CHAPTER 133

Hematopoietic Cell Culture

CHRISTINE S. OLVER

History of Hematopoietic Culture Long Term Bone Marrow Culture

Types of Hematopoietic Culture Long Term Culture Initiating Cell Assay
Progenitor Assays Growth Factors Used in Hematopoietic Culture

Acronyms and Abbreviations

BFU-E, burst-forming unit-erythroid; BMMC, bone marrow mononuclear cells; CFU, colony-forming unit; CFU-E,
colony-forming unit-erythroid; CFU-GEMM, colony-forming unit-granulocyte, erythroid, macrophage, megakary-
ocytic; CFU-GM, colony-forming unit-granulocyte/macrophage; EPO, erythropoietin; G-CSF, granulocyte colony
stimulating factor; GM-CSF, granulocyte/macrophage-colony stimulating factor; HC, hematopoietic cell; LTBMC,
long term bone marrow culture; LTC-IC, long term culture initiating cell.

HISTORY OF HEMATOPOIETIC CULTURE

H
ematopoietic cell (HC) culture involves the cul-
tivation of primary cells; those that have been
derived directly from the host. Sources of HC
include bone marrow, peripheral blood mononuclear
cells, spleen (rodents) and fetal liver (rodents). Generally,
HC culture is used to study progenitor cells, which are
lineage committed but morphologically indistinguish-
able cells. Because progenitors cannot be identified
microscopically, their descendents need to be studied
and identified in order to retroactively identify the pro-
genitor. This is done by culturing in a semi-solid
medium under conditions that allow a single progeni-
tor to form a clone of differentiated cells. Thus, progeni-
tor cells are also called colony-forming units (CFU).
Hematopoietic cell culture can enumerate these pro-
genitor cells in a crude suspension of hematopoietic
tissue cells. Besides enumeration, HC culture is used to
study the physiology, differentiation mechanisms and
growth factor control of hematopoiesis. Additionally,
liquid based HC culture is employed to establish long-
term cultures that recapitulate the microenvironment of
in vivo hematopoiesis. It is also used to expand either
lineage-committed cells or undifferentiated stem cells
for transplantation. This chapter describes the history
of HC culture, a description of its methods and uses,
the types of resulting cells, and the growth factors
required.
The discovery of progenitor cells in hematopoietic
tissue occurred almost 50 years ago8 using both in vitro
and in vivo assays. The colony-forming unit-spleen
(CFU-S) assay was originally developed in the early
1960s7,9 as a way to identify and enumerate bone marrow
progenitor cells. The CFU-S assay provided important
information on the nature of both hematopoietic pro-
genitor cells and less differentiated (and more pluripo-
tent) hematopoietic stem cells. Progenitor/stem cells
from bone marrow mononuclear cells (BMMC) injected
into lethally irradiated mice migrate to the spleen and
form macroscopic colonies of variably differentiated
hematopoietic cells. One cell is capable of forming a
colony of 106 differentiated cells, indicating that the
original cell is capable of massive division as well as
differentiation. These assays provided evidence that
these cells are of low frequency in bone marrow suspen-
sions (1 per 104 bone marrow cells), have no distinctive
morphologic features, have extensive proliferative and
differentiation capacity, and are capable of limited
self-renewal.
In vitro clonal progenitor assays were developed
shortly after CFU-S assays.1,5 Initially only granulocytic
progenitors were cultured, but then conditions for
erythroid colony formation were developed. A liquid
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1048 SECTION X: QUALITY CONTROL AND LABORATORY TECHNIQUES
culture system permitting long term culture of bone
marrow progenitors with an adherent stromal feeder
layer, termed long term bone marrow culture was first
developed by Dexter et al.4
TYPES OF HEMATOPOIETIC CULTURE
Progenitor Assays
Progenitor assays are also known as clonogenic assays
(Fig. 133.1). Hematopoietic mononuclear cells (derived
from blood, bone marrow, fetal liver or spleen) are cul-
tured in a semi-solid medium so that one dividing and
differentiating cell remains in one location as a colony
of variably differentiated cells. For example, an eryth-
roid progenitor will produce a colony containing
FIGURE 133.1 Schematic showing a typical
progenitor assay. (Courtesy of STEMCELL
Technologies.)
varying numbers of rubriblasts, rubricytes, and metaru-
bricytes. These colonies can be counted microscopically
in the culture dishes and are identified by their particu-
lar morphology (Fig. 133.2). Mature erythroid colonies
will appear orange-red because of their hemoglobin
content. The semi-solid medium may consist of agar,
methylcellulose, agarose, or plasma or fibrin clots. In
general, larger colonies of a single type are derived
from less differentiated progenitors (i.e. more prolifera-
tive potential). Also, mixed colonies (i.e. those with
morphologies of more than one lineage) are derived
from less differentiated progenitors (multipotential).
The enumeration of progenitors from a mixture of
HCs is usually done as a percentage of the original
cells plated, rather than as an absolute number
Table 133.1 lists and describes the various progenitors
derived from progenitor cell assays.
step 1
Prepare Cells
Process human cells by:
† ammonium chloride lysis
† density gradient separation
† progenitor cell enrichment with
EasySep®, StemSep®, RosetteSep® or
FACSorting (e.g. CD34+)
Wash cells (e.g. in Iscove’s MDM plus 2% FBS),
then count and adjust cell concentration.
2
MethoCult®
step
Add Cells to MethoCult®
Add cells to MethoCult® and vortex.
step 3
Plate and Incubate
Dispense cells into pre-tested petri dishes using
syringe and blunt-end needle. Incubate human
cells for 14–16 days in humidified incubator at
37∞C and 5% CO2.
step 4
Count Colonies
Count and evaluate colony types using inverted
microssope and gridded scoring dishes.
Alternatively, individual colonies may be plucked
for routine staining, PCR, or cytogenetic
analysis.
 CHAPTER 133: HEMATOPOIETIC CELL CULTURE 1049

