning regimen areexcluded from the analysis (remaining patients, n 5 24),a similar relationship is observed between graft VPC levels(high vs low) and length of stay (p 5 0.02), with the num-ber of organs having any toxicity (p 5 0.008) and there isa trend toward a relationship with the number of organshaving severe toxicity (p 5 0.07). In addition, a subanalysisof patients with non-Hodgkin’s lymphoma reveals the samefindings (n 5 16). Patients with increased graft VPCs hadshorter LOS (p 5 0.0005), fewer organs with any toxicity(p 5 0.006) and a trend toward a reduced number of organsystems with severe toxicity (p 5 0.064).

Mucositis, gastrointestinal tract, and liver injuries wereresponsible for the majority of severe organ toxicity (see

Table 2. Patient Characteristics

Characteristic

Total patients, n 29

Mean age (y), (median, range) 47 (47, 20–67)

Gender, male/female (% male) 23/6 (79)

Diagnosis, n (%)

Non-Hodgkin’s lymphoma 16 (55)

Hodgkin’s lymphoma 3 (10)

Multiple myeloma 8 (28)

Germ cell tumor 1 (3)

Multiple sclerosis 1 (3)

No. of patients receiving TBI (%) 5 (17)

Graft CD34þ cells/kg, mean (median, range) 7.4 (5.9, 1.1–26) � 106

Graft VPCs/kg, mean (median, range) 5.2 (1.1, 0–43) � 103

Days of PBSC collection, mean (median, range) 1.5 (1.0, 1–3)

Total days admitted to hospital, mean (range) 19 (11–35)

Nonrelapse mortality at day þ100, n (%) 2 (7)

PBSC 5 peripheral blood stem cell; TBI 5 total body irradiation; VPC 5

vascular progenitor cells.

509T. Iqbal et al. / Experimental Hematology 36 (2008) 506–512

0

10

20

30

≤ 2.0 > 2.0Graft VPCs x 10

3/kg

LO

S (d

ays)

p = 0.0017

Figure 1. Impact of graft vascular progenitor cells (VPC) on length of

hospital stay (LOS).

Fig. 2). Genitourinary and renal system injury was infrequentand did not contribute significantly to severe transplant-related toxicity. The profile of organ toxicity is shown inFigure 2 and illustrates reduced incidence of toxicity inall eight organ systems for the group with increased graftVPC content. Although the proportion of patients sufferingany toxicity or severe toxicity was not statistically differentfor any of the individual organ systems, it is remarkablethat severe toxicity only occurred in two organ systems(gastrointestinal and mucositis) among patients with highgraft VPCs and toxicity of any grade occurred in onlyfive of eight organ systems in this group, whereas toxicitywas observed in all organ systems in the group with lowgraft VPCs. It is interesting that no cardiac or nervous

0

10

20

30

40

50

60

70

80

Blad

der

Hea

rt

CN

S

Gut

Live

r

Muc

ositi

s

Lung

Ren

al

% w

ith

an

y g

rad

e to

xicity

A

0

10

20

30

40

50

60

70

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Blad

de

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CN

S

Gut

Live

r

Muc

ositi

s

Lung

Ren

al

% w

ith

to

xicity ≥ g

rad

e 2

B

Figure 2. Organ toxicity in patients with low graft vascular progenitor cell (VPC) levels (C) and high graft VPC levels (B): (A) Organ toxicity of any

grade, (B) organ toxicity of grade 2 or greater. CNS 5 central nervous system.

510 T. Iqbal et al./ Experimental Hematology 36 (2008) 506–512

system toxicity occurred at all in the group with high graftVPCs.

Nonrelapse mortality occurred in two patients, but wasnot statistically different between the two groups (p 5

NS), although both deaths occurred in patients with lowerVPC graft content. The causes of death were infectionand multiorgan failure in the two patients, respectively.

Toxicity not related to graft CD34 levelsThe potential impact of graft CD34þ levels on transplanttoxicity was also performed using data from all 29 patients.When the cohort is divided into two groups based on themedian CD34 level (i.e., below the median of 5.85 � 106

per kg or above the median), there is no statistically signif-icant difference in the length of hospital stay (p 5 0.09), themean number of organ systems with any toxicity (p 5

0.49), or the mean number of organs with severe toxicity(grade $ 2, p 5 0.07). In addition, if the cohort is dividedinto two groups based on the optimal CD34 level of 5.0 �106 per kg, the length of stay (p 5 0.35), mean organsystems with any toxicity (p 5 0.60) or with severe toxicity(p 5 0.29) are not different between the two groups. Fur-ther, no specific threshold of CD34þ cells was identifiedin this cohort that associated with a significant correlationwith any of the toxicity outcomes.

