Cell culture formats

Manfred Kubbies, Dept. Human Genetics, Univ. Würzburg, 2014

Relevant cell culture formats

Adherent monolayer culture (epithelial, mesenchymal cells, normal-/tumor cells)

Suspension culture (blood, bone marrow, normal-/tumor cells)

Spheroid culture (aggregate culture) (tumor cells, mixed culture normal-/tumor cells, suspension/agar type)

human fibroblasts human CD34 bone marrow cells

human T47-D breast tumor cells

Feeder layer cell culture (coculture substrate = feeder cells (often + primary cells, e.g. stem cells)

Microcarrier cell culture (alginate beads for suspension growth of adherent cells)

Adherent cells in monolayer culture

Human fibroblasts (skin) Human epithelial cells (trachea)

PromoCell, 2007, http://www.promocell.com

Adherent cells are detached from the vessel surface

and seeded into new cell culture flask at lower cell density.

Dead cells float in the cell culture supernatant and can simply be removed by rinsing.

Suspension culture mononuclear cells

Example for isolation of mononuclear cells from blood or bone marrow:

• Separation medium Ficoll (1.077 for lymphocytes), Percoll

• Overlay of dense separation medium with cells

• Centrifugation for a distinct period of time

• Harvest of cells from interphase band

• Seed cell into basal medium + supplements (RPMI1640/FCS/cytokines)

Human CD34 bone marrow progenitor-/stem cells (4/7 day culture)

Suspension cell cultures are passaged simply by dilution into new cell culture flasks.

Adherent monolayer cell culture

Plating efficiency

Percentage of adherent cells after seeding into a new cell culture

(not applicable for suspension cultures).

Cloning efficiency

Percentage of growing cell clones after diluted seeding of adherent

or suspension cells.

Split ratio

Dilution factor applied at passaging of a cell culture into new cell

culture vessels. The number of population doublings is calculated

from the number of passages and the respective split ratio.

Cell culture nomenclature from primary tissue

Example skin tissue: H 93-8 T7

H for

skin

Year of

preparation

1993

8th skin

in 1993

7th passage

Cell culture splitting

Split ratio: dilution factor of new seeded cell number / cell number harvested.

Total split ratio: split ratio plus size difference of a) cell culture plate surfaces

(adherent cells) or b) volume differences (suspension cells).

For calculation of the total number of population doublings of a cell culture take into account

the number of passages and the total split ratio.

Examples adherent cell culture:

Old and new cell culture vessel sizes identical (e.g. T75):

Cell number harvested: 4 x 106 cells / T75

Seeded cell number into new vessel: 5 x 105 / T75

Split ratio cell number: 1:8

Number of population doublings: 2n = 8, n = 3

Old and new cell culture vessel sizes different (e.g. from T25 into T75):

Cell number harvested: 2 x 106 Zellen / T25

Seeded cell number into new vessel: 2 x 105 / T75

Split ratio cell number: 1:10

Difference cell culture vessel surface: T25:T75 = 1:3

Total split ratio: 1:10 plus 1:3 = 1:30

Number of population doublings: 2n = 30, n = 4,9

The new seeding density can be calculated as: seeding density old cell culture / total split ratio

(e.g. 100 % confluency and total split of 8:1 = 12.5 % confluency new cell culture).

Bulk cultures and clones

Bulk cultures originate from primary cultures without any cell cloning step during cell propagation.

Mechanical cell selection and cell cloning

• Dilute plating of individual cells into microtiter plates (suspension and adherent cells;

no cell marker specificity)

• Dilute plating into culture vessels and harvest of cell clones by „cylinder trypsinization“

technique (adherent cells; no cell marker specificity)

• Cell cloning via FACS cell sorting into microtiter plates (suspension and adherent cells;

cell marker specificity)

Please note: some kind of cell cloning may occur even during rescue of „dead“ cell cultures !

Biochemical cell selection and cell cloning

Biochemical cell selection techniques: a) resistance genes (e. g. phosphotransferase APH/

neomycin, DHFR/methotrexate), b) marker genes (e.g. GFP, surface markers like l-NGFR).

• Dilute plating of individual cells selected by a biochemical selection marker into

microtiter plates (suspension and adherent cells; gene-of-interest specificity)

• Dilute plating into culture vessels and harvest of cell clones by „cylinder trypsinization“

technique (adherent cells; gene-of-interest specificity)

• Cell cloning via FACS cell sorting into microtiter plates (suspension and adherent cells;

cell marker and gene-of-interest specificity)

Please note: during biochemical selection the evolutionary selection pressure is only on the

resistance gene and not necessarily on the gene-of-interest !

