Experimental Hematology 28 (2000) 743–752

0301-472X/00 $–see front matter. Copyright © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.PII S0301-472X(00)00184-3

Standardization of hematopoietic stem cell assays: Asummary of a workshop and working group meeting sponsored

by the National Heart, Lung, and Blood Institute held at the NationalInstitutes of Health, Bethesda, MD on September 8–9, 1998 and July 30, 1999

Henry Chang

a

, LeeAnn Jensen

a

, Peter Quesenberry

b

, and Ivan Bertoncello

c

a

National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md., USA;

b

University of MassachusettsCancer Center, Worcester, Mass., USA;

c

Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne, Australia

(Received 8 November 1999; revised 15 March 2000; accepted 27 March 2000)

Introduction

In a 1981 review article, Herzig [1] concluded that therewas “...no reliable means of learning whether the hemopoi-esis reconstituting ability of a marrow with normal cellular-ity has been diminished by treatment received prior to ob-taining marrow samples for storage other than thedemonstration of the ability of transplanted marrow to re-store hemopoiesis after treatment that produces marrowaplasia.” Although significant advances in experimental he-matology have occurred in the 20 years since this review,Herzig’s statement largely remains true. Advances in ourunderstanding of the organization of the hematopoietic sys-tem and the development of powerful cell separation tech-niques and reagents to subset and hierarchically order thestem cell compartment have failed to produce consensusabout the best way to quantitate stem cells. Thus, a defini-tive assay to rapidly and reliably predict the long-term re-constitutive ability of stem cells in a clinical setting remainselusive.

To address these issues and identify obstacles that limitthe correlation among in vitro assays, in vivo assays, andtransplant outcomes, a stem cell assay workshop was held atthe National Institutes of Health on September 8–9, 1998, toreview the state of the art in surrogate stem cell assays. Par-ticipants at this meeting discussed the immunophenotypic,molecular, and functional characteristics of hematopoieticstem cells, with a view to the systematic establishment ofrational and reliable benchmarks for the rigorous compari-son and evaluation of surrogate assays. This initial work-shop was followed by a smaller working group meeting on

July 30, 1999, to develop a strategy for standardization ofsurrogate stem cell assays relevant to clinical transplantation.

In this summary of the workshop and working group pro-ceedings, the presentations and discussions of the participantshave been edited and reorganized for the sake of brevity andclarity. The participants are listed in the Appendix, and onlykey references to published work are given in the bibliography.

Description of the issues in stem cell assay development

To open the meeting, Peter Quesenberry summarized somemajor obstacles to the development of predictive stem cellassays. They may be characterized at the levels of:

(a) Intrinsic properties of the stem cell. Stem cell prop-erties may be selected for or altered merely by as-says. Stem cells may vary in their ability to enter cellcycle, to engage in asymmetric divisions, or to hometo and proliferate in the bone marrow.

(b) External influences. These factors may be soluble(e.g., cytokines) or cell-bound. Assay results also canbe affected by small perturbations in the cell cultureenvironment.

(c) Host factors. There may be immunologic disparitiesbetween the donor and the host and the transplantconditioning regimen may affect engraftment. Otherphysiologic influences include circadian rhythmsand estrous cycles.

(d) Definitions of engraftment. Short-term and long-term endpoints for hematopoietic reconstitution invivo are defined arbitrarily.

Ivan Bertoncello emphasized the impact of these factorsand the limitations of stem cell assays. Hematopoietic stemcells are operationally defined by their ability to sustain life-long, multi-lineage production of mature blood cells in a

Offprint requests to: Henry Chang, M.D., Division of Blood Diseasesand Resources, NHLBI, NIH, MSC 7950, 6701 Rockledge Dr., Room10170, Bethesda, MD 20892-7950, USA; E-mail: changh@nih.gov

744

H. Chang et al./Experimental Hematology 28 (2000) 743–752

steady state or after perturbation and by their capacity to re-generate the entire hematopoietic system long term aftertransplantation. Whereas a single cell or a few extremelyrare cells are able to fulfill these criteria, larger sets of prim-itive cells with varied proliferative histories, maturationalages, and differentiative potentials have been used to restore

life-long hematopoiesis when transplanted in large numbersin hosts treated with different preparative regimens. Sincethe engraftment potential of different subpopulations can bealtered by their intrinsic state as well as external factors, itmay be impossible to determine whether a surrogate assaymonitors or predicts the behavior of a compartment or indi-

Table 1. Glossary

Cell culture assaysCAFC cobblestone area forming cell (stroma-supported progenitor cell that displays a stem cell phenotype over 2–12 weeks in

culture)HPP-CFC high proliferative potential colony-forming cellLTC-IC long-term culture-initiating cell

Cell lines32D a murine, IL-3 dependent cell lineKG1a a human leukemia cell line that does not spontaneously differentiate to granulocytes or macrophages, or respond to

colony stimulating factors (CSFs)U-937 a histiocytic cell line derived from a lymphoma patient; [some stocks were contaminated with KG1 cells]

Cell surface molecules and markersCAS receptor crk-associated substrate (a focal adhesion protein) receptor [crk is a protooncogene in the tyrosine kinase pathway]CXCR4 a chemokine receptor for a stroma-derived factor (SDF-1)Ho Hoechst 33342 dye [low retention of this dye by stem cells]ICAM intercellular adhesion moleculeLFA-1 lymphocyte function-associated molecule-1lin lineage-associated antigens, e.g., CD2, CD3, CD10, CD11b, CD14, CD19, CD20, CD33, CD36, 7B9, and glycophorin-APECAM platelet-endothelial cell adhesion moleculeRho rhodamine 123 dye [low retention of this dye by stem cells]Sca stem cell antigen (also found on B-cells, stroma, embryonic stem cells, etc.)Thy-1 a stem cell antigen (also found on thymocytes, brain, and stroma cells)VLA-4 very late antigen-4 (an integrin)

