This presentation focuses briefly on neural stem cells, the road map of neurogenesis, the markers as well the controversy involved in neurogenesis - that arose in the year 2018.

2. WHAT ARE NEURAL STEM CELLS?
Multipotent cells with the
ability to self-renew and
proliferate in order to give rise
to progenycells

4. HISTORY OF NEURAL STEM CELLS
THE NEURON DOCTRINE – each
neuron is an individual entity that
forms the basic unit of neural circuitry.
This was presented by Santiago
Ramón y Cajal in 1894.
“Intheadult…thenervepathsaresomething
fixed,ended,and immutable.Everythingmay
die,nothingmayberegenerated.”
The first evidence of
adult brain neurogenesis
came from JOSEPH
ALTMAN in the 1960s
from his studies in rats.

5. INITIAL WORK ISOLATION
Initial works in 1958 (Messier and Leblond) and 1961 (Smart) employed
the use of tritiated thymidine for labeling brain cells of mice 
radioactivity in the subventricular zone (SVZ) of the brain, the
hippocampus, cerebral cortex and fornix .
In 1989, DR SALLY
TEMPLE was able to
isolate neural stem cells
from rodent brain and
culture them in vitro.

6. THE ROAD MAP – HISTORICAL PERSPECTIVE
EXPERIMENT BY YEAR FINDINGS
LeBlond and Smart 1961 Tritiated thymidine labeling of cells- Discovered
neuroglial cells in mouse brain
Altman and Das 1965, 1967 Neurogenesis in adult brain of rats and guinea pigs,
respectively
Kaplan and Hinds 1977 Adult-born neurons in dentate gyrus and olfactory bulb
coupling tritiated thymidine and electron microscopy
Goldman and Nottebohm 1983 Neurogenesis in song birds (canaries)
Rakic 1985 Neurogenesis in monkeys happened only in early age and
not in adults
Reynolds and Weiss 1992 Used certain growth factors
Eriksson et al. 1998 Neurogenesis in adult human hippocampus in cancer
patients
Kornack and Rakic 1999 Re-examined neurogenesis in macaque monkey and
confirmed neurogenesis in the DG
Sorrells et al. 2018 Neurogenesis occurs only in the young, and not in adults
Boldrini et al. 2018 Neurogenesis occurs in adults, including old individuals

7. ROSTRAL MIGRATORY STREAM
Experiment by Altman in rats showed radially-oriented
streaks of cells in apparent migration. They were most
prominent around the rostra1 wall of the anterior horn
of the lateral ventricle and connected the lateral and
olfactory ventricles. This L-shaped structure, which
first moves vertically and then horizontally,
penetrates the laminated olfactory bulb
and is known as Rostral Migratory Stream.
Smart in his experiment had established that the
subependymal layer extended in mice from the anterior
wall of the lateral ventricle rostrally into the olfactory
bulb.
RMS

8. MARKERS
Some researchers theorize that the SGZ is a conducive
environment for the proliferation of NSCs into granule
cells, from which they migrate to the granule cell layer.
Quiescent radial glia-like type I neural
progenitor cells (QNPs)
Type II Intermediate neural progenitor cells (INPs)
Type III Intermediate neural progenitor
cells (INPs)
Immature neurons
Mature granule neurons that integrate into
the hippocampal circuitry
SOXBLBP
2, NESTIN,
Ki67, NESTIN
Lose expression of neural stem
cell markers and gain expression
of neuronal markers
DCX, PSA-NCAM
NeuN, β-III TUBB,
CALBINDIN

9. MARKERS OF NEURAL STEM CELLS
Exclusively found in
vertebrates
Belongs to the family of
Intermediate Filament proteins
Role : Self renewal of neural
stem cells
NESTIN
Green Fluorescent Protein is an excellent marker of
neural stem cells, so when the Nes/GFP screening was
done, it was seen that the Nes –/– individuals had
very low expression of GFP indicative of the fact that
absence of Nestin caused neural stem cells to
differentiate

10. SOX2
 Transcription factor
 Role: Important for maintenance of neural stem cells
Sox2 helps in hippocampal development as well. When Sox2
was deleted in mice and postnatal observations were done. This
was observed :

11. The defective hippocampal development was seen to mimic the
mutation of Shh or of its receptor Smo.
The absence of Shh causes an arrest of postnatal hippocampal
development which closely resembles that of Sox2 knockout
mouse, strongly suggesting that Sox2 controls hippocampal
development via regulation of Shh.