Any cell cloning technique harbors the risk of genetic drift (founder effect).

This drift aspect is more apparent in tumor cells due to their mutator phenotype.

Chromosomal drift of recombinant tumor cell clones in vitro

Any cell cloning technique harbors the risk of genetic drift (founder effect).

This drift aspect is more apparent in tumor cells due to their mutator phenotype.

Cell cycle and aneuploidy analysis of plasmid-transfected tumor cells after biochemical G418/neomycin

selection and FACS cell cloning. MatLyLu: rat prostate tumor cells, RAW264.7: murine tumor macrophages.

FACS DNA analysis: PI staining. Grey peak: invariant chicken red blood cell DNA standard. The numbers

below the X-axis indicate the fluorescence ratios of the tumor G1 to the CRBC G1 peaks.

Mother cell line

(bulk culture)

Mother cell line

(bulk culture)

Clone 1 Clone 1

Clone 2 Clone 2 Clone 3 Clone 3 Clone 4

Master thesis, Bergmann AK, Univ. Graz

3.8 4.1

4.1

4.2

4.2

2.8

2.8 2.8 2.8

2.9 4.6

4.6 4.7 4.4 4.4

MatLyLu RAW264.7

In vitro cocultivation techniques

3D coculture Tumor cells AND (fibroblasts OR endothelial cells OR monocytes/macrophages OR T-cells).

Fibroblasts AND endothelial cells. Mesenchymal stem cells AND T-cells.

NK ADCC assay Monolayer cocultivation of tumor cells and NK cells at different effector/target ratios (e.g. E/T = 10:1) using

purified NKs, PBLs or whole blood. The NK ADCC assay can be performed in a spheroid/aggregate format.

Red/blue: endothelial cells

Green/blue: tumor cells

Blue: fibroblasts

Friedrich J, et al

Int.J. Radiat. Biol. 2007

Brown: endothelial cells

Blue: skin fibroblasts

Kunz-Schughart, et al

AJP Cell Physiol. 2006

Note: due to the cell line heterogeneity and existence of cell type optimal media conditions, the selection of media,

serum conditions, use of extra growth factors, etc. is of utmost importance applying coculture in vitro cell models.

Conkwest. Life Science Company 24h NK purity 80%; Raji labeled with CMFDA (green FL)

NK/Raji: 10/1 NK/Raji: 3/1

In vitro cocultivation techniques

Feeder layer Feeder cells are adherent cells which provide conditioned medium and a support matrix for cell attachment

and proliferation for difficult to cultivate cells. Feeder cells are treated with mitomycin C or irradiation: they

are metabolically active but do not proliferate. Examples: human keratinocytes on NIH-3T3 cells (Rheinwald,

1975), human embryonic stem cells on murine feeder cells; human B- and T-cells from CD34+ cells on

murine OP9 or MS-5 stromal cells or murine fetal thymus tissue.

Organotypic cocultures (sandwich technique) Sheets of cells of different differentiation status are overlayed and cultivated as multilayer cell system.

Examples: keratinocytes on fibroblasts (artifical skin), tumor cells on fibroblasts (invasion).

hu ESC on mu feeder mu ESC on mu feeder

Invitrogen life technologies CM Technologies

hu ESC on murine feeder

Sun Pharmaceutical Res Centre, Mumbai, India

Human dermal fibroblasts were placed on poly-

carbonate inserts (4 μM porous) in a six-well

culture plate. HaCaT keratinocytes cells were

seeded on the fibrin matrix and grown to con-

fluence. After 48 h of seeding, organotypic cul-

tures were raised to the air–liquid interface for

6–12 days to achieve epidermal stratification.

Alameda JP et al, Cell Death and Disease, 2011

Stratified 3D structures

Mueller-Klieser

Crit Rev Onc Hematol 2000

Physiological and morphological characteristics retained in spheroids are more similar to their in vivo

counterpart tissues compared to 2D monolayer cell cultures.

Stratified cellular composition: as a consequence of diffusion gradients of nutrients, oxygen and meta-

bolites viable cells are located at the periphery (3 to 5 cell layers) followed by a layer of quiescent cells

and necrotic cells in the center (e. g. tumor cells)

PC3 HRE-GFP + PI staining

(normoxia, 4 d culture)

Diffusion

problem !

pO2

growth

factors

pH

Kunz-Schughart

Cell Biol Int 1999

nutrients

Sensitivity of spheroid cultures to anticancer drugs is lower compared to monolayer cultures.

