James E. Trosko, Ph.D.

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Stem Cells as Tools for Environmental Sciences.

Junctional Communication, Alteration in Stem Cell Quality/Quantity and the

University East Lansing, Michigan 48824 E-Mail: james.trosko@ht.msu.

1 Stem Cells as Tools for Environmental Sciences. A Vision: An Integration of Basic Mechanisms of Stem Cell Biology, Gap Junctional Communication, Alteration in Stem Cell Quality/Quantity and the Barker Hypothesis. James E. Trosko, Ph.D. 246 Natl. Food Safety Toxicology Center Dept. Pediatrics and Human Development College of Human Medicine Michigan State University East Lansing, Michigan james.trosko@ht.msu.edu Home Page: NAS- Workshop, June, 2010 Human embryonic stem cells on feeder layer ; immunostained against Oct 4

2 OVERCOMING CRITICISMS THERE IS A DEVELOPING SPECULATION AND ASSUMPTION THAT AGENTS OR CIRCUMSTANCES ARE PROMOTERS OR ARE PROMOTING IF THEY LEAD TO DECREASE IN CELL-CELL COMMUNICATION BY AN INTERFERENCE WITH GAP-JUNCTION EXPRESSION. THIS IS A SERIOUS MISCONCEPTION THAT SHOULD BE ABANDONED! Emmanuel Farber, Risk Assessment for Possible Carcinogens: A Critical Look. Drug Metabolism Reviews 32: , 2000.

3 Ecologically relevant species Other animal research models Humans: Sensitive populations In vitro to in vivo comparisons/ extrapolations Cross-Species comparisons/ extrapolations In Vitro adult stem cell 3-D models (rodent or human) Mouse Organisms Organs RISK ASSESSMENT of TOXICANT INTERACTIONS AT DIFFERENT LEVELS OF THE BIOLOGICAL HIERARCHY Cells Gene Space Protein Space Metabolite Space Molecules

COWS & DOGS CAUSE HUMAN BREAST, PROSTATE CANCERS, COLON CANCERS BIOLOGICAL & CULTURAL EVOLUTION INTERACT TO

Urology 57: 31-38, 2001. 2. Thompson, D.E., et al.,cancer incidence in atomic bomb survivors. Radiation Res.

, et al, Developmental Exposures to estradiol and bisphenol A increases susceptibility to prostate

4 COWS & DOGS CAUSE HUMAN BREAST, PROSTATE CANCERS, COLON CANCERS BIOLOGICAL & CULTURAL EVOLUTION INTERACT TO CAUSE THESE CANCERS. REFERENCES: 1. Coffey, D.S. Similarities of prostate and breast cancer: Evolution, Diet, and Estrogens. Urology 57: 31-38, Thompson, D.E., et al.,cancer incidence in atomic bomb survivors. Radiation Res. 137: s17-s67, Ho, S.M., et al, Developmental Exposures to estradiol and bisphenol A increases susceptibility to prostate carcinogenesis... Cancer Res., 66: , Dolinoy,D.C., et al, Maternal genistein alters coat color... Environ.Health Perspect., 114: , MOM, IS THAT DAD?

5 STEM CELLS AND THEIR USE IN REGENERATIVE THERAPY AND DRUG DISCOVERY AND TOXICITY ASSESSMENT: A VISION TYPES OF STEM CELLS AND THEIR CHARACTISTICS Embryonic- pluripotent; ips pluripotent; adult, pluripotent, multipotent, bipolar, unipolar. While there seem to be universal markers (i.e., Oct4A and lack of connexin genes or functional gap junctions, Lgr5), more universal markers have to be validated, as well as organ specific markers ( estrogen receptor, albumin) Exist in unique organ specific niches ( Extra-cellular substrates; oxygen-tension; interaction with lineage communication and extracellular signals) Express universal and organ-specific markers Appear to express various drug transporter genes (i.e. ACBG-2) to be resistant to toxicants Unknown, at present, DNA repair capacities of each stem cell type compared to progenitor and terminally-differentiated cells. Challenge to the origin of ips cells ( re-programmed differentiated somatic cells or selected adult stem cells). Most environmental toxicants act, mechanistically, as epigenetic agents. Therefore, they will trigger various types of intracellular signals.. Do not know if the same chemicals can induce the same intra-cellular signals and responses to different types of stem cells.

