Stem Cells

Totipotent stem cells: can differentiate into any cell typein the body; generally cells from the first few divisions afterfertilization;

Pluripotent stem cells: descendents of totipotent stem cellswhich develop by about Day 4 after fertilization; can differentiate into any cell type other than totipotent cells

Multipotent stem cells: descendents of pluripotent stemcells and antecendents of specialized cells in particulartissues; Ex: hematopoietic stem cells found in bone marrow give rise to all forms of blood cells

Progenitor cells “unipotent stem cells”: can produce onlyone cell type; Ex: erythroid progenitor cells differentiateinto only red blood cells

Stem Cell Classifications

Leukocytes made in bone marrow and released into bloodstream.

Leukemia results from leukocytes becoming cancerous.

Treatment:

- Chemotherapy / radiation to destroy abnormal leukocytes - Bone marrow transplants from matching donor to provide healthy stem cells – go to bone marrow and replicate

Umbilical cord stem cells

- very high quality stem cells which are “immature” so that they are not as antigenic as more mature cells – no need for exact histocompatibility match

-in very small numbers; research to expand cultures

Useful in treating children with genetic diseases like sickle-cell anemia,thalassemia, and leukemia.

Acute Leukemia’sAcute Lymphoblast Leukemia (ALL)

Acute Myelogenous Leukemia (AML)Acute Biphenotypic Leukemia

Acute Undifferentiated Leukemia

Chronic Leukemia’sChronic Myelogenous Leukemia (CML)Chronic Lymphocytic Leukemia (CLL)

Juvenile Chronic Myelogenous Leukemia (JCML) Juvenile Myelomonocytic Leukemia (JMML)

Myelodysplastic SyndromesRefractory Anemia (RA)

Refractory Anemia with Ringed Sideroblasts (RARS) Refractory Anemia with Excess Blasts (RAEB)

Refractory Anemia with Excess Blasts in Transformation (RAEB-T)

Chronic Myelomonocytic Leukemia (CMML)

Stem Cell DisordersAplastic Anemia (Severe)

Fanconi Anemia Paroxysmal Nocturnal Hemoglobinuria (PNH)

Pure Red Cell Aplasia

Myeloproliferative DisordersAcute Myelofibrosis

Agnogenic Myeloid Metaplasia (myelofibrosis) Polycythemia Vera

Essential Thrombocythemia

Lymphoproliferative DisordersNon-Hodgkin's Lymphoma

Hodgkin's Disease

Other Inherited DisordersLesch-Nyhan Syndrome Cartilage-Hair Hypoplasia Glanzmann Thrombasthenia OsteopetrosisAdrenoleukodystrophy

Inherited Platelet AbnormalitiesAmegakaryocytosis / Congenital Thrombocytopenia

Inherited Metabolic DisordersMucopolysaccharidoses (MPS) Hurler's Syndrome (MPS-IH) Scheie Syndrome (MPS-IS) Hunter's Syndrome (MPS-II) Sanfilippo Syndrome (MPS-III) Morquio Syndrome (MPS-IV)Maroteaux-Lamy Syndrome (MPS-VI)Sly Syndrome, Beta-Glucuronidase Deficiency (MPS-VII) Adrenoleukodystrophy Mucolipidosis II (I-cell Disease)Krabbe Disease Gaucher's Disease Niemann-Pick Disease Wolman Disease Metachromatic Leukodystrophy

Histiocytic DisordersFamilial Erythrophagocytic Lymphohistiocytosis Histiocytosis-X Hemophagocytosis

Inherited Erythrocyte AbnormalitiesBeta Thalassemia Major Sickle Cell Disease

Inherited Immune System DisordersAtaxia-TelangiectasiaKostmann SyndromeLeukocyte Adhesion Deficiency DiGeorge Syndrome Bare Lymphocyte Syndrome Omenn's SyndromeSevere Combined Immunodeficiency (SCID) SCID with Adenosine Deaminase DeficiencyAbsence of T & B Cells SCID Absence of T Cells, Normal B Cell SCID Common Variable Immunodeficiency Wiskott-Aldrich SyndromeX-Linked Lymphoproliferative Disorder