DiscussionOur observations suggest that endothelial-like precursors inperipheral blood stem cells may participate in the repair oftissue injury and protect against organ dysfunction in autol-ogous transplant recipients. In our study population, we ob-served an important association between graft content ofvascular progenitors and length of hospital stay and with re-duced incidence of transplant-related toxicity. It is possiblethat VPCs within the cellular graft protect against treat-ment-related tissue injury or that VPCs accelerate the repairof organ dysfunction. Given the small number of patients,we were unable to control for competing variables thatmay increase the risk of toxicity, including patient age, ex-tent of prior chemotherapy or radiation treatments, variableconditioning regimens, and effects of underlying comorbid-ities or concomitant medication usage. In subanalyses ofour data, however, we observed the same pattern of associ-ation between graft VPC levels and toxicity for patientswho received only chemotherapy and for patients under-going transplantation for non-Hodgkin’s lymphoma. Theobserved association between graft VPCs and toxicityfollowing myeloablative chemo/radiotherapy encouragesus to contemplate the notion of cell therapy for repair oftissue damage that may include clinical problems such asmyocardial infarction or stroke.

High-dose chemotherapy with or without total body irra-diation can induce widespread tissue damage in multipleorgan systems. The Seattle criteria for assessing toxicity

following hematopoietic transplantation were first devel-oped in a cohort of patients undergoing both autologousand allogeneic transplantation [14], and have since beenused by many centers to describe transplant-related toxicity.The pathophysiology of chemotherapy-induced organ dam-age is variable and includes necrosis of epithelial tissues(i.e., mucositis, gastrointestinal toxicity), ischemic damagerelated to hypoperfusion of critical organs (cardiovascular,renal impairment), and idiopathic organ toxicity related tomedications (neurological, skin, and renal damage). TheSeattle Criteria alone remain inexact and do not reflectthe global impact on patient morbidity. Additional mea-sures of toxicity following transplantation, such as lengthof hospital stay, intensive care unit admission, and nonre-lapse death by day 100 may better reflect overall impactof the transplantation on patient morbidity and resource uti-lization. Rates of transplant-related mortality and intensivecare unit admission, however, were low in our cohort andwere not useful for a comparison of toxicity.

Repair of tissue injury is a complex process involving in-flammatory and angiogenic cytokines and the mobilizationand homing of mononuclear cells, vascular progenitor cells,and other mediators of tissue repair [19,20]. VPCs appearimportant in this process and may arise from multiple sour-ces, including bone marrow [21–23], the vessel wall[21,24], and from circulating monocytes [4]. Our own im-munohistochemistry data supports the important role ofVPCs derived from circulating monocytes, consistent withseveral recent reports [3,4]. VPCs can be mobilized fromquiescent vascular zones of the marrow [25] and collectedin peripheral blood stem cell collections used in autologoushematopoietic transplantation. Although standard mobiliza-tion schemes involving chemotherapy and cytokines, suchas granulocyte colony-stimulating factor can yield an eight-fold increase in circulating VPCs [8], the optimal conditionsfor collection of graft VPCs remain unclear. Additional cy-tokine stimulation using granulocyte-monocyte colony-stimulating factor, vascular endothelial growth factor [26],erythropoietin [27], estrogen [28], and 5-hydroxy-3-methyl-glutaryl-coenzyme A–reductase inhibitors [29] may also in-fluence VPC mobilization and collection. VPCs canparticipate in neovascularization as first demonstrated byAsahara et al. [30] in a mouse model of bone marrow trans-plantation, where the subsequent repair of hind limb ische-mia and myocardial infarction were observed in the murinerecipients. Takakura et al. [31] also demonstrated that endo-thelial progenitors mobilized from bone marrow to sites ofischemia differentiate into endothelial cells to form newvessels.

The precise definition and characterization of VPCs re-mains to be further clarified. Cell culture assays of endothe-lial-like clusters from blood or marrow may reflecta functional status of cells in the assay. The clonality of cellsin the clusters enumerated in this assay has not been estab-lished and the clusters may arise from aggregation of

511T. Iqbal et al. / Experimental Hematology 36 (2008) 506–512

heterogenous cells from multiple lineages. The correlation ofthe VPC cluster effect with various indicators of vascularhealth [18], however, underscores the relevance of the VPCassay. Results observed in our study, however, may not applyif a different VPC assay is used in place of the method by Hillet al. [18]. Ongoing efforts will continue to address theprecise etiology of cells giving rise to VPC clusters and theirrelevance in regenerative therapy. The characterization ofcellular subsets within hematopoietic grafts with the abilityto repair tissue injury has important potential implicationsfor cellular therapy of organ dysfunction.

Developing blood products for regenerative therapy willrequire greater insight regarding the precise mediators con-tributing to this possible beneficial effect. In order to reducethe toxicity of tissue injury associated with autologoustransplantation, cell therapy laboratories at transplantationcenters may consider assessment of graft VPCs, however,standardization of the methodology and additional studywill be needed. In the future, methods of augmenting graftcontent of VPCs may contribute to reduced toxicity in he-matopoietic transplantation and may allow older patients orthose with preexisting organ dysfunction to tolerate thetransplantation regimen. Studies in older patients, in partic-ular, would be most important.

AcknowledgmentsWe wish to acknowledge the assistance of Dr. Mukarram Ali Zaidifor assistance with statistical analysis and of Claire Yuhua Li fortechnical assistance with the immunohistochemistry. In addition,we wish to acknowledge the compassion of the physicians, nurses,and other health-care professionals involved in the care of thesepatients. Funding for this project was provided in part by TheOttawa Hospital Foundation, Bone Marrow Transplant Fund forResearch and Education and by Canadian Blood Services. Theauthors declare no conflicts of interest.

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