Resistance due to limited diffusion and/or presence of quiescent cells.

Tumor spheroids mimic avascular tumor areas and micrometastases.

Multicellular 3D spheroid / aggregate culture

Spheroid / aggregate culture

MCF7

3D spheroid

MDA-MB-361

3D aggregate

Spinner culture

Gyrator rotation

Hanging drop

Liquid-Overlay Trypsinized cells are seeded onto agar or matrigel coated cell

culture vessels.

Cell seeding and growth of colonies in softagar

(is not a spheroid but a cloning assay !)

Softagar assays are useful for studies of tumorigenic and

oncogene-transfected cells.

Trypsinized cells are seeded into spinner flasks and kept

in suspension by continuously stirring.

Trypsinized cells are seeded into an Erlenmeyer flask and

kept in suspension by shaking.

Trypsinized cells are seeded into a small volume and

cultivated in an inverted microtiter plate or glass slide.

Poly-HEMA culture Trypsinized cells are seeded onto poly-HEMA coated cell

culture vessels.

Spheroids in suspension culture

MDA-MB-361 MDA-MB-435 MCF7-ADR

HEK 293 IGR-OV 1 A549

MDA-MB-468 SK-BR3 MDA-MB-231

Colo 205

HepG2

PC3

Reproducible geometry of single spheroids per single microtiter plate well (96 well plate). (Ivascu A. et al, J. Biomolecular Screening, 11(8):922, 2006)

Stratified cell sheets in spheroids. Prolonged cell culture durations increase the cell

death rate mainly in the spheroid core region. In parallel, the cell cycle progression

slows down in comparison to a monolayer cell culture.

500 µm

1 d

ay

10

d

ays

Cell death and

culture duration

(MDA-MB-231)

500 µm

Cell death and

p53 knockdown

(MCF-7)

p5

3 w

t P

53

ko

Spheroids and cell death

Zahir and Weaver, Curr Opinion in

Genetics and Development 2004

Significant gene expression differences between cells grown as monolayer or spheroid culture.

Additional variability due to exogenous factors like base-membrane extract or collagen I.

Environmental gene expression control in 2D and 3D cultures

Non-malignant mammary epithelial cells were either grown on collagen I or rBM (matrigel*) as monolayers or spheroids.

RNA expression analysis was conducted using Affymetrix gene chips.

* matrigel: basement membrane preparation form murine Engelbreth-Holm-Swarm sarcoma tumor; enriched in laminin, collagen IV,

heparansulfate proteoglycan and growth factors (e.g. EGF, FGF, TGFß).

Softagar cloning assays: tumorigenicity analysis

Softagar assays are cloning assays indicative of survival and proliferation of cells in semisolid medium.

Although growing cell clones generate spheroids or aggregates in softagar, this assay is not comparable

to homo- or heterotypic spheroid or aggregates generated in liquid medium (suspension assay).

In vivo tumorigenicity and in vitro soft agar assay are two ways to assess the neoplastic properties of a tumor cell line. Normal

cells will not grow in soft agar. Cells that do grow in soft agar are considered to be anchorage-independent and more tumori-

genic, however, not all cancer cell lines will form tumors in nude or SCID mice. Typical test formats: a) comparison of mother

and oncogene-transformed daughter cell lines (e.g. 3T3 cells) or b) exposure of cell lines to carcinogenic compounds.

Cell are mixed at low density with softagar in complete cell culture medium and cultivated for up to 4 weeks. Colonies are

counted by microscopic inspection (with or without staining).

CrkII is a cellular protein which induces anchorage independent

growth of NIH 3T3 cells. Iwahara T et al, PNAS 101:17693, 2004

Softagar cloning assays: bone marrow stem cell differentiation

CD34 bone marrow or umbilical cord progenitor/stem cells enriched from Ficoll isolates or purified via FACS or magnetic

cell sorting. Inocculate cells in semisolid methylcellulose, agar or collagen matrix in Iscove´s medium +/- FBS supplemented

with cytokines (e. g. SCF, flt-3, IL-3, GM-CSF, EPO, etc.). Cell density: Ficoll ~ 105 cells/ml, purified CD34+ ~ 2x103

cells/ml. Culture duration: ~ 14 days. Count and identify colonies by morphology. Examples below: 100x magnification.