Cytokines and protooncogenesEPO erythropoietinflk-2 fetal liver kinase-2flt-3 fms-like tyrosine kinase-3 (a.k.a. fetal liver tyrosine kinase)FL flt-3 ligandfms homolog to feline McDonough strain sarcoma virus (encodes receptor for M-CSF)G-CSF granulocyte colony-stimulating factorGM-CSF granulocyte-macrophage-colony-stimulating factorIL- interleukin-[followed by number]c-kit homolog to the Hardy-Zuckerman 4 feline sarcoma retrovirus (HZ4-FeSV); product of mouse W gene locus, binds SCFM-CSF macrophage-CSFMIP-1

a

macrophage-inhibitory protein-1 alphaSCF (KL) stem cell factor, c-kit ligand, or Steel factorSteel factor same as SCFTPO thrombopoietin

Developmental genes

D

a ligand that inhibits the notch receptor-bearing cell, but allows migration of the delta-containing celldishevelled a signal transduction gene involved in cell polarityjagged a ligand for the notch receptor that helps to maintain stem cellslunatic fringe a modulator of the notch signal pathway in segmentationnotch a surface receptor that can signal the nucleus to repress differentiationnumb a transcription factor that is asymmetrically distributed and may help to determine cell fate by interference with notch signalsscfr stem cell frequency regulator genes that control stem cell pool sizewnt a family of genes involved in cell fate determination in tissues

Mouse strainsNOD/SCID non-obese diabetic/severe combined immunodeficiencyW, W

v

white dominant spotting (v

5

viable

.

1 week to maturity). This mutation on chromosome 5 causes reduced pigmentation, sterility, and macrocytic anemia. The phenotype is similar to the steel (SL) mouse, but the W mutant has a defect in the c-kit protooncogene, a tyrosine kinase receptor for stem cell growth factor (which is defective in SL mice). W mice have severe anemia from day 12 of gestation and die within the first week of birth. Viable dominant spotting (W

v

/W

v

) mice can survive to maturity. Heterozygous (W/

1

or W

v

/

1

) mice have white spotting and are fully viable and fertile.

H. Chang et al./Experimental Hematology 28 (2000) 743–752

745

vidual cells within that compartment. Furthermore, surro-gate in vitro assays that measure the function of a smallsample often monitor the function of only a few highly se-lected cells from the total stem cell pool. Other phenotypicand functional characteristics, such as homing, engraftment,and renewal, may not be examined, so the ability to predictthe fate of transplanted cells and transplant outcomes is in-herently limited [2].

In addition to these issues, Ruud Hulspas described otherproblems with the standardization of assay techniques.There have been a wide variety of markers, protocols, andsurrogate assays, which should be correlated with the abilityof stem cells to proliferate and differentiate in vivo [3]. Cellpreparations may differ among laboratories because stemcell purification methods cannot be easily or precisely repli-cated. Failure to control variables such as mouse strains, an-tibody specificity or cocktails, buffer composition, prepara-tion time, and flow cytometry settings have made thecomparison of data impossible. Dr. Hulspas cited an exam-ple where two technicians processed and sorted aliquots ofthe same cell preparation following identical protocol butobtained different results. Slight discrepancies in the pH ofmedia and buffers or the overnight refrigeration of cells, forinstance, can alter cellular fluorescence or stem cell func-tion. For CD34

1

cell analysis, disparities of about 1 log in asingle sample are not uncommon when measured by differ-ent laboratories, yet variations of 10 to 20% or less arerequired for this assay to be useful. Major obstacles to stan-dardization of stem cell assays are the preference of investi-gators for their own assays and the difficulty of sharingtechniques and limited cell samples for cross comparison. Apotential solution to some of these problems is to include astandard or reference assay in published data.

In vivo assays

Measurement of long-term, multi-lineage hematopoietic re-constitution in vivo is used to validate other surrogate as-says of stem cell potential. While impractical for routineevaluation in clinical transplantation, competitive repopula-tion assays in mice have provided a rationale for the studyof the potential of human hematopoietic stem cells in NOD/SCID mice and fetal sheep.

David E. Harrison described a competitive repopulationassay in mice. Whereas long-term engraftment followinghuman transplants may take years to assess, this measure-ment requires 6 to 8 months in mice. In this assay, stemcells from a test donor mouse are mixed with a standard,congenic, “competitor” marrow, distinguished by geneticdifferences in hemoglobin and glucosephosphate isomerase(GPI). The mixture is infused into an irradiated, congenichost to determine the relative growth of the transplantedcells, whose counts stabilize after 21 days. After 3 to 4months, repopulation has been achieved by functional,primitive hematopoietic stem cells (PHSCs), proportional in

number to the bone marrow cells infused. This repopulatingability is expressed relative to the standard competitor as re-populating units (RUs), where each unit is equivalent to thatof 10

5

adult marrow cells. Since this method compares therepopulating ability of a group of cells, limiting dilution canbe used to estimate small numbers of functional stem cellsin a set of 20 or more mice with a Poisson model. Althoughthe total number of PHSCs stays constant with age, this as-say has revealed differences between different mousestrains [4]. Marrow cells from young BALB/cByJ andDBA/2J mice, for instance, repopulate better than cells fromolder mice, with relative values of 1.67 in fetal liver, to astandard of 1.0 in marrow from young animals, to .63 withmarrow from older animals. For C57BL/6J mice, the oldercells repopulate as well as younger ones, and when the re-cipient marrow is used for secondary transplants, there is lessloss of repopulating ability than with marrow from the formerstrains. Both the competitive repopulation and dilution as-says can measure long-term repopulating ability of geneti-cally marked PHSCs in vivo without lengthy cultures, andthe relative growth potential of these populations is com-pared to the same standard. The disadvantages for these as-says are that they require 3 to 6 months for completion andthey cannot be done in humans.