Plasma Cell DisordersMultiple Myeloma Plasma Cell Leukemia Waldenstrom's MacroglobulinemiaAmyloidosis

Other MalignanciesEwing Sarcoma NeuroblastomaRenal Cell CarcinomaRetinoblastoma

Phagocyte DisordersChediak-Higashi Syndrome Chronic Granulomatous Disease Neutrophil Actin Deficiency Reticular Dysgenesis

Umbilical Stem Cord Cell Applications

78 cell lines being cloned / 17 cell lines available for NIH-funded research

NIH Human Embryonic Stem Cell RegistryResearch on human embryonic stem cell lines may receiveNIH funding if the cell line meets the following criteria:

- removal of cells from the embryo must have been initiated before August 9, 2001, when the President outlined this policy;

- the embryo from which the stem cell line was derived must no longer have had the possibility of developing further as a human being.

- the embryo must have been created for reproductive purposes but no longer be needed for them. Informed consent must have been obtained from the parent(s) for the donation of the embryo

- no financial inducements for donation are allowed.

LONDON - Scientists who were awarded Britain's first license for human cloning say they had succeeded in creating a cloned embryo for the first time in the country. The Newcastle University scientists said Thursday they had successfully produced an early stage embryo cloned from a human cell using nuclear transfer. Britain, which four years ago became the world's first country to license cloning to create stem cells, is aiming to join South Korea on the leading edge of the research, which many scientists believe may lead to new treatments for a range of diseases.

British scientists said to clone human embryo

May 2005

Stem cell experts seek rabbit-human embryo

· Hybrid will hasten research, say scientists· Grey area exposed in regulation procedures

January 2006

- 5 year window during which research can be conducted.

- Most of the stem cells will be obtained from 6-day old fertilized human eggs.

-currently 118,379 embryos in storage (55% may be used in the future in IVF treatment); 25% are no longer needed.

France now places itself in between the UK and the USA. In the UK stem cell research and cloning are both allowed. The US only allows stem cell research in privately funded laboratories - cloning is prohibited.

                                             

                                  

                                             

                                  

                                             

                                  

                                             

                                  

“France welcomes human stem cell research…”

"permissive"

United Kingdom, Belgium, Sweden, Iran, Israel, India, Singapore, China, Japan, South Korea, South Africa

"flexible" - derivations from fertility clinic donations only, excluding SCNT, Research is permitted only on remaining embryos no longer needed for reproduction Australia, Brazil, Canada, France, Spain, The Netherlands, Taiwan

- various embryonic stem cell derivation techniques including somatic cell nuclear transfer (SCNT), also called research or therapeutic cloning

- completely prohibited to permitting research on a limited number of previously established stem cell lines Austria, Ireland, Norway, Poland, Germany, Italy, and the United States.

“restrictive”

a, Classical derivation of embryonic stem (ES) cells destroys the embryo from which they are derived.

a, Classical derivation of embryonic stem (ES) cells destroys the embryo from which they are derived. b, Modified method does not compromise the embryo, but is not donor specific.

a, Classical derivation of embryonic stem (ES) cells destroys the embryo from which they are derived. b, Modified method does not compromise the embryo, but is not donor specific. c, Donor-specific pluripotent stem cells made using nuclear transfer (NT) techniques. [Therapeutic cloning]

a, Classical derivation of embryonic stem (ES) cells destroys the embryo from which they are derived. b, Modified method does not compromise the embryo, but is not donor specific. c, Donor-specific pluripotent stem cells made using nuclear transfer (NT) techniques. d, Altered nuclear transfer (ANT) method blocks expression of the cdx2 gene until the blastocyst stage, making embryo unable to implant.