BFU-E CFU-GEMM CFU-Eo

CFU-G CFU-Meg CFU-GM

Nissen-Druey C, et al, Acta Hematologica Vol.113(1), 2005

Cancer stem cells/tumor initiating cells: evolution and tumor heterogeneity

Dick, Blood 112:4793, 2008 (mod MK)

and behaviour might be predictable

….. there is increasing support for the cancer stem-cell hypothesis, which, if correct, provides an explanation for the limitation of many

current breast cancer models ….. The cancer stem cell hypothesis is a different model: tumors originate in tissue stem and/or progenitor

cells through the dysregulation of the normally tightly regulated process of self renewal. They retain stem-cell properties which initiates/

drives carcinogenesis and differentiation, albeit aberrant, that contributes to tumor cell heterogeneity (Kakarala and Wicha, J Clin Oncol,

2008)……Do they really exist? True stem cell markers for tumor cells missing. Currently only operationally defined: low cell numbers re-

quired for tumor xenograft establishment. Can stem cells be cultivated ex vivo? The 3D environment (niches) as well as the cell culture

medium matters (Calabrese et al, Cancer Cell 2007)……..

Dontu et al, Genes & Dev. 17:1253, 2003

Dontu et al, J Mam Gland Biol Neopl 10:75, 2005

Breast cancer CSCs/TICs: in vitro differentiation / self renewal

Serum containing culture Serumfree culture

Differentiation Self renewal

Cell culture plates/flasks:

Ultra low attachment surface (Corning)

Serumfree spheroid culture:

MEBM*

B27 supplement

10 ng/ml b-FGF

20 ng/ml EGF

antibiotic/antimycotic

Serum containing monolayer culture:

MEBM*

10 % FCS

antibiotic/antimycotic

B27 supplement

* mammary epithelial basal medium (Lonza)

Ponti et al, Cancer Res, 65(13):5506, 2005

…….enzymatic digestion was also required and tissue fragments were incubated at 370C for 2 hours in a 1:1 solution of

collagenase/hyaluronidase. After filtration through a 30 µm pore filter, single cells were plated at 1,000 cells/mL in serum-

free DMEM-F12, supplemented with 10 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor

(EGF), 5 µg/mL insulin, and 0.4% bovine serum albumin (all from Sigma). Cells grown in these conditions as nonad-

herent spherical clusters of cells (usually named ‘‘spheres’’ or ‘‘mammospheres’’) were enzymatically dissociated every 3

days by incubation in a trypsin-EDTA solution for 2 minutes at 370C. Differentiation was induced by culturing mammo-

sphere derived cells for 8 d on collagen-coated dishes in DMEM-F12 supplemented with 5% fetal bovine serum without

growth factors.

Breast cancer CSCs/TICs: preparation and in vitro cell culture

Serumfree spheroid culture

Serum containing

monolayer culture

EMT genetics, dedifferentiation, morphologies of breast tumor cells in vitro

Sommers CL et al,

Breast Cancer Res. Treat., 1994

(mod MK)

2D

3D

The epithelial to mesenchymal transition (EMT) is a highly conserved cellular program that allows polarized, well-differentiated

epithelial cells to convert to unpolarized, motile mesenchymal cells. EMT is critical for appropriate embryogenesis and plays a

crucial role in tumorigenesis and cancer progression.

Estrogen

receptor

E-

cadherin

Desmo-

plakin

Zonula

occludens

Vimentin Integrins

E-type

M-type

Stem cell culture: human embryonic stem cells

Human ES cells: (a) grown on mouse feeder cells.

Scale bars: 200 µm.

hES cells are transferred to non-adherent culture

plates spontaneously form differentiated

structures termed embryoid bodies.

They possess cells of all three germ layers and are

frequently used to isolate differentiated cells under in

vitro conditions. Scale bars: 200 µm.

Stojkovic M, et al, Reproduction 128:259, 2004

Generation of hu ESC culture

Stem cell culture: human embryonic stem cells

Cell culture conditions

Human ESC grow slowly (generation time e.g. 36 h). The cell culture formats are still quite complex. LIF inhibits

differentiation of murine but not human ESCs.

Continuous culture of isolated ICM cells and hES cells in an undifferentiated state may require the presence of feeder layer

cells (irradiated or mitomycin C treated): MEF (primary mouse embryonic fibroblasts), STO (mouse embryonic fibroblast cell

line), fetal muscle, skin and foreskin cells, adult Fallopian tube epithelial cells, adult marrow cells.

Feeder free: dishes coated with animal-based ingredients with the addition of MEF-cell-conditioned medium (Stojkovic M, et

al, Reproduction 128:259, 2004) or human-derived ECM (hdECM) and conditioned medium, which have both been derived

from human foreskin feeder cells (Escobedo-Lucea C, et al, Human Embryonic Stem Cell Protocols, 2010).