John Dick discussed the immunodeficient mouse assay,which is based on the reconstitution of nonobese diabeticmice with severe combined immunodeficiency disease(NOD/SCID mice) [5]. The SCID mouse-repopulating cells(SRCs) are more primitive than progenitors detected by invitro assays for colony-forming cells (CFCs) and long-termculture-initiating cells (LTC-ICs). SRCs are rarely trans-duced with retroviruses, which distinguishes them fromCFCs and LTC-ICs. By limiting dilution, the frequency ofSRCs is about one in 617 human cord blood CD34

1

CD38

2

cells, and 1 SRC transplanted into a mouse produces about400,000 progeny after 6 weeks. There is approximately oneSRC in 3.0

3

10

6

adult bone marrow cells, one in 6.0

3

10

6

mobilized peripheral blood cells from normal donors, and 1in 9.3

3

10

5

cord blood cells. When CD34

1

CD38

2

cordblood cells are cultured in serum-free media for 4 days, thetotal number of CD34

1

CD38

2

cells increases fourfold, witha 10-fold rise in CFCs and a twofold to fourfold increase inSRCs. However, longer expansion (for 9 days) results in theloss of all SRCs, despite further increases in total cell num-ber, CFC content, and CD34

1

cell numbers. The CD34

2

population, which does not proliferate in long-term cultures,also has been assayed in NOD/SCID mice for SRCs. Al-though such mice often develop thymomas after 6 months(which makes lengthy studies difficult), they can be repopu-lated long-term with CD34

2

CD38

2

lin

2

cells. However,the frequency of SRCs in these cells is low (about one in1.3

3

10

5

). To exclude the possibility of contamination byCD34

1

cells, CD34

2

cells were expanded in serum-free orhuman umbilical vein endothelial cell (HUVEC)–condi-tioned media for 4 days. With the latter media, fewer cells

746

H. Chang et al./Experimental Hematology 28 (2000) 743–752

became CD34

1

, yet the efficiency of engraftment improvedto 1 to 2–3

3

10

4

cells. It is not known if selective death ofdifferentiated cells or increased expression of homing mole-cules during culture led to these results.

Esmail Zanjani has developed a xenotransplant model inwhich the engraftment potential of human stem cells is as-sessed in fetal sheep. Recently, he examined the repopulat-ing potential of human CD34

2

cells [6] by transplantingthese cells into primary and secondary recipients and em-ploying a stem cell exhaustion strategy. In primary recipi-ents, human CD34

1

and CD34

2

cells persisted for about 20months posttransplant. As few as 75 CD34

1

CD38

2

cellswere required for engraftment, but upon expansion in vivo,the relative percentage of CD34

1

CD45

1

cells has been con-sistently higher for the group receiving CD34

2

grafts thanfor the animals receiving the CD34

1

grafts (9.6

6

2.8% vs2.4

6

0.7%, n

5

8). While human cell activity in the groupreceiving CD34

1

grafts remained relatively unchanged, asignificant increase in donor cells in the group receivingCD34

2

grafts started to occur 14 months post-transplant.The long-term potential of CD34

2

cells also was demon-strated by re-transplantation of human CD34

2

cells from thebone marrow of chimeric primary animals into secondaryrecipients, which resulted in multilineage engraftment thatpersisted for at least one year. The human cells in animalsthat became chimeric after receiving 4

3

10

4

CD34

1

cellswere depleted following four cycles of treatment with hu-man IL-3

1

GM-CSF, but no significant effect was seen inanimals that were chimeric after receiving 6

3

10

4

CD34

2

cells. These observations in primary and secondary hostssuggested that the latter cells can undergo self-renewal andmay be more primitive than the CD34

1

cell population.

In vivo determinants of stem cell number and function

Christa Muller-Sieburg discussed the effect of genetic di-versity on the size of the stem cell compartment [7]. In theoutbred human population, there is more than 100-fold vari-ation of LTC-ICs that cannot be explained by donor, age, orsex. For inbred mice, LTC-IC levels vary little within astrain, but inter-strain differences are dramatic. DBA/2 micehave the highest levels of LTC-ICs, and C57BL/6 micehave a far lower number. The strains 129 and FvB also havefairly high levels of LTC-IC. To examine the genetic basisfor variation in stem cell pool size, Dr. Muller-Sieburg stud-ied inbred mice and showed multigenic control of the phe-notype. Two candidate loci were identified, whose allelepatterns were significantly associated with the quantitativevariation in stem cell frequency. These loci have beennamed stem cell frequency regulator (Scfr) genes. Both arelocated on chromosome 1; each comprises about 10 centi-morgans, and both are syntenic to parts of chromosome 1 inhumans. Confirmation of the location of the Scfr-1 locuswas obtained through congenic mouse experiments. Thecongenic strain B6.C-H25 has a C57BL/6 background and

carries a segment of chromosome 1 derived from BALB/c.Current efforts are directed toward further mapping the lociby testing reduced congenics. In other experiments with chi-meric mice, Dr. Muller-Sieburg showed that intrinsic prop-erties of stem cells contributed 80% toward LTC-IC fre-quency, and the environment accounted for the rest.

