Stem Cells and the Stem Cells and the Cardiovascular SystemCardiovascular System

20 October 201120 October 2011

Robert Siggins, Ph.D.Robert Siggins, Ph.D.

stemcellschool.com

Outline and ObjectivesOutline and Objectives

• What are stem cells (SCs)?What are stem cells (SCs)?

• Which SCs contribute to the adult CV Which SCs contribute to the adult CV system?system?

• What are the physiological and What are the physiological and pathophysiological roles for SCs in the pathophysiological roles for SCs in the CV system?CV system?

Stem Cell “Theory”

• 1855 First eluded to by (Remak) Virchow Omnis cellula e cellula

– RediOmne vivum ex ovo

• 1875 ”Embryonic rest” proposed by Cohnheim (cancer stem cells)

• 1917 HSC proposed by Pappenheim

• 1961 McCulloch and Till describe CFU-s

Definition of SCs

• Totipotent, pluripotent, or multipotent

• Self-renewal

• Clonogenic

• Niche

http://icanhascheezburger.com

autismpedia.org

Totipotent = ability to give rise to all cells of the body and all cells that constitute the extraembryonic tissues (i.e. placenta)

Pluripotent = ability to make all cells of the body (all three germ layers)

Multipotent = ability to differentiate into more than one cell type (usually germ layer restricted)

Self-renewal

pluripotentpluripotent

stemcellresources.org

Embryonic SCs (ESCs) are

Tissue-restricted SCs are Multipotent!Tissue-restricted SCs are Multipotent!

stemcellresources.org

toonpool.com

Physiol Rev 85:1373-1416, 2005

UEA-1 (ulex europaeus agglutinin-1) or SSEA-4 (stage-specific UEA-1 (ulex europaeus agglutinin-1) or SSEA-4 (stage-specific embryonic antigen-4) eliminates teratoma-initiating cells!embryonic antigen-4) eliminates teratoma-initiating cells!

Definition of SCs

• Totipotent, pluripotent, or multipotent

• Self-renewal

• Clonogenic

• Niche

http://xarquon.jcu.cz/edu/hematologie/03kmenove_bunky/03stem_lineages.htm

Symmetric and Asymmetric Divisions

Cell Extrinsic and Intrinsic Models

Regulated by niche attachment

Regulated by niche cell polarity

Regulated by intrinsic polarity

Asymmetric Divvision

Definition of SCs

• Totipotent, pluripotent, or multipotent

• Self-renewal

• Clonogenic

• Niche

Cardiac Stem Cells

(CSCs)

Large clone of c-kit+ cells

(green) generated by a single rat c-kit+ CSC.

Physiol Rev 85:1373-1416, 2005

Cardiac Stem Cells

(CSCs)

Clonogenic cells in

differentiating medium

acquire the myocyte

phenotype (α-sarcomeric actin, red).

Physiol Rev 85:1373-1416, 2005

Cardiac Stem Cells

(CSCs)

Clonogenic cells acquire

the SMC phenotype (α-smooth

muscle actin, magenta).

Physiol Rev 85:1373-1416, 2005

Cardiac Stem Cells

(CSCs)

Clonogenic cells acquire

the EC phenotype

(vWF, yellow).

Physiol Rev 85:1373-1416, 2005

Definition of SCs

• Totipotent, pluripotent, or multipotent

• Self-renewal

• Clonogenic

• Niche

Niches are specialized

microenvironmnets comprising support

cells, soluble factors, ECM,

SCs, and progenitor cells

Every niche is Every niche is unique!unique!

http://stemcells.nih.gov/info/

PNAS 103:9226-9231, 2006

Cardiac niches and putative supporting cells. Apical (A, B, and F) and atrial (C–E) niches contain CSCs and LCCs. (A) c-kit+ (green) Ets1+ (white; EC progenitors). (B) MDR1+ cells (white) GATA-4+ (magenta dots); α-sarcomeric actin+GATA-4+ (red; myocyte precursors). GATA-4+ cardiac progenitors (arrowheads). (C) Sca-1+MEF2C+ (yellow & white dots; myocyte progenitors);