6 STEM CELLS AND THEIR USE IN REGENERATIVE THERAPY AND DRUG DISCOVERY AND TOXICITY ASSESSMENT: A VISION TYPES OF STEM CELLS AND THEIR CHARACTISTICS -Continued How Embryonic stem cells can be used : To induce abnormal cellular responses that might lead to embryo-lethality. To interfere with the normal differentiation/proliferation for embryonic stem cells ( which could lead to the Barker hypothesis- too many or too few stem cells later in life. A-bomb breast cancer data; progeria premature aging; bisphenol A induction of prostate cancer; obesity in bipolar patients) Not on plastic; not in 2-D., not in high oxygen; not on generic matrices 3-D, low oxygen; absence of artificial substrates; Practical approach to use normal adult stem cells of several major organs ( e.g., brain; heart, liver, kidney; lung, breast, prostate, skin; pancreas; muscle) From a male and female, with monitoring Oct4A; organ specific markers; biological markers- cell proliferation; cell differentiation, cytotoxicity, senescence.

7 STEM CELLS AND THEIR USE IN REGENERATIVE THERAPY AND DRUG DISCOVERY AND TOXICITY ASSESSMENT: A VISION TYPES OF STEM CELLS AND THEIR CHARACTISTICS Continued Good News/ Bad News- How to test with stem cells & how to interpret data. Valproic acid anti-pyschotic & stimulator of adipose-derived stem cell proliferation. Thalidomide- sedative, teratogen and anti-cancer therapeutic (antiangiogenesis) Phenobartibital- anti-convulsive drug, rodent tumor promoter, induces hepatocellular carcinomas pre-weaning; post weaning induces adenomas. Retinoids; Inducer of differentiation- anti-cancer drug; tumor promoter; teratogen. Not good for detection of chemical toxicants due to drug resistance of expressed ABCG2. Many more examples

8 TOPICS TO BE DISCUSSED BARKER HYPOTHESIS: Adult Diseases are Linked To Pre-natal and Early Post-Natal Life.

9 T.A. Jacks and R.A. Weinberg Cell, Vol. 111, , December 27, 2002 However, the 3D culture system has the distinct advantage that it takes into account physiologically relevant interactions while still being amenable to facile genetic manipulation and biochemical analysis.

10 Systems Integration of Intracellular Signaling and Intercellular Signaling Of Stem Cells, Progenitor and Terminally-Differentiated Cells In Tissues. Stem Cell Niche Stem Cell A B C D ENDOGENOUS FACTORS:Hormones, GF s, Cytokines, etc EXOGENOUS FACTORS: Toxicants; O2 E N D O G Progenitor E Cells N A O B Gap C U Junction D S A B C D Differentiated Cell A B C D Extra-Cellular Martix

11 Integration of Extra-, Intra-, and Intercellular Communication Between Cell Types to Cell Fate Toxicants Growth Factors PLC FAS JAK caspases [ STAT STAT NFAT MAPKKK MAPK WNT IKB-K IKB/ NF-KB C-RAF Tight Junction Gap junction JAK STAT C-RAF B-catenin Cell adhesionc-raf [ MEKK MEKK1 SEK SAPK MAPK ( Extra-cellular Matrix AKT P13 K B-catenin cadherin cell fates ////////// senescence proliferation differentiation apoptosis stem cell niche maintenance adaptive responses

12 STEM CELL NICHE: Interactions of Extra- (ECM; O 2, Hormones, GF s; Neurotransmitters), Intra-( finite signaling pathways) and Gap Junctional Inter- Cellular Communication (ions, Small MW Molecules). HUMORAL (O2, GFs, Cytokines) No GJ IC Extra- EXTRA- STROMAL CELLS Structural & Physical Stress Paracrine ( GF s) Extra- ECM ( i.e. Liminin 517) Neurotransmitters Extra- NEURON Extra-

13 Genomic Expression Changes in Cells of a Tissue A B C D E A B C D E Terminallydifferentiated cell A B C D E Stem cell niche A progenitor cell A B C B C D E Senescent cell D E Apoptotic cell

14 HME-11: Two types (Type I and Type II) of normal human breast epithelial cells Pure cultures of Type I cells

15 Differentiation Pathway of Normal Human Breast Epithelial Cells In Vitro Type I Type I Type II-1 Type I Type II-1

16 Type I Stem Cell Young Type II Cell Mature Type II Cell

17 Type I Stem Cell Young Type II Cell Mature Type II Cell

19 Human Adult Kidney Stem Cell Grown on X-Rayed Human Fibroblast Mat

20 EVIDENCE SHOWING NO GJIC IN HUMAN ADULT KIDNEY STEM CELLS ON HUMAN FIBROBLASTS

21 FOUR DIFFERENT HUMAN CARCINOMA CELL LINES GROWN ON NORMAL X-RAYED HUMAN FIBROBLAST MATS

22 Trosko (Dir.) CANCER & STEM CELL RESEARCH GROUP MICHIGAN STATE UNIVERSITY Pediatrics WB-cells grown on Matrigel Cell- Differentiating System WT-WB cells (high differentiation) DN-Cx43WB cells (low differentiation)