Peter Quesenberry discussed the variations in stem cellphenotype and engraftment associated with cell cycle [8].Most marrow hematopoietic stem cells are quiescent, asshown by studies in which mice were fed oral bromodeoxy-uridine (BrdU) for a month. About 70% of the cells were la-beled, but about 30% of them did not cycle. Less than 2% oflin

2

Hoechst(low)/Rhodamine(low) [lin

2

Ho

low

Rho

low

] and10 to 15% of lin

2

Sca

1

murine cells were in S-phase; how-ever, more than 50% of the former were high-proliferativepotential colony-forming cells (HPP-CFCs). Exposure ofHPP-CFCs to IL-3, IL-6, and IL-11,

6

Steel factor drovethem into S-phase as shown by incorporation of

3

H-thymi-dine. The first cell division took 36 to 40 hours with morethan 60% of cells in S-phase, but each of five subsequent di-visions occurred every 12 hours. If male mouse marrow wasstimulated with cytokines for various periods and injectedwith fresh female marrow into an irradiated BALB/c mouse,a competition assay revealed cell cycle variation in engraft-ment potential. Engraftment was better when male cellswere cultured for 40 hours (presumably G

1

), but poorer at32 hours (presumably late S/early G

2

). A defect in stem cellhoming may explain these observations, but the results aresupported by studies where 5-fluorouracil (5-FU) was usedto treat murine marrow donors before transplantation intononmyeloablated hosts. After 6 days of 5-FU, the marrowengrafted poorly, but the marrow repopulating potential re-turned to normal by 12 to 35 days after 5-FU treatment. Inother experiments, male mouse marrow was transplantedinto female nonablated hosts, followed by a short dose ofhydroxyurea (900 mg/kg) at 0, 3, 6, 12, and 15 hours. Onlyat the 12-hour point were more than half of the cells thatcontributed to long-term hematopoiesis killed. Presumablythey were in S-phase and sensitive to hydroxyurea. Thus,variations in stem cell cycle can affect PHSC assay, cellharvesting, and engraftment outcomes.

David Bodine described the use of competitive repopu-lation assays to define stem cell populations for gene thera-pies. Long-term repopulating cells were characterized aslineage negative (lin

2

), c-kit

hi

cells. Approximately 30 to 50of these cells were able to reconstitute a w/w

v

congenicmouse, and about 500 of these cells were required to com-pete almost equally with 2

3

10

6

unfractionated bone mar-row cells. Large stem cells contained more amphotropicretroviral receptor mRNA than small cells, which madethem more amenable to nonspecies-specific transduction[9]. However, receptor mRNA in the small cells could beupregulated sixfold to 10-fold by incubation with IL-3 (10ng/mL), IL-6, and SCF for up to 144 hours. Transfected hu-man cells also can be assayed in NOD/SCID mice, but ex-

H. Chang et al./Experimental Hematology 28 (2000) 743–752

747

tinction of expression of transduced genes remains a prob-lem. This effect may be lessened by the use of internalpromoters, rather than reliance on the retroviral LTR. Hu-man progenitor cells are hard to transduce, but Dr. Bodinehas found that frozen and thawed cord blood has a 10-foldhigher level of amphotropic retroviral receptor mRNA thanfresh samples.

Stroma-based in vitro assays

Rob Ploemacher discussed in vitro stroma-based assays forstem cells, which may be more reliable than phenotypicanalyses [10]. These assays were developed because stemcells proliferate better with stromal contact or in stroma-conditioned medium (SCM), irrespective of the addition ofcytokines. He tried to correlate cobblestone-area-formingcell (CAFC) numbers with in vivo stem cell assays thatmonitor spleen colony formation (CFU-S), marrow-repopu-lating ability (MRA), and long-term repopulating ability(LTRA) using sex-mismatched hemopoietic chimerism. Heshowed that cells selected by wheat germ agglutinin (WGA)lectin were not able to initiate long-term in vitro cultures ona stromal layer in vitro, but could produce CFU-S at day 12.The WGA-dim cells contained the repopulating ability, witha CAFC frequency at 4 to 5 weeks of culture that correlatedwith LTRA at 6 to 12 months post-transplant. These isola-tion conditions allowed for a 590- to 850-fold enrichment ofLTRA over normal bone marrow. It has not been possible totake any single cobblestone area and reconstitute an irradi-ated animal, probably because most of the primitive stemcells that produce the cobblestone area lose their repopulat-ing ability. These functional assays, both in vitro (CAFC,LTC-IC) and in vivo (repopulation of NOD/SCID mice),have not been validated in the human transplant setting. Dr.Ploemacher also used these methods to study human cordblood. He pooled cord blood samples and cultured themfor 2 to 12 weeks in Flt-3L, SCF, TPO, and IL-6. TheCD34

1

CD38

2

cells (which also contained CD34

1

CD19

1

pre-B-cells) underwent 10

3

- to 10

6

-fold expansion, but thenumber of CAFCs increased only 10- to 100-fold after 6weeks. When 2-week cultures were injected into NOD/SCID mice, the number of cells required to repopulate themafter 6 weeks fell from 100,000 to less than 10,000. Thus,this assay may be helpful in validating the functional capac-ity of stem cell expansion products.

Catherine Verfaillie described a method to identify sin-gle cells that have the capacity for self-renewal and multi-lineage differentiation. Called the myeloid-lymphoid initiat-ing cell assay, it adapts the LTC-IC protocol with alymphoid differentiation step [11]. Single human bone mar-row CD34

1

lin

2

HLA-DR

2

or CD38

2

cells were obtainedby FACS and cultured on stroma from AFT024 murine fetalliver cells in 96-well plates. The cytokines Flt-3L, IL-7, andSCF were added to the medium for 4 to 6 weeks. Then thecontents of each well were trypsinized and split into eight

parts, with each part placed into corresponding wells ofeight secondary plates. Four plates were given myeloidstimuli, and four were given lymphoid stimuli. After an-other 5 to 7 weeks, the secondary colonies were tested formyeloid or lymphoid markers, such as CD15 (myeloid),CD56 (NK cells), CD19 (B cells), CD1a or CD11b (den-dritic cells), and T-cell epitopes. This assay demonstratedthe existence of multipotent progenitor cells, which areabout 10 times less frequent than the usual LTC-ICs. Theexperiments take about 15 weeks and are difficult to per-form because of the risk of contamination.