Sca-1+von Willebrand factor+ (green; EC precursor). (D–F) Connexin 43 and E- and N-cadherin in niches containing c-kit+ (D & F, green) and Sca-1+ (E, yellow) CSCs and LCCs. GATA-4+ (D, magenta dots) and MEF2C+ (E & F, yellow dots). Connexin 43 (yellow dots), E-cadherin (green dots), and N-cadherin (white dots) Nuclei are stained by propidium iodide (blue).

Cardiac niches and putative supporting cells. (G–J) Connexin 43 and 45 and N- and E-cadherin in atrial niches containing c-kit CSCs–LCCs (green) are represented by yellow dots located between two CSCs–LCCs (arrowheads), a CSC–LCC and a fibroblast (arrows), and a CSC–LCC and a myocyte (double arrowheads); see Insets. Nuclei are stained by propidium iodide (blue). PNAS 103:9226-9231, 2006

Formation of Gap Junctions

PNAS 103:9226-9231, 2006

PNAS 103:9226-9231, 2006

Myocyte turnover and life span (A and B) Apical section illustrating bright (arrows), intermediate (open arrowheads), and dim (arrowheads) BrdU+ myocyte nuclei (yellow) after 10 weeks of chasing. Myocytes are stained by cardiac myosin (B, red).

PNAS 103:9226-9231, 2006

(C) % of BrdU-bright, -int, and -dim myocyte nuclei. (Right) Bars document the total number of BrdU+ myocytes and total number of myocytes in the atria, base–mid region, and apex.

Myocyte Turnover

PNAS 103:9226-9231, 2006

Myocyte Turnover

PNAS 103:9226-9231, 2006

Myocyte Turnover

PNAS 103:9226-9231, 2006

Myocyte Lifespan

ESCs versus Adult SCs (ASCs)

• ESCs– Embryolically

derived– Not tissue-specific– Pluripotent– Requires in vitro

fertilized egg for derivation (ethical concerns)

• ASCs– Tissue resident– Tissue-specific– Multipotent– No ethical

problems with their clinical application

nature.com

americanprogress.org

http://stemcells.nih.gov/info/media/

Outline and ObjectivesOutline and Objectives

• What are stem cells (SCs)?What are stem cells (SCs)?

• Which SCs contribute to the adult CV Which SCs contribute to the adult CV system?system?

• What are the physiological and What are the physiological and pathophysiological roles for SCs in the pathophysiological roles for SCs in the CV system?CV system?

Identification of Mitotic Spindles in Dividing Myocytes from Infarcted Hearts

(A) blue = organization of tubulin in the mitotic spindle (arrows)(B) green = metaphase (arrowheads)(C) green and blue = combination of tubulin and metaphase chromosomes (arrows and arrowheads)(D) red = sarcomeric α-actin, blue = tubulin, and green = chromosomes in metaphase (arrows and arrowheads)

N Engl J Med 344:1750-1757, 2001

A B

C D

A Myocyte in the Process of Cytokinesis

Arrows = actinRed = staining of myocyte sarcomeric α-actingreen = PI labeling of chromosomes N Engl J Med 344:1750-1757, 2001

Effects of MI on the Number of Mitotic Myocytes

N Engl J Med 344:1750-1757, 2001

N Engl J Med 346:5-15, 2002

The Y Chromosome in Transplanted Hearts. •Myocytes (A and B)•Smooth-muscle cells (C and D), Endothelial cells in both coronary arterioles (E and F), and capillary endothelial cells (G and H)•Blue areas = PI staining in nuclei, Green areas indicate Y chromosome •The red areas indicate sarcomeric α-actin in B, of smooth-muscle α-actin in D, and of factor VIII in F and H.

