Strategies for enhancing neural cell efficacy in the host cns

1. 29th Annual National Neurotrauma Symposium Reconstructing the Injured Brain & Spinal Cord with Stem Cells July 13, 2011 – Hollywood Beach, Florida Strategies for Enhancing Neural Stem Cell Efficacy in the Host CNS Evan Y. Snyder, MD, PhD, FAAP Professor; Director, Program for Stem Cells & Regenerative Biology Director, Stem Cell Research Center Sanford-Burnham Institute for Medical Research Depts. of Pediatrics (Newborn Intensive Care) & Neurology/Neuroscience, University of California, San Diego Biomedical Sciences Graduate Program, University of California, San Diego Steering Committee, Sanford (San Diego) Consortium for Regenerative Medicine Associate Member, Sanford Pediatric Research Center

3. Neurotraumatic Conditions (“The Chaperone Affect”)

4. Stroke/hypoxic-ischemic injury

5. Spinal Cord Injury / Traumatic Brain Injury

6. Not just diffusible factors but cell-cell contact via gap junctions, as well

7. Improved “homing”/pathotropism

8. IN VITRO --“Disease in a Dish”

9. Modeling development, injury, disease, ?therapy • hESCs & HIPSCs into “neural tubes” ==> ultimately into hNSCs) • neural-vascular obligate co-patterning/co-dependence – driven by neural crest • high-throughput drug discovery • large scale comprehensive profiling

11. including stem cell-mediated ‘rescue’/ ‘protection’ / ‘regeneration’ / altered gene expression / creation of more supportive milieu Complementing ‘cell replacement’

12. Host NSC NSC

14. Host NSC influence differentiation fate NSC

16. …Or might be biased to yield this neural cell type in a neuron deficient environment

18. Park et al, 2006

19. Park et al, (2006)

20. Park et al, Nature Biotech (2002)

22. Neurons (oligos) (astros) (NPs)

24. Host NSC NSC

25. G G GDNF GDNF N N N Ret Ret GDNF GDNF GDNF GDNF GDNF GDNF Host NSC

26. Host NSC NSC

27. Host NSC NSC Diffusible Factors

28. Host NSC NSC Lysosomal Enzymes

29. Host NSC NSC Anti- Inflammation

30. Host NSC NSC Protection

31. Host NSC NSC Anti- Scarring

32. Host NSC NSC Pro- Mobilization

33. Host NSC NSC Pro- Neurite Outgrowth

34. Host NSC NSC Pro- Angiogenic

36. Parkinson’s Disease (Nat Biotech ‘02; PNAS ‘07; Stem Cells ‘09)

37. Neurogenetic degeneration (Nat Med ‘07; Stem Cells ‘09)

38. Some aging-related degeneration

39. Spinal cord injury & Head trauma (PNAS ‘02; PNAS’10)

40. Stroke / Hypoxic-Ischemia (Nat Biotech ‘02; Exp Neurol ’06; PNAS’11)

41. Cerebellar Degeneration (J Neurosci ‘06; PNAS ‘06; PNAS’10)NSC-mediated‘rescue’/ ‘protection’ / ‘regeneration’/anti-inflammation-- complementing ‘cell replacement’Neural stem cells display an

42. (Infarcted) (Intact) Park et al, Nature Biotech (2002)

43. Park et al, Nature Biotech (2002)

45. Diminished Scarring in stroke following human NSC Implantation (supported by scaffold) Kook In Park

46. Host NSC NSC Cell-Cell Contact

47. Host NSC NSC Cell-Cell Contact (gap junctions)

48. Host NSC NSC Cell-Cell Contact (gap junctions)

49. Early neuronal membrane properties emerge in NSCs during differentiation in slice Ability to generate action potential Jaderstad et al, PNAS ‘10