Beverly Torok-Storb described an approach to dissectthe role of stroma in stem cell assays [12]. Because the mar-row microenvironment is complex, her overall strategy hasbeen to clone functionally distinct stromal cell lines and toexamine their gene products. In addition to ELISA assays,she has collaborated with a biopharmaceutical company(Genetics Institute, Cambridge, MA, USA) to screen for dif-ferential expression of about 250 human genes with oligo-nucleotide arrays. Two human cell lines were described.The first one, HS-5, was immortalized with a replication-defective recombinant retrovirus (LXSN-HPV16 E6E7),which contained human papillomavirus E6/E7 genes. Thesegenes interfere with the tumor-suppressor proteins p53 andretinoblastoma (Rb) and prevent cell cycle arrest withoutsignificant transformation. HS-5 cells resemble fibroblasts,lack contact inhibition, and produce extracellular matrix.They secrete G-CSF, GM-CSF, M-CSF, MIP-1, IL-1, IL-6,IL-8, and IL-11. Media conditioned with these cells supportthe proliferation of hematopoietic progenitors, but the great-est increase in CFU from CD34

1

cells is seen when kit-ligand (KL) or flt3-ligand (FL) is added. When humanCD34

1

CD38

2

cells are cultured in HS-5–conditioned me-dium plus 100 ng/mL FL for 15 days and injected into fetalsheep, they engraft and can be transplanted serially. A clini-cal protocol to expand CD34

1

cells for autotransplantion isin progress. Another cell line, HS-27a, does not support thegrowth and differentiation of CD34

1

38

lo

cells in serum-freemedia and does not secrete detectable levels of cytokines.However, it does promote the formation of “cobblestone”areas. HS-27a expresses the human gene hJagged1, whoseproduct can bind to the notch1 receptor to repress differenti-ation. Contact between HS-27a and the hematopoietic pre-cursor 32D cell line also activates notch1 and preventsG-CSF–induced differentiation. These cells may be used tostudy notch/jagged signaling and possibly to identify pep-tides or antibodies that can function as agonists or antago-nists of this pathway.

Clonogenic in vitro assays

Ivan Bertoncello described the advantages and limitationsof the HPP-CFC assay as a surrogate measure of stem cellpotential [13]. HPP-CFC are defined operationally by theirrelative resistance to near-lethal doses of 5-FU, their obliga-

748

H. Chang et al./Experimental Hematology 28 (2000) 743–752

tory requirement for multiple cytokines, and their formationof macroscopic colonies (

.

.5 mm diameter with

>

50,000cells). In mice, HPP-CFCs co-fractionate with PHSCs andregenerate after 5-FU treatment, similar to PHSCs withlong-term reconstitution potential. Although experimentsshow that HPP-CFCs are among the most primitive hemato-poietic cells detected in clonal agar culture and are a reliablesurrogate assay of stem cell activity, investigators need tobe aware of their limitations. HPP-CFCs are heterogeneousand comprise a hierarchical order of at least four sub-popu-lations of primitive hematopoietic progenitors characterizedby their growth factor preferences [14]. Consequently,whereas all PHSCs are HPP-CFCs, not all HPP-CFCs arestem cells. The most primitive HPP-CFCs, which areclosely related if not identical to PHSCs, require three toseven cytokines to express their full potential. On the otherhand, HPP-CFCs stimulated by mixtures of two cytokinesare relatively mature and are the immediate precursors ofcommitted progenitor cells. Some investigators fail to ap-preciate that the presence of HPP-CFCs, grown in the pres-ence of multiple cytokines, does not necessarily indicate anobligatory requirement for each of these cytokines. In orderto determine precisely the nature of HPP-CFCs, one mustanalyze their growth in the presence of each permutationand combination of cytokines. Sometimes colony size canbe an unreliable index of HPP-CFC content and stem cellpotential. During bone marrow regeneration in vivo or ex-vivo expansion of stem cell progeny, the kinetic status ofcells in the assay may lead to the generation of large colo-nies, simply because colony-forming cells are more likely togrow more rapidly. Growth factor preferences of these cellsneed to be determined carefully. Likewise, culture condi-tions and overplating of cells can lead to underestimation ofHPP-CFC content. Despite these caveats, the HPP-CFC as-say seems to be one of the most informative, reliable, andversatile short-term in vitro assays of stem cell potential.

Stem cell characteristics

A major problem in the identification of stem cells is thatthey are considered to be quiescent until triggered to prolif-erate. Jeffrey Moore discussed this stem cell phenotype asdefined by a new legume lectin called FRIL [15]. FRIL (Flt-3receptor-interacting lectin) was identified by its ability tostimulate proliferation of 3T3 fibroblasts transfected withthe Flt-3 receptor, but not those with the related fms recep-tor or untransfected cells. When growth conditions of these3T3 cells were designed to permit only Flt-3 receptorligands to rescue cells from death, fractionation and purifi-cation of phytohemagglutinin-leukocyte–conditioned me-dium (PHA-LCM) showed that the new agent came fromred kidney bean extracts (which also contain the mitogenPHA). However, in contrast to PHA and other mitogeniclectins, FRIL did not stimulate the secretion of IL-6 or othercytokines or exhaust the culture medium by inducing cell

proliferation and differentiation. Instead, a small populationof cells in a dormant state usually persisted at the end of theculture period. FRIL preserved human cord blood progeni-tor cells in suspension cultures for up to a month withoutexogenous cytokines or stroma and maintained the numberof cord blood SCID repopulating cells (SRC) for 3 to 13 daysin suspension culture. If the latter cells were transplantedinto NOD/SCID mice, they gave rise to lymphoid, myeloid,and erythroid progeny. Subsequent exposure of FRIL-cul-tured cells to combinations of early-acting cytokines (with-out FRIL) expanded the number of total mononuclear cells,progenitors, and possible SRC. The eventual clinical appli-cations may include: (1) synchronization of the stem cellcycle to improve gene transfer and later engraftment, (2) ex-pansion of the number of stem cells and improvement oftheir quality for autologous and allogeneic transplantation,and (3) protection of stem and progenitor cells from the tox-icity of S-phase specific drugs for cancer, to allow for higherdoses or longer duration of chemotherapy.