The Y Chromosome in Transplanted Hearts. In I, J, and K, the bright blue, fluorescent areas and the arrows indicate the presence of Ki-67, and the yellow areas show the Y chromosomes.

N Engl J Med 346:5-15, 2002

ASCs of the CV system

• Isolated based on functional assessments or surface phenotypes

• Cardiac Stem Cells (CSCs) and Endothelial Progenitor Cells (EPCs)

• Possible Vascular Progenitor Cell (VPC); resident in vessel walls

Surface Markers for EPCs

• Mouse– VEGFR2, LinNeg, c-kit, Sca-1, VE-cadherin

• Human– CD133, VEGFR2, VE-cadherin, CD34

Cell 127:1137-1150, 2006

Vasculogenesis

• Aggregation of de-novo-forming angioblasts into a primitive vascular plexus

• Hemangioblast Flk1+ cells produce ECs and hematopoietic cells

• Earliest marker of angioblast precursors Flk1/VEGFR-2

• Complex remodeling processgrowth, migration, sprouting and pruning lead to development of functional circulatory system

Outline and ObjectivesOutline and Objectives

• What are stem cells (SCs)?What are stem cells (SCs)?

• Which SCs contribute to the adult CV Which SCs contribute to the adult CV system?system?

• What are the physiological and What are the physiological and pathophysiological roles for SCs in the pathophysiological roles for SCs in the CV system?CV system?

N Engl J Med 363:1638-1647, 2010

N Engl J Med 363:1638-1647, 2010

N Engl J Med 363:1638-1647, 2010

N Engl J Med 363:1638-1647, 2010

Evidence for Cardiomyocyte Renewal in Humans

Olaf Bergmann, Ratan D. Bhardwaj, Samuel Bernard, Sofia Zdunek, Fanie Barnabe-Heider, Stuart

Walsh, Joel Zupicich, Kanar Alkass, Bruce A. Buckholz, Henrik Druid, Stefan Jovinge, Jonas Frisen

Science 324:98-102, 2009

Fig. 1. Cell turnover in the heart. (A) Schematic figure demonstrating the strategy to establish cell age by 14C dating. The black curve in all graphs shows the atmospheric concentrations of 14C over the decades since 1930 [data from (14)]. The vertical bar indicates the date of birth of the individual. The measured 14C concentration (1) is related to the atmospheric 14C concentration by use of the established atmospheric 14C bomb curve (2). The average birth date of the population can be inferred by determining where the data point intersects the x axis (3).

Science 324:98-102, 2009

Fig. 1. Cell turnover in the heart. 14C concentrations in DNA of cells from the left ventricle myocardium in individuals born after (B) or before (C) the nuclear bomb tests correspond to time points substantially after the time of birth, indicating postnatal cell turnover. The vertical bar indicates the date of birth of each individual, and the similarly colored dots represent the 14C data for the same individual.

Science 324:98-102, 2009

Fig. 1. Cell turnover in the heart. For individuals born before the increase in 14C concentrations, it is not possible to directly infer an age because the measured concentration can be a result of 14C incorporation during the rising and/or falling part of the atmospheric curve, and thus the concentration is indicated by a dotted horizontal line.

Science 324:98-102, 2009

Science 324:98-102, 2009

Fig. 2. Isolation of cardiomyocyte nuclei. (A to C) Flow cytometric analysis of cardiomyocyte nuclei from the left ventricle of the human heart with an isotype control antibody or antibodies to the cardiomyocyte-specific antigens cTroponin I or T. Boxes denote the boundaries for the positive and negative sorted populations. (D) cTroponin I and T are present in the same subpopulation of heart cell nuclei.

Science 324:98-102, 2009

Fig. 2. (E) Western blot analysis of flow cytometry–isolated nuclei demonstrates nearly all detectable cTroponin T (analyzed with two different antibodies) and I protein in the cTroponin T–positive fraction. Brain and heart tissue were used as negative and positive controls, respectively. (F) The cardiac troponin T–positive population is enriched for the cardiomyocyte-specific transcription factors Nkx2.5 and GATA4. Both fractions contain similar amounts of the nuclear protein histone 3 (loading control).