TABLE 133.2 Some Hematopoietic Cytokines and their

Functions

Cytokine/
Growth Predominant Progenitors Other Cells
Factor Stimulated Affected

M-CSF Macrophages Primitive

progenitors
GM-CSF Granulocytes/macrophages Primitive
progenitors
G-CSF Granulocytes Primitive
progenitors
IL-3 Granulocytes, macrophages, Erythroid (with
BFU-E megakaryocytes, EPO)
eosinophils, mast cells
Stem cell Primitive progenitors in N/A
factor synergy with other
cytokines
EPO CFU-E to erythroblasts Megakaryocytes
(synergistic)
TPO Megakaryocyte colonies Primitive
CFU-GM progenitors

FIGURE 133.2 Photomicrograph of a CFU-GM colony and a
BFU-E colony in a culture plate. (Courtesy of STEMCELL
Technologies.)
TABLE 133.1 Hematopoietic Progenitor Cells and the
Contents of the Subsequent Colonies
hematopoietic cells are generated. Lineage-committed
cells will be released from the stroma as non-adherent
cells and may then be enumerated using progenitor
assays.6 LTBMC is primarily used to study the humoral
and cellular mechanisms of hematopoiesis.3
Long Term Culture Initiating Cell (LTC-IC) Assay

Progenitor Differentiated Cell Colony Size
CFU-GEMM Granulocytes, macrophages, >500 cells
rubriblasts, megakaryocyte
BFU-E Rubriblasts >200 cells
CFU-GM Granulocytes, macrophages >20 cells
CFU-E Rubriblasts 8–200 cells
Long Term Bone Marrow Culture (LTBMC)
This is a liquid culture system that consists of both an
adherent and non-adherent cell population and reca-
pitulates in vitro the in vivo relationships of bone
marrow stromal and hematopoietic cells. Proliferation
and differentiation of HCs occurs in liquid culture and
production of progenitors can be assessed by culturing
cells harvested from the liquid cultures in a subsequent
progenitor assay. These cultures can be maintained for
several months. The adherent layer consists of typical
bone marrow stromal cell components, including
fibroblasts, adipocytes, endothelial cells, and macro-
phages.11 The cultures are initiated by a suspension of
BMMCs and incubated until an appropriate stromal
layer is formed. The cultures are re-infused with another
BMMC suspension, and fed with media twice weekly.
The cells of the microenvironment (stromal layer)
produce growth factors and can support the growth of
multipotent progenitor cells from which all mature
An LTC-IC is a cell that can give rise to all of the pro-
genitor cells described above over a 5–8 week period
when cultured on an appropriate adherent “feeder cell”
layer.10 The LTC-IC assay measures the frequency of
these cells by performing progenitor assays after 5
weeks in culture and then inferring the number of LTC-
ICs in the original culture. The majority of CFCs are in
the adherent layer.2 The frequency of LTC-ICs in human
marrow is approximately 1 per 104 BMMC. LTC-ICs
comprise 1 in 50–100 purified human CD34+ bone
marrow cells.
GROWTH FACTORS USED IN
HEMATOPOIETIC CULTURE
The growth of progenitors in semi-solid medium is not
autonomous and therefore a variety of “growth factors”
are required for their growth and differentiation. The
reader is directed to an excellent review of hematopoi-
etic cytokines by Metcalf.8 Each type of progenitor (e.g.
CFU-GM, BFU-E, etc) responds to a certain set of growth
factors, and thus many growth factors are named for
the predominant lineage whose growth they stimulate.
The growth factors were originally called “colony stim-
ulating factors” (CSFs) because of this functional effect
on progenitor assays. The CSFs were derived from
media conditioned by immortalized cell lines or by
mitogen stimulated primary cultures of mouse lung or
1050 SECTION X: QUALITY CONTROL AND LABORATORY TECHNIQUES
lymphocytes. Over a brief period of time, these growth
factors were identified and cloned (Table 133.2).
Although growth factors may have a predominant
effect, they often synergize with other factors, or even
affect the functions of mature hematopoietic cells. If
growth factors are not added to the growth medium,
many can be supplied using an adherent “feeder layer”
of cells which produce them.
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human marrow cultures reveals the preferential location of primitive hemo-
poietic progenitors in the adherent layer. Blood 1983;62:291–297.
3. Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation
of haemopoietic stem cells in vitro. J Cell Physiol 1977;91:335–344.
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