Anthony Ho examined asymmetric cell divisions of he-matopoietic progenitors, in which one daughter cell remainsa stem cell, but the other becomes committed to differenti-ate [16]. He used index sorting of various CD34

1

subsetsand time-lapse photography with the cell membrane label-ing dye, PKH26, to monitor divisional history and corre-lated it with growth pattern and cloning efficiency. The flu-orescence intensity of this dye is reduced by half followingeach cell division and can be used to gauge replication history.The first mitosis of fetal liver CD34

1

CD38

2

cells occurred af-ter 36 to 38 hours, but then, in about 20–30% of cells, asym-metric divisions took place every 12 hours. The cells mayhave moved apart slightly. With progressive ontogenicsources of CD34

1

CD38

2

cells, the percentage of cellsthat underwent asymmetric division dropped. When CD34

1

CD38

2

fetal liver cells were exposed to single cytokinessuch as FL, TPO, and SCF, and cell divisions were moni-tored every 3 to 12 hours for up to 8 days, cloning efficiencydecreased (

z

9–30%). A cocktail of SCF, IL-3, IL-6, GM-CSF, and EPO was better (

z

68% cloning efficiency), butthe ratio of asymmetric divisions versus the total number ofcells still fell. However, the fraction of asymmetric divi-sions compared to the total number of divisions (asymmet-ric division index

5

ADI) remained constant at about 40%.The results indicated that mitotic rate and cloning efficiencycould be altered with various combinations of growth fac-tors, but that the ADI is not. This observation suggest that,although the pattern of commitment can be skewed by ex-trinsic signals, the proportion of cells undergoing asymmet-ric divisions is determined by intrinsic factors.

Henry Chang suggested that an assay for stem cells couldbe based on the hypothesis that stem cells have a full com-plement of DNA, as manifested by long telomeres. It maybe difficult to detect this property and then select viablecells to perform functional stem cell assays. Current meth-ods allow the reverse, flow cytometric isolation of stem

H. Chang et al./Experimental Hematology 28 (2000) 743–752

749

cells based upon surface markers, followed by an assay fortelomere length of single non-viable cells. Since telomerestend to shorten with each mitosis, an alternate strategywould be to use stem cell quiescence as a surrogate stemcell characteristic. Donor cells could be stained with stablybound vital dyes, such as PKH26 or PKH2, which do not re-quire mitosis for incorporation and which become dilutedwith cell division. The cells would be cultured and coloniesharvested to identify quiescent cells that have not divided.After sorting the cells the most highly fluorescent would becandidate stem cells since they would have divided theleast. A similar approach has been reported where hemato-poietic stem cells were pre-selected by incubation of humanmarrow with IL-3 and kit ligand, and the stimulated cellswere killed by 5-FU [17]. The cells that retained PKH26fluorescence were CD34

1

and c-Kit

1

and required a stromalfeeder layer to proliferate in vitro.

Cell homing and migration

A difference between the data generated using in vitro as-says and that from in vivo assays may be due to the fact thatthe latter assays also depend on homing of the PHSC to thebone marrow. Homing may be difficult to distinguish fromcell trapping. Visser and colleagues isolated progenitor cellswith wheat germ agglutinin and labeled them with PKH26before injection into mice [18]. The cells were found in thespleen and bone marrow, but surprisingly, about 75% ofthem were not accounted for. This loss occurred within 1day, so it probably was not due to stem cell differentiationinto nonhematopoietic tissues. Further research is needed tounderstand this phenomenon. Dye-stained, sca-1

1

, c-kit

1

,lin

2

cells that homed to the spleen could be transplanted andcould repopulate secondary recipients faster than those thathave homed to the marrow [19], but the latter cells (pre-pared by elutriation) appeared to possess long-term recon-stituting ability [20]. Also, one expansion protocol dimin-ished the homing of dye-stained cells 10-fold. In the future,it would be of interest to find nascent colonies in tissue sec-tions, isolate them with techniques such as laser-capture mi-crodissection, and characterize the most highly stained cellsmolecularly.

Bernhard Palsson has begun to look at the effect of stemcell migration with a custom-built automated time lapse mi-croscope system (ATLMS). It includes a fluorescence mi-croscope with infinity-correlated objectives and a motorizedstage with micromanipulators [21]. The system is enclosedin a sealed plastic chamber with proportional/integral/deriv-ative (PID) temperature control and a CO

2

sensor. A cooledcharge coupled device (CCD) camera captures the imagesto be processed on a computer workstation. Dr. Palssonshowed pictures of CD34

1

cells extending processes up tohundreds of microns long that could be visualized withPKH26 or PKH2 fluorescent dyes. At any time, about 5% ofprimary CD34

1

cells, but up to 60 to 70% of the human leu-

kemia cell line, KG1a cells, extend these long podia. Thisphenomenon likely depends on microenvironmental vari-ables, such as distance to neighboring cells and local cyto-kine or substrate concentrations. The podia display inte-grins, including CD11a, CD18, CD29, CD49d, and CD49e,plus other adhesion molecules, such as CD44, CD54, andCD62L. They formed on surfaces coated with fibronectin,laminin, and collagen IV, but not as well on plastic. Eventhough their function is not understood, these cell podiacould be important for sensing gradients. A migration assaywas developed by tilting the microscope 7

8

to watch KG1acells migrate uphill. After a fixed time, the cells that had mi-grated past a defined “finish line” were counted. This assaywas used to compare the effects of various cytokines andsubstrates and showed that the cells actively migrate unlessthey are preparing to divide. The setup may be used to studyasymmetric cell divisions in addition to interactions withstromal cells.