50. Spreading Calcium Waves Jaderstad et al, PNAS (2010)

51. Role of calcium fluctuations Apoptosis Astrocytic control of cerebral micro-circulation Synaptic information processing by astrocytes Cell to cell signalling Neuron ↔ neuron Glia ↔ glia Glia ↔ neuron Cell cycle regulation Cell migration timing Differentiation

52. Expression of first Connexin 43 & then Connexin 26 are important components in gap junction formation DAP1 GFP Tuj1 Cx26 Jaderstad et al, PNAS (2010)

53. Gap Junctions

54. Functional Gap-junctional Intercellular Channels Jaderstad et al, PNAS ‘10

55. Pharmacological Suppression of Gap Junction Function(via carbenoxolone [CBX] or 18--glycyrrhetinic acid [18--GA]) Blocks Dye Transfer to Host Cells in Engrafted Slice Cultures Jaderstad et al, PNAS ‘10

56. Pharmacological Suppression of Gap Junction Function(via carbenoxolone) Blocks Glutamate-Initiated Calcium Influx in Host Cells of Engrafted Slice Cultures Jaderstad et al, PNAS (2010) Eric Herlenius

57. Cell Death(i.e. % of Propidium Iodide+ host striatal cells)decreases when NSCs making gap junctions are present Without NSCs With NSCs PROPIDIUM IODIDE+ cells (With NSCs) GFP/ DAPI / PROPIDIUM IODIDE (No NSCs) Jaderstad et al, PNAS ‘10

58. Even SmallSuppression of Connexin 43 Expression(via siRNA)--& henceGap Junction Formation--Abrogates a Component ofNSC’s Beneficial Impact on Host Cells(i.e., it’s anti-gliotic scar effect) Jaderstad et al, PNAS ‘10

59. Jaderstad et al, PNAS ‘10

61. Parkinson’s Disease (Nat Biotech ‘02; PNAS ‘07; Stem Cells ‘09)

62. Neurogenetic degeneration (Nat Med ‘07; Stem Cells ‘09)

63. Some aging-related degeneration

64. Spinal cord injury & Head trauma (PNAS ‘02; PNAS’10)

65. Stroke / Hypoxic-Ischemia (Nat Biotech ‘02; Exp Neurol ’06; PNAS’11)

66. Cerebellar Degeneration (J Neurosci ‘06; PNAS ‘06; PNAS’10)NSC-mediated‘rescue’/ ‘protection’ / ‘regeneration’/anti-inflammation-- complementing ‘cell replacement’Neural stem cells display an

67. 50 µm 0.5 cm o. Cerebellar Purkinje Cell Neuron Degeneration Mutants Li et al, PNAS & J Neurosci 2006

68. Calbindin Purkinje Cell Layer evident following early transplantation of neural stem cells

69. Intercellular Contacts of NSCs with Nervous Mutant Purkinje Neurons bgal (NSC) Calb (PN) Dapi (nuclei) Li, et al, PNAS; J. Neurosci. (2006)

70. Host Nr Purkinje Neurons (PNs) Remain At Nearly Wild Type Numbers Following Early NSC Transplantation Li, et al, PNAS; J. Neurosci. (2006)

71. 70 60 y = 0.5032x - 4.8333 50 2 R = 0.688 (n=8) P<0.05 40 30 Rotarod test (seconds) 20 10 0 0 20 40 60 80 100 Purkinje cell survival (%) Positive Correlation between NSC-Rescued Host PNs & Improved Motor Behavior Li, et al, PNAS; J. Neurosci. (2006)

72. Influence of NSCs upon Nr PNs (via cell-cell contact) appears to be mediated by gap junctions (particularly those employing Cx 43) Jaderstad et al, PNAS ‘10