Examples of various colony morphology types observed during reprogramming and propagation of iPSC colonies on feeders

and matrigel. (d, e) Human iPSCs derived from the fibroblasts of a normal subject are growing on feeder cells. These

images display one human iPSC colony with surrounding SNL feeder cells. Human iPSC colonies can have a circular or

oval/tear-drop shape. (f) The same human iPSC line can be grown under feeder-free conditions. One human iPSC colony

growing on a matrigel substrate is shown. The high nuclear to cytoplasmic ratio and prominent nucleoli typical for iPSCs is

more easily appreciated in colonies growing on Matrigel than on feeder cells. 10× phase contrast objective. Neely MD, et al, Cell Culture Techniques, Neuromethods, vol. 56, 2011

Stem cell culture: human embryonic stem cells

Examples ESC cell culture media

Klimanskaya I, et al, Cloning and Stem Cells, 2004. hES cells were maintained on mitomycin C–treated mouse embryonic

fibroblasts (MEFs) in growth medium: knockout high glucose DMEM* supplemented with 500 U/ml of penicillin, 500 µg/ml of

streptomycin, 1% nonessential amino acids solution, 2 mM of GlutaMAX-I, 0.1 mM ß-mercaptoethanol, 4 ng/ml bFGF, 10

ng/ml human LIF, 8% of serum replacement* (Invitrogen) and 8% plasmanate# (Bayer Research Triangle Park, NC).

* composition not disclosed from Invitrogen life technologies. # Plasmanate contains 5 g selected plasma proteins buffered

with sodium carbonate and stabilized with 0.004 M sodium caprylate and 0.004 M acetyltryptophan. The plasma proteins

consist of approximately 88 % normal human albumin,12 % alpha and beta-globulins and not more than 1 % gamma-globulin.

Lerou P, Human Pluripotent Stem Cells: Methods and Protocols, 2011. Dulbecco’s modified Eagle’s medium/Nutrient mixture

F-12 (DMEM/F12) supplemented with 20 % knockout serum replacement, 2 mM L-glutamine, 0.1 mM MEM-NEAA (non-

essentiell amino acids), 100 µM ß-mercaptoethanol and 10 ng/ml human recombinant bFGF.

ESC characterization

High levels of telomerase activity, normal karyotype and differentiation potential under in vitro and in vivo conditions.

These characteristics preferably shown over extended periods of culture. hES cell lines express cell surface (SSEA3,

SSEA4, TRA1-60, TRA1-81, GTCM2, TGT343) and intracellular markers (Nanog, OCT4, Rex1). But note: not all hES cell

lines have the same genetic profile even when cultured under the same conditions …….comparing three different hES cell

lines it was found that 52% of genes examined were expressed in all three independently derived hES cell lines and the

expression of 48 % genes was limited to just one or two hES cell lines……. In addition, not all derived hES cell lines

maintain their pluripotency under the same conditions, their potential for large-scale culture and growth under feeder-free

protocols, or their ability to form teratomas after injection into SCID mice. Moreover, their capacity to differentiate

spontaneously into different cell types under in vitro conditions is variable (Stojkovic M, et al, Reproduction 128:259, 2004).

Stem cell culture: differentiation of human embryonic stem cells

Examples ESC differentiation factors

To hematopoietic cells:

cocultivation on hematopoietic stroma cells, SCF+IL-3+IL-6.

To neuronal cells:

murine bone marrow stroma cell line PA6 cocultivation, noggin

treatment.

To keratinocytes:

BMP-4.

To cardiomyocytes:

activin A and BMP-4, cardiomyogenin.

To dendritic cells:

BMP-4, VEGF, SCF and GM-CSF.

To hepatocytes:

activin A, FGF-4 and BMP-2.

To pancreatic cells:

betacellulin, activin A and retinoic acid and nicotinamide.

Sartipy P, et al, Drug Discovery Today 12:688, 2007

Hepatocyte-like cells derived from hES cell line SA002. The hES

cells were differentiated for 4 weeks on mouse embryonic fibro-

blasts and subsequently transferred to a 96-well plate, coated with

collagen I for further culture. Sartipy P, et al, 2007

Stem cell culture: human induced pluripotent stem cells (iPSC)

Summary of feeder- and serum-free production of

human-induced pluripotent stem (hiPS) cells.

Timetable diagram of protocol.