Malcolm Moore spoke about stem cell motility, whichhelps infused cells find their way to the bone marrow. Dr.Moore has used transwells with 3

m

m pores to develop anassay, where a human marrow-derived endothelial cell line(BMEC-1) is grown to confluence in the top chamber, and amouse stromal cell line (MS-5) is layered in the bottomchamber as a source of the chemokine, stroma-derived fac-tor-1 (SDF-1) [22]. About 5 3 104 CD341 cells isolatedfrom peripheral or cord blood are added to the top chamberfor 3, 12, 24, or 48 hours, and the cell number, the progeni-tor cells, and the CAFC content of all the cells that passthrough the chamber are enumerated. The migratory cellscomprised about 50 to 60% of the input and were mostlyBFU-E’s. Dr. Moore found that the rate of transit correlatedwith the expression of CXCR4 receptors on CD341 CD382

cells. Recombinant SDF-1 (100 ng/mL) also can be placedin the lower chamber to produce chemotaxis, and an anti-body to the CXCR4 receptor (12G5) will block this effect.A comparison of cord blood cells and G-CSF–mobilized pe-ripheral blood cells showed that the former were more effi-cient in migration, 75% vs 40% over 48 hours, respectively.However, the expression of CXCR4 was only slightlyhigher in cord blood than in adult blood, so it is likely otherfactors are involved in stem cell migration. The expressionof SDF-1 or unique sinusoid adhesion molecules has notbeen determined in bone marrow. Knockout mice for SDF-1and CXCR4 are characterized by hematopoiesis in the liver,but not in bone marrow, which supports the role of thesemolecules in stem cell migration. The effect of cytokines onstem cell motility also was examined. When progenitorswere expanded in vitro for 7 to 14 days with FL, KL, andTPO, migration increased twofold. However, if IL-3 (50 ng/mL) was added, committed progenitors were increased, butthere was a 30% drop in chemotaxis as compared to con-trols. The clinical outcome of engraftment may therefore bebased on a complex relationship between homing and pro-liferation of stem cells.

750 H. Chang et al./Experimental Hematology 28 (2000) 743–752

Stem cell gene expressionRecent advances in genomics, bioinformatics, and cDNAmicroarray technologies have provided an opportunity tomolecularly define stem cells on the basis of specific geneticmarkers or unique gene expression patterns. Ihor Lemischkadescribed the analysis of gene expression in hematopoieticstem cells. His laboratory has built a comprehensive databaseof molecular phenotypes of stem cell and its microenviron-ment, for both humans and mice. It uses conserved stem cellproperties as an approach to identify relevant regulatorymolecules. Differential gene expression technologies, such assubtraction hybridization, differential display, and high-densitymicroarrays, were coupled with sophisticated bioinformaticstools to study highly purified stem cells [23]. Murine fetalliver (AA4.11 lin2/lo Sca-11 c-kit1) cells and murine bonemarrow (Rholo or Rhohi plus Thylo lin2 Sca-11 c-kit1) cellswere used, along with human bone marrow (CD341 lin2

CD382) cells. Full-length stem cell cDNA libraries weregenerated and depleted of sequences from more maturehematopoietic cells. These subtracted libraries were enrichedup to 200-fold for uniquely expressed gene products andwere analyzed by high-throughput DNA sequencing fromthe 59 end. These DNA and derived protein sequences con-stitute a stem cell database, which contains approximately4,000 entries to date. Bioinformatic analysis has revealed: 1)homologies to regulatory molecules in developmental path-ways, including those of invertebrates, 2) members of relatedprotein families, e.g., cell-surface receptors and DNA-bindingfactors, and 3) potential roles in biological processes, suchas apoptosis and cell signaling. It also has been possible toobtain virtual expression profiles with Expressed SequenceTag (EST) databases, which are now publicly available forabout 20 to 40% of the potential coding regions of themouse and human genomes. Clusters of unique ESTs havebeen arrayed at high density (.18,000 unique sequences)on commercially available membranes. Hybridization wasperformed with cDNA probe populations obtained fromRholo, Rhohi, and lin1 cells purified from murine bone marrow,which represent primitive, intermediate, and mature he-matopoietic cells, respectively. The procedures were optimizedfor as few as 10,000 to 20,000 cells. Comparative hybrid-izations were performed with the Rholo vs the Rhohi and theRholo vs lin1 subsets, using total complex cDNA as probes.Multiple differences in gene expression were identified. Al-though the chips are quantitative to at least fivefold, the sen-sitivity of the approach can be increased by PCR-based sub-traction to generate less complex probe populations, enrichedfor specifically expressed sequences. The substractionswere bidirectional, to obtain divergent probe populations forboth primitive and more mature cells. Hybridization of theseprobes to high-density arrays revealed mutually exclusivepatterns and uncovered many more differentially expressedgene products. Venn diagrams of gene expression patternscan be constructed for Rholo sequences not expressed inRhohi or in lin1 subsets. Linkage to cell cycle genes in yeast

and developmental molecules such as wnt 5⁄10, notch, di-sheveled, and lunatic fringe in Drosophila was noted.

Jan Visser compared the gene expression in CD341 andCD342lin2kit1sca1 murine cells [24]. He used a variety ofassays, such as differential display, RT-PCR, and gene ex-pression fingerprint analysis to identify, extract, and clonespecifically expressed cDNAs. About 1,000 stem cells gave2 to 3000 bands, which represented about 10,000 genes ex-pressed. Long-term repopulating cells (mostly CD342)were identified by limiting dilution, and more than 2,000 oftheir expressed genes were screened. Of 22 genes that wereunique to long-term repopulators, tissue transglutaminase,type II was expressed exclusively in the earliest stem cells.Further progress depends on cell purity, good directionaldatabases, and the ability to associate genes with the 39 un-translated regions and alternative splice forms of the RNAs.Dr. Visser also tested about 60 other known genes for ex-pression in stem cells and found that CD3-epsilon wasturned off in short-term repopulating cells. Further studieswith a panel of such markers are needed to establish clinicalutility.