Science 324:98-102, 2009

Fig. 2. (G) Gene expression analysis of flow cytometry–isolated nuclei shows high expression of cardiomyocytespecific genes in the cTroponin T–positive fraction (cTroponin I and T, Nkx2.5), whereas marker genes for endothelial cells (vWF), fibroblasts (vimentin), smooth muscle (ACTA2), and leukocytes (CD45) are highly expressed in the cTroponin T–negative fraction (H). Bars in (G) and (H) show the average from three independent experiments (TSD).

Science 324:98-102, 2009

Fig. 3. Cardiomyocyte turnover in adulthood. (A) The 14C concentrations in cardiomyocyte DNA from individuals born before the time of the atmospheric radiocarbon increase correspond to time points after the birth of all individuals. The vertical bar indicates year of birth, with the correspondingly colored data point indicating the Δ14C value. (B) 14C concentrations in cardiomyocyte DNA from individuals born after the time of the nuclear bomb test.

Science 324:98-102, 2009

Fig. 3. (C) Average DNA content (2n = 100%) per cardiomyocyte nucleus from individuals of different ages. Ploidy was measured by flow cytometry. (D) 14C values corrected for the physiologically occurring polyploidization of cardiomyocytes during childhood for individuals born before and after the bomb-induced spike in 14C concentrations, calculated on the basis of the individual average DNA content per cardiomyocyte nucleus. The 14C content is not affected in individuals where the polyploidization occurred before the increase in atmospheric 14C concentrations.

Science 324:98-102, 2009

Fig. 4. Dynamics of cardiomyocyte turnover. (A) Individual data fitting assuming a constant turnover (see supporting online text) reveals an almost linear decline of cardiomyocyte turnover with age (R = −0.84; P = 0.001). A constant-turnover hypothesis might therefore not represent the turnover dynamics accurately. (B) Global fitting of all data points (see supporting online text, error sum of squares = 1.2 × 104) shows an age-dependent decline of cardiomyocyte turnover.

Science 324:98-102, 2009

Fig. 4. (C) The gray area depicts the fraction of cardiomyocytes remaining from birth, and the white area is the contribution of new cells. Estimate is from the best global fitting. (D) Cardiomyocyte age estimates from the best global fitting. The dotted line represents the no-cell-turnover scenario, where the average age of cardiomyocytes equals the age of the individual. The black line shows the best global fitting. Colored diamonds indicate computed data points from 14C -dated subjects. Error bars in (A) are calculated from the errors on 14C measurements. Error bars in all other graphs are calculated for each subject individually and show the interval of possible values fitted with the respective mathematical scenario.

http://stemcells.nih.gov/info/

Postnatal neovascularization in physiological or pathophysiological events is through the processes of angiogenesis and vasculogenesis. Angiogenesis = EC activation. Vasculogenesis = BM EPC mobilization and activation

Isolation of Putative Progenitor Endothelial Cells

for Angiogenesis

Takayuki Asahara, Toyoaki Murohara, Alison Sullivan, Marcy Silver, Rien van der Zee, Tong Li,

Bernhard Witzenbichler, Gina Schatteman, Jeffrey M. Isner

Science 275:964-967, 1997

Fig. 1. Attachment, cluster formation, and capillary network development by progenitor ECs in vitro (A) Spindle shaped attaching cells (ATCD34+) 7 days after platingMBCD34+ (50 cells/mm2) on fibronectin in standard medium (14). (B) Number of ATCD34+ cells 12 hours and 3 days after culture of MBCD34+ on plastic alone (CD34+/non), collagen coating (CD34+/Col), or fibronectin (CD34+/Fn), and MBCD34- on fibronectin (CD34-/Fn).