Deriving “Controversy-Free” Embryonic Stem Cells

Transplant to recipient to produce normal individual – routinely done in Fertility Clinics to test genetics before IVF

8-cell embryo

Production of stem cell lines 30% successful

lox P sites work inpairs and flank a DNAsegment (target)

Cre is an enzyme;binds to lox P sites & cuts them in half &splices the two halvestogether after removingtarget DNA

Viral Recombination System

Viral enzyme and bacteriophageDNA sequences (P1 bacteriophage)

Exon 3 mutant

Exon 3

Exon 3

Exon 3

Exons 2 & 3were sub-cloned& lox-P flankingsites inserted between Exons 2 & 3

Electroporesedin to ES cells

HomologousRecombination

Cre loop-outaccomplishedremoving mutation & leaving functionalExons 2 & 3

a, Primary tail-tip fibroblasts were infected with a conditional lentiviral RNA interference (RNAi) construct targeting Cdx2 before nuclear transfer (NT). Blastocysts deficient for Cdx2 were morphologically abnormal and unable to implant but gave rise to NT-ESCs.

b, The shRNA, which targets nucleotides 1890–1908 located in the 3' UTR of Cdx2, was cloned into the conditional RNAi vector generating pSicoR-Cdx22Lox. This vector carries the Cdx2 shRNA construct and an enhanced green fluorescence protein (EGFP) gene flanked by two LoxP sites (2Lox), which allows for Cre-mediated deletion of the shRNA and the EGFP sequences.

Generation of nuclear transfer-derived pluripotent ES cell from cloned Cdx2-deficient blastocystsAlexander Meissner and Rudolf Jaenisch, Nature 439, 212-215 (12 January 2006)

“Altered nuclear transfer” (ANT) approach.

- Cdx2 gene encodes trophectoderm-specific transcription factor activated in 8-cell embryo, essential for establishment of trophectoderm.

- Cdx2-deficient blastocysts form ICMand were capable of generatingpluripotential ES cells in tissue culture.

“Lack of Cdx-2 is not a deficiency but an insufficiency.”

Cdx2deficient

Cdx2 functional

“Holy Grail” of Stem Cell Production

Adult stem cells

“Transplantation of embryonic stem cells into the infarcted mouse heart:formation of multiple cell types.” Dinender K. Singla et al. (2006)

Fig. 1. ES cells differentiate into cardio-myocytes, smooth muscle cells andendothelial cells. A beating area was stained with anti-myosin (a), anti-eGFP(green fluorescent protein vector) (b) and TOPRO (nuclear stain)(c). The merged image (c) shows co-expression of myosin and eGFP in cardiomyocytes.

Smooth muscle cells were stained with anti-smooth muscle α actin(d), anti-eGFP(e) and TOPRO (f). The merged image (f) shows overlap of expression of smooth muscle α actin and eGFP.

Endothelial cells that have formed a capillary-like structure were stained with anti-von Willebrand Factor (glycoprotein produced by platelets to hold

platelets together)(g), anti-eGFP(h) and TOPRO (i). The merged image (i) shows co-expression of von Willebrand Factor and eGFP.

NEUROSCIENCE

Development of functional human embryonic stem cell-derived neurons in mouse brain. Alysson R. Muotri et al.   December 20, 2005 | vol. 102 | no. 51 | 18644-18648

- hESC cells transfected to express EGFP (enhanced green fluorescent protein);

-hESC cells injected into lateral ventricle of brain

- pregnancy allowed to continue

Day 14 mouse embryo

@ 1 – 2 months post injection mouse brains were sectioned at 100 µm and viewed with fluorescent confocal microscope at 0.5 µm;

@ 18 months post injection brain slices prepared to measure electropotential of EGFP-expressing neurons;

Copyright ©2005 by the National Academy of Sciences

Muotri, Alysson R. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 18644-18648

Fig. 2. Widespread chimerism of hESC in the mouse developing brain visualized by EGFP fluorescence