Baharvand H, et al, Human Embryonic Stem Cell

Protocols, Methods in Molecular Biology, 2010

Stem cell culture: human induced pluripotent stem cells (iPSC)

Signal pathway inhibitors which improve in vitro human iPSC cultivation:

iPSC reprogramming efficiency, in vitro survival, cell expansion, feeder free cultivation

Thiazovivin Rho/ROCK

Promotes durability

PD0325901 ERK/MEK

Inhibits differentiation

SB431542 TGFß/ALK5

Improves reprogramming

CHIR99021 GSK3

Enhanced proliferation/viability

Defined Neural Induction Medium (N2B27)

Combine equal amounts of NBM with DMEM/F12 and

supplement with: 0.3% glucose, 2 mM L-Glut, 1% B-27

supplement, 1% ITS-A,1% N-2 supplement, and

0.5% Pen/Strep.

NBM: Neurobasal medium (Invitrogen)

ITS-A: insulin-transferrin-selenium solution (Invitrogen).

ITS-A supplement consists of 1 g/l insulin, 0.67 mg/l

sodium selenite, 0.55 g/l transferrin, and 11 g/l sodium

pyruvate.

N-2 Supplement (Invitrogen):

1 mM human transferrin, 86.1 mM recombinant insulin,

2 mM progesterone, 10.01 mM putrescine, 3.01 mM

selenite.

Neural differentiation of iPS and hES cells. Pluripotent iPS cells undergo neural induction via noggin treatment, PA6 coculture system, or directly on

laminin substrate in defined neural induction media. Following neural induction, neural progenitors are mechanically harvested and expanded as

neurospheres. From a neurosphere state, progenitors can be plated on substrate conditions and media to bias their differentiation to mature neurons,

glia or neural crest cells, as shown by expression of S100b, b-III tubulin and p75, respectively.

Denham N and Dottori M. Neurodegeneration:

Methods and Protocols, 2011.

Stem cell culture: human induced pluripotent stem cells (iPSC)

Stem cell culture: genetic instability of embryonic stem cells in vitro

The application of human embryonic stem cells (HESCs) to provide differentiated cells for regenerative medicine will

require the continuous maintenance of the undifferentiated stem cells for long periods in culture. However, chromosomal

stability during extended passaging cannot be guaranteed, as recent cytogenetic studies of HESCs have shown

karyotypic aberrations. The observed karyotypic aberrations probably reflect the progressive adaptation of self-renewing

cells to their culture conditions.

Adaptation to culture of human embryonic

stem cells and oncogenesis in vivo. Baker DEC, et al, Nature Biotechnology 25:207, 2007

Stem cell culture: genetic instability of iPS cells in vitro

Significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibro-

blasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS

cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human

iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling hu ES cells.

Copy number variation and selection during reprogramming to pluripotency. Hussein SM, et al, Nature 471:58, 2011

Reprogramming

to iPS cells

In vitro cell culture;

clonal expansion

Further readings: Somatic coding mutations in human induced pluripotent stem cells. Gore A, et al, Nature 471:63, 2011. Here we show that 22

human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point

mutations in the regions sampled . The majority of these mutations were ……. enriched in genes mutated or having causative effects in cancers.

Regulation of the hypoxia signaling pathway

HIF1α and HIF2α mRNAs are constitutively expressed in many cells. In a favorable environment these proteins are being

degraded via the VHL-proteasome pathway.

In an unfavorable environment or due to oncogenic pathway activation the HIF1α or HIF2α protein is being stabilized. They

interact with the constitutively expressed HIF1ß (DNA binding) and p300 (RNA transcription) proteins and initiate mRNA

transcription of HRE-promotor carrying genes.

HIF hypoxia

p53mut

PTENmut

ras

PI3K(?)

Akt(?)

mTOR

src

HIF1α

HIF1ß

p300

FIH-1

low nutrient supply

HIF1a and HIF2a

inducer

HIF protein

interaction

partner

Carroll VA, et al

Exp Rev Mol Med

2005 Typical oxygen partial pressures in vivo (mmHg)

Atmosphere 155, spleen 66, subcutis 50, gastric mucosa 47, cervix 36,

skeletal muscle 28, myocardium 25, liver 24, brain 24, breast cancers 17,

adenocarcimas 10-12, cervical cancer stage 0 20 and stage II 5.

Hypoxia: biochemistry of the HIF prolyl-hydroxylation cycle

Prolyhydroxylase PHD-2 hydroxylates HIF-1α

at the prolin residues 402/564 using 2-oxo-

glutarate, HIF-1α and oxygen (Fe2+ is required

as substrate). 2-oxoglutarat is being converted

to succinate and CO2.

Low oxygen or Fe2+ depletion inhibits

the prolyl-hydroxylation of HIF1α.