73. Blocking Gap Junction Formation(with Cx43 RNAi)abrogatesNSC-mediated neuronal rescue Jaderstad et al, PNAS (2010)

74. WHAT ARE THE GAP JUNCTIONS DOING?

76. TPA in its role as upstream of a number of signal transduction pathways

77. e.g., neurotrophic factor handling; mitochondrial function

78. Nervous mutant has abnormally high TPA levels

79. Abnormalities emulated by  TPA

80. NSC intercession (somehow) restores (lowers) level to normalA different way of viewing “gene therapy” -- homeostatic pressure to return an endogenous gene to equipoise -- not too high & not too low Surviving Purkinje Neurons (as % of wild type control nr NSCs A logarithmic-parabolic relationship between tPA activity & PN survival in cerebellarorganotypic slice cultures Li, et al, PNAS; J. Neurosci. (2006)

81. Nervous Cerebellum NSC tPA  Plasminogen Plasmin Mitochondrial VDAC Neurotrophins BDNF, NT3 PKC Energy  Metabolism Dendrite & Axon Development PN Survival More Balanced More Normal Motor Behavior Li, et al, PNAS; J. Neurosci. (2006)

83. NSCs Restore/Improve Expression of Key Neurotrophic factors in Nr Cerebella cultured NSCs undifferentiated nsc C a nt3/bgal/dapi bdnf undif.nsc dif.nsc b BDNF NT3 undifferentiated nsc c young wt nr/nr nr+nsc adult wt nr/nr nr+nsc trkc/dapi trkb BDNF NT3 wtnr wt nr/nrnr + nsc (downstream targets of TPA/plasmin system) A EGL BDNF/Calb / Dapi NT-3/Calb / Dapi B Wt nr/nrnr+nsc BDNF NT-3 NOTE: receptors notabnormal Li, et al, PNAS; J. Neurosci. (2006)

85. wt nr + NSC nr/nr 256±63 nm diameter 274±82 nm diameter 585±95 nm diameter Mitochondrial Morphological Abnormalities Reversed Following Early NSC Transplanation

86. Nervous Cerebellum tPA Inhibitor tPA Plasminogen Plasmin Mitochondrial VDAC Neurotrophins BDNF, NT3 PKC Energy Metabolism Dendrite & Axon Development PN Survival PKC Antagonist More Normal Motor Behavior Li, et al, PNAS; J. Neurosci. (2006)

87. Jaderstad et al, PNAS (2010)

88. Gap junction formation (Cx43) reciprocally between hNSC-derived neurons in cervical region of contused adult rat spinal cord associated with improved respiratory function Jaderstad et al, PNAS (2010)

89. Summary • Direct cell-cell contact between stem cells & other neural cells, mediated by gap junction coupling, represents not only an early form of inter-cellular communication, establishing a template for more mature electrochemical synaptic interfaces, but also mediateshomeostasis-promoting actions (including metabolic/ mitochondrial) • Exogenous stem cells may “rescue” endangered cells by this mechanism • Mechanismof rescue is not known • Could involve the passage of beneficial molecules • Could involve the containment of toxic molecules from inter-cellular spread • Is likely a pervasive mechanism throughout the stem cell“world” & in development (& disease/therapy)

90. Summary of Stem Cell-Mediated Therapeutic Actions TISSUE (e.g., CNS) Damage or Degeneration Stem Cell Engraftment (e.g., NSC) Paracrine support by secreted factors Replacement of lost Cells Induction of beneficial [Ca++]i signaling patterns Intercellular exchange of ions & molecules RESCUE REPLACEMENT

91. Host NSC NSC

96. Host NSC Molecules That effect HOST NICHE NSC

98. Neurotraumatic Conditions (“The Chaperone Affect”)

99. Stroke/hypoxic-ischemic injury

100. Spinal Cord Injury / Traumatic Brain Injury

101. Not just diffusible factors but cell-cell contact via gap junctions, as well

102. Improved “homing”/pathotropism

103. IN VITRO --“Disease in a Dish”

104. Modeling development, injury, disease, ?therapy • hESCs & HIPSCs into “neural tubes” ==> ultimately into hNSCs) • neural-vascular obligate co-patterning/co-dependence – driven by neural crest • high-throughput drug discovery • large scale comprehensive profiling