An approach tostandardization of surrogate stem cell assaysThe workshop defined the need for researchers to collabo-rate and compare the results of molecular, cellular, and ani-mal assays with engraftment data. A working group wasformed and held a roundtable discussion of July 30, 1999.As a first step, a practical approach was proposed to stan-dardize the measurement of engraftable human hematopoi-etic cells.

Differences between three major cell sources—bonemarrow, mobilized peripheral blood, and umbilical cordblood—were considered with regard to how they could beassayed to predict clinical outcome. Bone marrow may havea higher content of accessory cells that need to be character-ized qualitatively and quantitatively to determine their ef-fect on stem cell proliferation and quiescence. When in-fused, the donor cells could interact with host stromavariably, depending on factors such as histoincompatibilitystatus or cytokine release following a myeloablative condi-tioning regimen. Since bone marrow aspirates from livingdonors are limited in quantity and are frequently diluted byperipheral blood, cadaveric marrow samples were proposedfor such studies. However, this material is unsuited for hu-man transplants and may limit correlation of in vitro with invivo data.

The second PHSC source was human peripheral blood,mobilized by treatment with G-CSF or GM-CSF and har-vested by apheresis. Even if normal donors are treated uni-formly, their mobilized products are not equivalent, whichprobably reflects genetic or cyclical differences in the do-nor. A group of donors should be studied to account for in-dividual variation. Mobilized peripheral blood may contain

H. Chang et al./Experimental Hematology 28 (2000) 743–752 751

more mature precursor cells and tends to engraft earlier thanbone marrow, but this stem cell source should provide suffi-cient cells for testing.

The third stem cell source considered was umbilical cordblood, which is readily available. Because samples aresmall, they cannot be distributed widely and specimens mayhave to be pooled.

At the 1998 meeting, the variation in surrogate humanstem cell assay endpoints as compared to animal modelswas mentioned. Not only are there large variations amongindividuals, but significant differences are noted when sam-ples from the same person are assayed. Since cell cycle sta-tus and other factors are not known at the time of collection,at least 8 to 12 donors in each category may be needed tocompare different assay systems. It was felt that five large,individual samples of cadaveric marrow, five samples ofmobilized peripheral blood, and possibly five large pools ofcord blood could be obtained initially from both clinical andcommercial sources. These samples would be frozen inDMSO and stored centrally. This repository later could beexpanded to include fetal liver, purified stem, and accessorycell populations. Standardized reagents (e.g., antibodies,GMP-quality biologics) should be utilized for the perfor-mance of assays in the participating laboratories. After dis-tribution, each cell aliquot would be checked for viabilitybefore use.

Although assays may differ between laboratories, an im-portant goal would be to correlate the results at each sitewith transplant outcome. Xenogenic transplant models inmice and fetal sheep would be included because competi-tive repopulation and limiting dilution studies are possible.Statistical analysis of the data would be performed to try toidentify the most predictive assays. At a later stage, the bestassays could be standardized. Training courses, an interac-tive Web site, or the exchange of laboratory personnel couldbe provided to instruct investigators in the performance ofthe assays of choice.

AcknowledgmentsWe would like to thank Barbara Alving and Helena Mishoe for re-view of the manuscript and suggestions.

The opinions expressed in this paper are not those of the N.I.H.or the U.S. government.

Appendix

The speakers for the first meeting on September 8–9, 1998,were: Peter Quesenberry, M.D., from the University ofMassachusetts Cancer Center in Worcester, MA, USA; andIvan Bertoncello, Ph.D., of the Peter MacCallum Cancer In-stitute, Melbourne, Australia (Co-Chairpersons); David Bodine,Ph.D., from the National Human Genome Research Institutein Bethesda, MD., USA; John Dick, Ph.D., of the Hospitalfor Sick Children in Toronto, Canada; David E. Harrison,

Ph.D., from the Jackson Laboratory in Bar Harbor, ME.,USA; Anthony D. Ho, M.D., Ph.D., from the MedizinischeKlinik und Poliklinik V in Heidelberg, Germany; RuudHulspas, Ph.D., from the University of Massachusetts MedicalCenter in Worcester, MA, USA; Ihor Lemischka, Ph.D.,from Princeton University in Princeton, NJ, USA; JeffreyMoore, Ph.D., from Phylogix in Scarborough, ME., USA;Malcolm Moore, Ph.D., from Memorial Sloan-KetteringCancer Center in New York, NY, USA; Christa Muller-Sieburg, Ph.D., from the Sidney Kimmel Cancer Center inSan Diego, CA, USA; Bernhard Palsson, Ph.D., from theUniversity of California in La Jolla, CA, USA; RobPloemacher, Ph.D., from the Erasmus University in Rotterdam,the Netherlands; Beverly Torok-Storb, Ph.D., of the FredHutchinson Cancer Research Center in Seattle, WA, USA;Catherine Verfaillie, M.D., of the University of MinnesotaHospital Center in Minneapolis, MN, USA; Jan Visser,Ph.D., from the New York Blood Center in New York, NY,USA; and Esmail Zanjani, Ph.D., from the Veterans AffairsMedical Center, Reno, NV, USA.

A working group met on July 30, 1999, and includedDrs. Stephen Bartelmez, Ph.D., from the Seattle BiomedicalResearch Institute in Seattle, WA, USA; Ian McNiece,Ph.D., from the University of Colorado Health SciencesCenter in Denver, CO, USA; Makio Ogawa, M.D., Ph.D.,from the Medial University of South Carolina, Charleston,SC, USA; Robertson Parkman, M.D., from the Children’sHospital of Los Angeles, Los Angeles, CA, USA; and Ger-ald Spangrude, Ph.D., from the University of Utah MedicalCenter, Salt Lake City, UT, USA; as well as Drs. Quesen-berry, Bertoncello, Bodine, Hulspas, Lemischka, M. Moore,Muller-Sieburg, Torok-Storb, Verfaillie, Visser, and Zan-jani from the previous meeting.

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