PHD-2

O2, Fe2+ CO2 +

2-OG 2-oxoglutarate

FH fumarate hydratase

GLUT1 glucose transp. type 1

HI: hypoxia-inducible factor

IDH isocitrate dehydrogenase

PHD HIF prolylhydroxylase

SDH succinate dehydrogenase

VHL von Hippel–Lindau protein

IDH X

Isocitrate

Linehan

Nat. Rev. Urology,

2010

In vitro HIF induction in cells

The HIF transcription factor activity is increased by HIF1a or HIF2a protein stabilization. This can be

induced by lower oxygen concentrations (incubator), prolylhydroxylase inhibition (chemical compounds)

or Fe2+ chelation (hypoxia mimetics: dipyridyl, desferrioxamine). Furthermore, HIF1a and/or HIF2a

stabilization may be due to oncogene activation or environmental stress like nutrient depletion.

Note: hypoxia mimetics are toxic to cells at longer incubation periods (e. g. 48 h).

HIF1α induction

by environmental stress (different HRE-GFP tumor spheroids, 21 % pO2)

Red (PI, dead cells), green (GFP)

21 % pO2

1 % pO2

HIF1α induction

by hypoxia (PC3 HRE-GFP monolayer, 24 h)

MDA-MB 435 HCT116 MDA-MB 435

PC3 PC3 PC3

Cell culture format complexity: environmental stress factors

cell free

extracts

biochemical

assays

single cells

2D cultures

adherent

suspension

spheroids

3D culture

homo-/

heterotypic

confrontation

in vivo

xenografts

xeno/syngeneic

subcutaneous

orthotopic

clinical

studies

in vivo/

ex vivo

assays

Increasing complexity

Standardized protocols

Hypoxia

Serum stress (lower serum conc.)

Energy stress (lower glucose conc.)

3rd dimension +/- coculture

Combi of environmental stress factors

Generation of transient and stable in vitro transfectants

Transient transfection:

lipofection, electroporation, injection, non-integrating viruses (e.g. AdV)

Stable transfectants:

biochemical selection after lipofection/electroporation/injection, integrating viruses (e.g. retrovirus)

Lipofection: DNA, RNA Transduction: viruses (DNA, shRNA) Electroporation: DNA, RNA, proteins Injection: DNA, RNA, proteins Calcium-precipitation: DNA (plasmids) Penetratin / TAT technique: peptides / proteins Scrape-loading: low molecular weight compounds

Alteration of cell viability? Lipofection rates: adherent vs suspension cells?

Please note:

a) risk of genetic drift of transfectants (evolution of survival of the fittest)

b) loss of expression of gene-of-interest (selection is on plasmid resistance gene)

Investigation of wt or ko genes or siRNA which

a) inhibit cell proliferation or b) lower cell viability.

There will be no development of recombinant bulk cultures or cell clones,

exept the gene of interest has been inactivated e.g. by epigenetic silencing

Generation of recombinant, stable transfected cell

lines with lethal or proliferation inhibitory genes

Examples

Overexpression of cell cycle inhibitory proteins (e. g. p16, inhibitor of CDK4).

Recombinant cells will be arrest in G1-phase – no proliferation.

Overexpression of pro-apoptotic proteins (e. g. caspase-3, protease).

Recombinant cells will die by apoptosis.

How to investigate physiology of cell cycle inhibitory or pro-apoptotic genes?

Generate gene recombinant cell lines using inducible promotors (e. g. TET system)

mod from: BD Bioscience/Clontech

Tet-ON / Tet-OFF plasmids: gene + GFP reporter

Tet-OFF: tetracyclin inactivates transactivator

Tet-ON: tetracyclin activates transactivator

Bidirectional Tet-Plasmid

GENE

GENE

transactivator

Genes and its proteins: fluorescent reporter molecules

Promotor activity (e.g. activators, inhibitors), tracing/localization of fusion genes (e.g. co-localization)

LacZ beta-galactosidase (E.coli): fluorescein-b-di-galactoside = fluorescein + 2 galactoside

Chloramphenicol transferase (E.coli): bodipy-chloramphenicol + acetyl-CoA =

acetyl-bodipy-chloramphenicol + CoA

Luciferase (firefly): luciferin + ATP + O2 = oxyluciferin + AMP + PPi + CO2

Beta-lactamase (E.coli): coumarin-fluorescein (conjugate) = coumarin + fluorescein

Green fluorescent protein (jellyfish): no fluorescent substrate necessary; GFP autofluorescent

GFP fluorescence in live and dead cells

(modMK, Steff A. et al, Cytometry 45:237, 2001)

Dead cells loose their intracellular GFP

label due to membrane leakage

HIF1a induction by environmental stress (PC3 HRE-GFP spheroid, 4 d, 21 % pO2)

bright field

PI, dead cells

GFP, HIF1 induction

GFP (log)

PI (l

og

)

untreated

dexamethasone

PI (l

og

)

GFP (log)

l-NGFR: a immunological reporter molecule not lost in dead cells

FSC/SSC gate includes dead cells and fragments. Eg5=kinesin, luc=luciferase. Hela cells.