3D reconstruction of1 µm confocal sections

astrocytes

oligodendrocytes neurons

Copyright ©2005 by the National Academy of Sciences

Muotri, Alysson R. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 18644-18648

Fig. 3. Maturation of neuron-derived hESC

Enlarged images ofdendrites

Copyright ©2005 by the National Academy of Sciences

Muotri, Alysson R. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 18644-18648

Fig. 4. hESC differentiate into functionally mature neurons in the mouse brain

Whole cell recording from neuron

- hESC can differentiate in to neuronal and glial cells in proper environment

- Demonstrates regional specificity

- Evidence for differentiation in striatum of rat model for Parkinson’s disease

- Demonstrates conservation of differentiation signals from mouse to man

Conclusions:

- aggregates of cells derived from embryonic stem cells.

-aggregation imposed by hanging drop or plating upon non-tissue culture treated plates;

-prevents cells from adhering to a surface to form the typical colony growth.

Upon aggregation, differentiation is initiated and the cells begin to a limited extent to recapitulate embryonic development.

Though they cannot form trophectodermal tissue (which includes the placenta), cells of virtually every other type present can develop.

Embryoid bodies

“Correction of a Genetic Defect by Nuclear Transplan- tation and Combined Cell and Gene Therapy” William M. Rideout, III et al. (5 April 2002)

Culture tail tip cells

Nuclear transfer intoenucleated oocyte

Activate and cultureto blastocyst

Isolate isogenicRag2 -/+ ES cells

Repair Rag2 genein ES cells

Differentiateinto embryoid bodies

Dissociate embryoidbodies and infect withHoxB4iGFP

Expand HSC cultureand transplant

Fig. 21.25, pg. 711

Therapeutic Coning combined with Gene Therapy Rag2- severe immune deficiency

Complete absence of mature B& T cells, and immunoglobulins

- Today’s Stem Cell Research – Stem Cell Research Medical and Health News

A new drug, SP-sc04, triggers normally dormant brain neuronal stem cells to differentiate into adult neuron cells.

Recently discovered primitive stem cell very similar to embryonicstem cells; adult stem cells can differentiate into nearly any type oftissue, even spermatogonia.

March 2006

Moraga Biotechnology Corporation: Primitive adult stem cells found in peripheral blood -discovery of Blastomere-Like Stem Cells (BLSCs) circulating in the peripheral blood of mammals.

-these adult stem cells were able to differentiate into most tissues and organs of the body.

April 2006

Stem cells can repair damaged spinal tissue and help restore function in rats with spinal cord injuries, according to a new study. The findings may eventually lead to insights that result in new treatments for humans with spinal cord injuries.

Michael Fehlings, MD, PhD, and his colleagues at the Krembil Neuroscience Center at Toronto Western Research Institute and the University of Toronto also identified a critical window during which stem cell transplants may be effective, says the study, which appears in the March issue of The Journal of Neuroscience.

"This work breaks new ground by showing that therapeutically useful stem cells can be derived from the adult brain of rodents, and that these cells can be caused to differentiate into the types of cells that are useful for repairing the damaged spinal cord," says Oswald Steward, PhD, director of the Reeve-Irvine Research Center for Spinal Cord Injury at the University of California, Irvine.

Fehlings' team used cells from the brains of adult mice labeled with a fluorescent marker, enabling them to trace the cells after they were transplanted into rats whose spines had been crushed. Stem cells transplanted up to two weeks after the initial injury survived thanks to a cocktail of growth factors and immune-suppressing drugs the team developed. More than one-third of the transplanted cells traveled along the spinal cord, were incorporated into damaged tissue, developed into the type of cell destroyed at the injured site, and produced myelin, an insulating layer around nerve fibers that transmits signals from the brain.