Oct-4 Expression in Skin Tissue Section Human A1 B1 Dog A2 B2 Figure

subsequently stained with Oct-4 primary antibody and avidin-hrp and

Oct-4 expression was shown in brown located in the nucleus (blue

23 Oct-4 Expression in Skin Tissue Section Human A1 B1 Dog A2 B2 Figure 8. Oct-4 protein expression in the stem cells of human and dog skin basal layers. Human (A) and dog (B) skin tissue sections were deparaffinized and subsequently stained with Oct-4 primary antibody and avidin-hrp and finally visualized with DAB (showing in dark brown color). Oct-4 expression was shown in brown located in the nucleus (blue hematoxylin stain) of the cells suggesting those few Oct-4 positive cells are stem cells (arrows). A2, B2 were higher magnification images.

24 Stem Cell Theory of Carcinogenesis JW Jung Dept. Vet. Pub. Health Seoul Nat l Univ. Trosko J.E.

25 Oct 4+, Cx- (Stem Cell) Nutrients Low O Level MITOGENIC SIGNAL Growth Factors & Cytokines Oct 4+, Cx- (2 Stem Cells) NICHE SUBSTRATE SYMMETRIC CELL DIVISION Oct 4+, Cx- (Stem Cell) Nutrients High O Level MITOGENIC SIGNAL Growth Factors & Cytokines Gap Junction (Differentiated Cell) Oct 4-, Cx+ (Progenitor Cell) Oct 4-, Cx- (Stem Cell) NICHE SUBSTRATE ASYMMETRIC CELL DIVISION

26 PROSTATE CANCER STEM CELLS: symmetric cell division (day 2-4) Oct4+ Oct4+ Oct4+

27 PROSTATE CANCER STEM CELLS: asymmetric cell division (day 6) Oct4+ Oct4 Oct4 Oct4+

29 Oct-4 Expression in Human Breast Epithelial Cells (HBEC) Type1 Young Type 2 Mature Type 2 Immortal weakly tumorigenic highly tumorigenic A1 B1 C1 D1 E1 F1 A2 B2 C2 D2 E2 F2 A3 B3 C3 D3 E3 F3 G Oct-4 GAPDH

30 GENETIC CONTROL OF CELL INTERACTIONS IN CHIMERAS C.L. MARKERT, Devel.. Genet., 4: , 279, 1984 Cells interact and communicate during embryonic development and through inductive stimuli mutually direct the divergent courses of their differentiation. Very little cell differentiation is truly autonomous in vertebrate organisms. The myriad cell phenotypes present in mammals, for example, must reflect a corresponding complexity in the timing, nature, and amount of inductive interactions. Whatever the nature of inductive stimuli may be, they emerge as a consequence of specific sequential interactions of cells during embryonic development.cont.

31 GENETIC CONTROL OF CELL INTERACTIONS IN CHIMERAS C.L. MARKERT, Devel. Genet., 4: , 1984 The first embryonic cells, blastomeres, of mice and other mammals, are all totipotent. During cleavage and early morphogenesis, these cells come to occupy different positions in the three-dimensional embryo. Some cells are on the outside, some inside. The different environments of these cells cause the cells to express different patterns of metabolism in accordance with their own developing program of gene function. These patterns of metabolism create new chemical environments for nearby cells and these changed environments induce yet new programs of gene function in responding cells Cont.

32 GENETIC CONTROL OF CELL INTERACTIONS IN CHIMERAS C.L. MARKERT, Devel. Genet., 4: , 1984 Thus, a progressive series of reciprocal interactions is established between the cellular environment and the genome of each cell. These interactions drive the cell along a specific path of differentiation until a stable equilibrium is reached in the adult.thereafter little change occurs in the specialized cells and they become remarkably refractory to changes in the environment. They seem stably locked into the terminal patterns of gene function characteristic of adult cells. The genome seems no longer responsible to the signals that were effective earlier in development. Cont.

33 GENETIC CONTROL OF CELL INTERACTIONS IN CHIMERAS C.L. MARKERT, Devel. Genet., 4: , 1984 Of course, changes can occur in adult cells that lead to renewed cell proliferation and altered differentiation as seen in neoplasms, both benign and malignant, but such changes are very rare indeed when one considers the number of cells potentially available for neoplastic transformation. Possibly mutations in regulatory DNA of dividing adult cells can occasionally lead to new and highly effective program gene functions that we recognize as neoplastic or malignant. However, most genetic changes in adult cells can probably lead to cell death since random changes in patterns of gene activity are not likely to be beneficial.

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