12. β- CATENIN
Role: Determines neural stem cell
state
Wnt signaling is a conserved cell
signaling system that is thought to
play multiple roles in NSC self-
renewal, neurogenesis, embryonic
patterning, and homeostatic tissue
regeneration in the developing and
adult brain.
CELL PROLIFERATION CELL DIFFERENTIATION

13. OTHER NEURAL STEM
CELL MARKERS
GFAP – marks astrocytes
NeuN, βIII-tubulin – mark neurons
GalC – marks oligodendrocytes
DIFFERENTIATION
MARKERS
Musashi-1, Musashi-2, Pax3, Pax6,
Hes1, Hes5, CD133, CD15, Vimentin

14. CONTROVERSY
Sorrells et al. found a sharp
drop in neurogenesis as the
human brain ages
Boldrini et al. find persistent
adult neurogenesis in humans
into the eighth decade of life

15. SORRELLS
Maps of Ki-67+ (green) cells in the DG from samples of individuals that
were between 22 gestational weeks and 35 years of age; GCL in blue.
Ki-67+SOX1+ and Ki-
67+SOX2+ cells
(arrows) are distributed
across the hilus and GCL
and the number of
double-positive cells
decreases between 22
gestational weeks and 1
year of age.

16. DCX+PSA-NCAM+ cells in the
DG from birth to the age of 77
showing that the number of
young neurons declines in the
human DG

17. BOLDRINI
(A and B) Co-expression of
Sox2 and nestin in QNPs.
(E) GFAP+ cells with apical
process (white arrow) nestin+
INP (yellow arrow).
(F) Nestin/Ki-67+ INP.

18. (M) Sox2+ cell decline in anterior-mid DG with aging.
(N and O) Nestin+ and Ki-67+ cells do not decline with older age in anterior, mid, or
posterior DG.

19. (A) PSA-NCAM and DCX expression in an
immature neuron between subgranular zone
(SGZ) and granule cell layer (GCL), two
PSA-NCAM+/DCX cells, and 4’,6-
diamidino-2-phenylindole (DAPI)+ nuclei.
(G–H) Stable DCX+
immature neurons,
NeuN+ mature granule
neurons

20. ISSUES RAISED
Post Mortem Delay -
PMD.
Boldrini’s study sample
size age group
DCX as a marker?
Sorrells used patients who had
epilepsy
Use of 10% formalin
Gerd
Kempermann

21. There have been studies conducted after this controversy as
well (in 2019) that lean towards Boldrini’s conclusion that
neurogenesis does persist through an older age in healthy
individuals as well as in patients suffering from mild
cognitive impairments (MCI) and Alzheimer’s Disease.
Tobin et al. observe
persistent hippocampal
neurogenesis in aging
brains with no cognitive
impairments, mild
cognitive impairments,
and Alzheimer’s disease.
Elena P. Moreno-Jiménez et
al. observe persistence of
adult hippocampal neurons
during both physiological and
pathological aging in humans

22. REFERENCES
1. Smart, I., & Leblond, C. P. (1961). Evidence for division and transformations of neuroglia cells in the
mouse brain, as derived from radioautography after injection of thymidine-H3. The Journal of Comparative
Neurology, 116(3), 349–367. doi:10.1002/cne.901160307
2. Temple, S. (1989). Division and differentiation of isolated CNS blast cells in microculture. Nature,
340(6233), 471–473. doi:10.1038/340471a0
3. Park, D., Xiang, A. P., Mao, F. F., Zhang, L., Di, C. G., Liu, X. M., Shao, Y., Ma, B. F., Lee, J. H., Ha, K. S.,
Walton, N., & Lahn, B. T. (2010). Nestin is required for the proper self-renewal of neural stem cells. Stem
cells (Dayton, Ohio), 28(12), 2162–2171. https://doi.org/10.1002/stem.541
4. Gilyarov, A. V. (2008). Nestin in central nervous system cells. Neuroscience and Behavioral Physiology,
38(2), 165–169. doi:10.1007/s11055-008-0025-z
5. Neradil, J., & Veselska, R. (2015). Nestin as a marker of cancer stem cells. Cancer Science, 106(7), 803–
811. doi:10.1111/cas.12691
6. Thiel, G. (2013). How Sox2 maintains neural stem cell identity. Biochemical Journal, 450(3), e1–
e2. doi:10.1042/bj20130176
7. Favaro, R., Valotta, M., Ferri, A. L. M., Latorre, E., Mariani, J., Giachino, C., Lancini, C., Tosetti, V.,
Ottolenghi, S., Taylor, V., Nicolis, S. K. (2009). Hippocampal development and neural stem cell maintenance
require Sox2-dependent regulation of Shh. Nature Neuroscience, 12(10), 1248–1256. doi:10.1038/nn.2397
8. Okano, H., Kawahara, H., Toriya, M., Nakao, K., Shibata, S., & Imai, T. (2005). Function of RNA-binding
protein Musashi-1 in stem cells. Experimental Cell Research, 306(2), 349–
356. doi:10.1016/j.yexcr.2005.02.021
9. Kaneko, Y., Sakakibara, S., Imai, T., Suzuki, A., Nakamura, Y., Sawamoto, K., Ogawa, Y., Toyama, Y.,
Miyata, T., Okano, H. (2000). Musashi1: An Evolutionally Conserved Marker for CNS Progenitor Cells
Including Neural Stem Cells. Developmental Neuroscience, 22(1-2), 139–153. doi:10.1159/000017435
10. Mizrak, D., Brittan, M., & Alison, M. (2007). CD133: molecule of the moment. The Journal of Pathology,
214(1), 3–9. doi:10.1002/path.2283