105. Stem Cells as “Instructional Tools”: Models of Embryogenesis, Organogenesis, Homeostasis-preserving Mechanisms Intrinsic Developmental Programs

106. PluripotentStem Cells as “Diseases-in-a-Dish” Blastocyst Blastocyst (can be from a diseased embryo diagnosed by PGD) Embryonic stem (ES) cells Neural stem cell (NSC) Morphogens ECM Fibroblasts Transcription factors; Proteins; Plasmids; RNA; Small molecules; microRNA Patient skin cells Induced pluripotent stem (iPS) cells

107. Stem Cells As In Vitro Models of Disease “Disease-in-a-Dish” * “diseased” cells, i.e., cells obtained from patients with particular diseases* “normal” cells that are stressed or perturbed in prescribed ways - To study underlying mechanisms of disease- To identify drug targets diagnostics prognostics- To identify protective agents and/or mechanisms- To identify & test drugs or “leads”-to-drugs hIPSCs particularly appealing for studying cell types that are difficult to obtain from a living patient

108. - - Screening Drug-like Small Molecules, in a Robotic High-Throughput Manner, to Reveal Mechanisms, Identify Novel Therapeutic Targets, and/or Discover New Drugs Add compounds Incubate plates Score hits on automated microscope

109. High-content / High-throughputDrug Discovery Proof-of-Concept: Screening a random library in an unbiased manner for neuroprotective small molecules IlyasSingec, Evan Snyder, Susanne Heynen, Michael Jackson

110. In the fully developed human body plan, the vascular & nervous systems are (must) be co-patterned How does that come about? ?An early program in embryogenesis that is sustained throughout development & growth

111. Some Implications Prototype of how 2 germ layers co-pattern (in an obligatory manner?) to give rise to the “real” body plan where lineages do not exist in isolation but must coordinate with each other Ectoderm & mesoderm& endoderm all must talk to each other “Cross-talk” as one of “Nature’s strategies” for organogenesis Important to recognize when thinking about repair ?Help explain “mysteries” in “normal development” e.g., brain vascularized before blood flow -- is this how? Developmental anomalies -- ?Help explain associations in some syndromes Neural with vascular / vascular with neural (e.g., Sturge-Weber? T-S? NF?) Reinforces notion that “repair strategies” may need to reinvoke “developmental strategies” When thinking about “regeneration” must be cognizant of “all lineages” Many pathologic entities injure multiple systems -- e.g., stroke, trauma, infection, inflammation

113. Trophic Support(endogenous or following genetic enhancement)

114. Neuroprotection(e.g., against oxidative stress; apoptosis)

115. Reducing inflammation

116. Restore intracellular mitochondrial-mediated homeostasis

117. Promote intercellular transfer of molecules via gap junctions

118. e.g., aid degradation of protein aggregates

119. Secrete or transfer other yet-to-be identified molecules

120. that might alter gene expression within damaged host cell

121. AS VEHICLES FOR EXOGENOUS GENE DELIVERY

123. Thanks IlyasSingec Ted Teng Eric Herlineus Richard Sidman Andrew CrainHelen Blau & Renee Reijo-Pera Brian TobeMassimo Pandolfo & SatyanChintawar Kook In Park ZeweiHuang NejmiDilmac David Cheresh Mahesh Lachyankar Peter Black & XandraBreakefield Jochen MaurerSteve Ashwal & Andy Obenaus SaharNissim Jeff Macklis Runquan Zhang Ralph Weissleder & Khalid Shah Karen AboodySamiaKhoury & Jaime Imitola Dustin Wakeman Jeff Rothstein & Bob Brown Jean-Pyo Lee Bill Weiss & Anders Persson Jitka & Vaclav Ourednik Gene Redmond & John Sladek Jeff Lindquist Bob Langer & Erin Lavik Xuejun (Jun) Parsons Fran Platt & Tom Seyfried Franz-Josef Muller Ernest Arenas & Carmen Salto Jianxue Li Tony Atala & Rene Yiou