Fig. 1. Attachment, cluster formation, and capillary network development by progenitor ECs in vitro Network formation (C) and cord-like structures (D) were observed 48 hours after plating coculture of MBCD34+, labeled with DiI, with unlabeled MBCD34- cells (ratio of 1:100) on fibronectin.

At 12 hours after coculture, MBCD34+-derived cells had formed multiple clusters (E and F). After 5 days, uptake of acLDL-DiI was detected in ATCD34+ cells at the periphery but not the center of the cluster (G and H).

Flow cytometric evaluation of EPC progression to EC-like phenotype, analyzing leukocyte and EC markers.

Fig. 3. Progenitor ECs express ecNOS, Flk-1/KDR, and CD31 mRNA and release NO(A) Complementary DNA (from 106 cells) was amplified by PCR (40 cycles) with paired primers (23) (B) NO release from ATCD34+ and ATCD34- cells cultured in six-well plates was measured as described (24). NO production was measured in a well with incremental doses of VEGF and Ach. HUVECs and bovine aortic ECs were used as positive controls, and human coronary smooth muscle cells (HCSMCs) as negative control. The values are means ± SEM of 10 measurements for each group.

Fig. 4. Heterologous (panels A to L), homologous (M), or autologous (panels N and O) EC progenitors incorporate into sites of angiogenesis in vivo. (A and B) DiI-labeled MBCD34+ (red, arrows) between skeletal myocytes (M), including necrotic (N) myocytes 1

week after injection; most are colabeled with CD31 (green, arrows). Note a preexisting artery (A), identified as CD31-positive, but DiI-negative. (C and D) Evidence of proliferative activity among several DiI-labeled MBCD34+-derived cells (red, arrows), indicated by coimmunostaining for antibody to Ki67 ( Vector Lab, Burlingame, California) (green). Proliferative activity is also seen among DiI-negative, Ki67-positive capillary ECs (arrowheads); both cell types contribute to neovasculature.

Fig. 4. (E) DiI (red) and CD31 (green) in capillary ECs (arrows in E and F) between skeletal myocytes, photographed through a double filter 1 week after DiI-labeled MBCD34+ injection. (F) A single green filter shows CD31 (green) expression in DiI-labeled capillary ECs

integrated into the capillary with native (DiI-negative, CD31-positive) ECs (arrowheads in E and F). (G) Immunostaining 1 week afterMBCD34+ injection showing capillaries comprising DiI-labeled MBCD34+-derived cells expressing Tie-2 receptor (green). Several MBCD34+-derived cells (arrows) Tie-2 positive and integrated with some Tie-2–positive host capillary cells (arrowheads) identified by the absence of red fluorescence. (H) Phase-contrast photomicrograph of the same section shown in (G) indicates the corresponding DiI-labeled (arrows) and -unlabeled (arrowheads) capillary ECs.

(I and J) Six weeks after administration, MBCD34+-derived cells (red, arrows) colabel for CD31 in capillaries between preserved skeletal myocytes (M). (K and L) One week after injection of MBCD34-, isolated MBCD34--derived cells (red, arrows) are observed between myocytes but do not express CD31. (M) Immunostaining of β-Gal in a tissue section harvested from ischemic muscle of C57BL/6J,129/SV mice 4 weeks after the administration of MBFlk-1+ isolated from transgenic mice constitutively expressing β-Gal. (Flk-1 cell isolation was used for selection of EC progenitors because of the lack of a suitable antibody to mouse CD34.) Cells overexpressing β-Gal (arrows) were incorporated into capillaries and small arteries; these cells were identified as ECs by anti-CD31 and BS-1 lectin (16).

(N and O) Section of muscle harvested from rabbit ischemic hindlimb 4 weeks after administration of autologous MBCD34+ cells. Red fluorescence in (N) indicates localization of MBCD34+-derived cells in capillaries seen (arrows) in the phase contrast photomicrograph in (O). Each scale bar is 50 μm.

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Comments?

Ideas?

rsiggi@lsuhsc.edu