G1

S G2M H33342

L-N

GF

R (

PE

)

H33342

l-NGFR / luc-shRNA

H33342

L-N

GF

R (

PE

)

H33342

G1

S G2M

l-NGFR / Eg5-shRNA

l-NGFR useful as gene marker: signal domain truncated p75 low-affinity nerve growth factor receptor.

Detection by fluorochrome labeled l-NGFR antobodies.

The l-NGFR marker protein is not lost in dead cells and therefore

still useful for identification of genetically modified, dead cells.

Cytotoxicity: the degree to which an agent excerts a specific destructive action on cells

The execution of such destructive action may be for example the lysis of cells by immune phenomena or the activity of

antineoplastic agents that kill cells. The term cytotoxicity is indicative of cell death effector functions. The process of

cell proliferation inhibition is not a cytotoxic phenomenon unless is goes in parallel or is followed by cell death.

Points to consider using selection or marker genes for identification of recombinant cells

Biochemical selection pressure via cytotoxic compounds is only on the selection marker but not on the gene of interest.

The GFP marker protein is lost in dead cells and therefore no longer useful for identification of genetically modified cells.

The l-NGFR marker protein is not lost in dead cells and therefore still useful for identification of genetically modified, dead cells.

Cytotoxicity of selection and marker genes for identification of recombinant cells

Compounds used for biochemical selection of recombinant cells are cytotoxic,

unless the recombinant cells expresses larger quantities of the resistance gene

e. g. dhfr-gene expression in methotrexate treated cells (DHFR needed for dTMP generation)

e. g. neomycin (= phosphotransferase) expression in G418 treated cells (inactivates cytotoxic G418 by phosphorylation)

The GFP marker protein is not cytotoxic in recombinant cell lines (mostly slight G1-phase slowdown)

The l-NGFR marker protein is not cytotoxic in recombinant cell lines (barely G1-phase slowdown)

Cell fusions

Cell fusion techniques Bring cells into close proximity using e. g. polyethylene glycol (PEG), electrofusion, sendai virus.

Detect and separate fused from unfused cells using biochemical selection or cell marker.

Cell selection markers useful to identify fused cells Dual expression of survival genes in hybrids (e.g. HAT medium hybridomas)

Dual expression of cell surface markers in hybrids (e.g. each cell lines adds a unique marker)

Lower fluorescence quenching in hybrids (e.g. fuse fluorochrome labeled with unlabeled cells)

HAT medium selection (hypoxanthine-aminopterin-thymidine). The trick is that aminopterin blocks DNA de novo synthesis, which is absolutely required

for cell division to proceed, but hypoxanthine and thymidine provide cells with the raw material to evade the blockage of the "salvage pathway", provided

that they have the right enzymes/genes (e.g. hypoxanthine-guanine phosphoribosyltransferase).

B-cells are fused with HGPRT negative, immortalized myeloma cells. Fused cells are incubated in the HAT medium. Aminopterin in the medium blocks

the DNA de novo synthesis pathway. Hence, unfused myeloma cells die, as they cannot produce nucleotides by the DNA de novo or salvage pathway.

Unfused B cells die as they have a short lifespan. In this way, only the B cell-myeloma hybrids survive.

HGPRT- or HGPRT-/HGPRT- or

TK- or TK-/TK-

HGPRT+/HGPRT- or

TK+/TK-

HGPRT+ or HGPRT+/HGPRT+ or

TK+ or TK+/TK+

immortal

HAT sensitive myeloma fused

hybrids

mortal

splenic B-cells

Genotype

Cell type

HAT fate

Explanation

dies survives dies

Unable to synthesize DNA.

1) TK mutation causes loss-of-

function in the salvage pathway.

2) Aminopterin blocks „de novo“

pathway

Immortal and restored

DNA synthesis.

1) Immortality from plasmacytoma

2) Rescued ability to synthesize

DNA from restored TK function.

Mortal.

1) Functional DNA synthesis

2) Dies because of limited

replication potential.