March 30, 2006: Stem Cell Treatment Succeeds In Spinal Cord-injured Rats

- A single dose of adult donor stem cells given to animals that have neurological damage similar to that experienced by adults with a stroke or newborns with cerebral palsy can significantly enhance recovery from these types of injuries.

- Using a commonly utilized animal model for stroke, researchers administered a dose of 200,000-400,000 human stem cells into the brain of animals that had experienced significant loss of mobility and other functions. The stem cells used in the study were a recently discovered stem cell type, referred to as multipotent adult progenitor cells, or MAPCs.

April 12, 2006: Stem cell transplants improve recovery in animal models for stroke, Cerebral Palsy

April 5, 2006: Using Stem Cells To Repair Torn Tendons

- adult stem cells can make new tendon tissue;

- engineered mouse mesenchymal stem cells, which reside in the bone marrow and fat tissue, to express a protein called Smad8 and another called BMP2, each involved in the formation of bone and cartilage.

- filled small sponges with these cells and implanted the sponges into torn Achilles tendons of rats;

- found that the cells not only survived the implantation process, but were able to invade the injury and repair the tendon for at least 7 weeks after implantation.

March 2007:Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic diseaseLee et al., Nature Medicine 13:439-447.

Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of diseases similar to Parkinson’s, ALS, and Alzheimer’s. Implanted stem cells migrated and integrated throughout the brain; - replaced damaged nerve cells and transmitted nerve impulses - boosted enzyme activity reducing lipid accumulations - dampened inflammation

February 2007:Mice cloned from skin cells. Li et al. PNAS 104:2738-2743

- use nuclei from hair follicle stem cells and other skin keratinocytes as NT donors

- skin a source of readily accessible stem cells, the nuclei of which can be reprogrammed to the pluripotent state by exposure to the cytoplasm of unfertilized oocytes.

Male mice have a constant supply of sperm-generating stem cells.

- Developed a transgenic mouse line labelling sperm stem cells identified by spermatogonia-specific marker (Stra8) with green fluorescent dye [only ~ 0.3% of mouse testis cells are stem cells]

- Expanded cells and exposed to a variety of culture conditions which demonstrated their embryonic stem cell nature and ability to form embroid bodies [multipotent adult germline stem cells (maGSCs)]

Pluripotency of spermatogonial stem cells from adult mouse testisKaomei Guan et al. (24 March 2006)

a, Epiblast-like colony formed under culture condition I.

b, ESC-like colonies appeared under culture condition II.

c, A typical colony of established culture under condition IV at passage 30.

d–g, Double immunostaining of maGSCs in culture condition IV (d–f) or condition II (g) with antibodies against GFP (green, d–g) and SSEA-1 (red, e), Oct4 (red, f) or SSEA-3 (red, g).

h, i, Alkaline phosphatase staining. SSCs cultured under condition IV (maGSCs, h) are strongly positive for alkaline phosphatase,

j, k, RT–PCR analyses of transcription factors essential for undifferentiated cells in SSCs cultured under conditions I, II, III and IV (j) and during differentiation of embryoid bodies after plating at day 5 (d5; k). M, 100-bp DNA markers; numbers to the right indicate the sizes of the resolved DNA fragments (in bp). Scale bars, 50    m (a–c, h, i), 25   m (d–g).

Analyses were performed at different stages during the differentiation of embryoid bodies after plating at day 5 (d5). M, 100-bp DNA markers. d0, maGSCs before embryoid body formation.

Organization of the sarcomeric proteins:

 (a) actinin

(b) sarcomeric MHC

(c) cardiac troponin T in isolated cardiomyocytes

(d) Connexin 43 staining in a cluster of uninucleate cardiac cells stained for sarcomeric-actinin (e, red)

(g) Original traces of ventricle-like action potentials in a cardiomyocyte derived from maGSCs.

(h) Nebulin-positive myotubes at differentiation

(k) Vwf-positive endothelial cells (red)

(l) Smooth muscle-actin-positive cells (red) of tube-like structure in embryoid body outgrowths