23. 11. Pfenninger, C. V., Roschupkina, T., Hertwig, F., Kottwitz, D., Englund, E., Bengzon, J., Jacobsen, S. E., Nuber, U. A.
(2007). CD133 Is Not Present on Neurogenic Astrocytes in the Adult Subventricular Zone, but on Embryonic Neural Stem Cells,
Ependymal Cells, and Glioblastoma Cells. Cancer Research, 67(12), 5727–5736. doi:10.1158/0008-5472.can-07-0183
12. Sun, Y., Kong, W., Falk, A., Hu, J., Zhou, L., Pollard, S., & Smith, A. (2009). CD133 (Prominin) Negative Human Neural Stem
Cells Are Clonogenic and Tripotent. PLoS ONE, 4(5), e5498. doi:10.1371/journal.pone.0005498
13. Kageyama, R., Shimojo, H., & Ohtsuka, T. (2018). Dynamic control of neural stem cells by bHLH factors. Neuroscience
Research. doi:10.1016/j.neures.2018.09.005
14. Kim, E. J., Ables, J. L., Dickel, L. K., Eisch, A. J., & Johnson, J. E. (2011). Ascl1 (Mash1) Defines Cells with Long-Term
Neurogenic Potential in Subgranular and Subventricular Zones in Adult Mouse Brain. PLoS ONE, 6(3),
e18472. doi:10.1371/journal.pone.0018472
15. Bengoa-Vergniory, N., & Kypta, R. M. (2015). Canonical and noncanonical Wnt signaling in neural stem/progenitor cells.
Cellular and Molecular Life Sciences, 72(21), 4157–4172. doi:10.1007/s00018-015-2028-6
16. Israsena, N., Hu, M., Fu, W., Kan, L., & Kessler, J. A. (2004). The presence of FGF2 signaling determines whether β-catenin
exerts effects on proliferation or neuronal differentiation of neural stem cells. Developmental Biology, 268(1), 220–
231. doi:10.1016/j.ydbio.2003.12.024
17. Ohtsuka, T., Sakamoto, M., Guillemot, F., & Kageyama, R. (2001). Roles of the Basic Helix-Loop-Helix GenesHes1andHes5in
Expansion of Neural Stem Cells of the Developing Brain. Journal of Biological Chemistry, 276(32), 30467–
30474. doi:10.1074/jbc.m102420200
18. Cao, S., Du, J., Lv, Y., Lin, H., Mao, Z., Xu, M., Xu, M., Liu, M., Liu, Y. (2017). PAX3 inhibits β-Tubulin-III expression and
neuronal differentiation of neural stem cell. Biochemical and Biophysical Research Communications, 485(2), 307-
311. doi:10.1016/j.bbrc.2017.02.086
19. Liu, Y., Zhu, H., Liu, M., Du, J., Qian, Y., Wang, Y., Ding, F., Gu, X. (2011). Downregulation of Pax3 expression correlates with
acquired GFAP expression during NSC differentiation towards astrocytes. FEBS Letters, 585(7), 1014–
1020. doi:10.1016/j.febslet.2011.02.034
20. Gómez-López, S., Wiskow, O., Favaro, R., Nicolis, S. K., Price, D. J., Pollard, S. M., & Smith, A. (2011). Sox2 and Pax6 maintain
the proliferative and developmental potential of gliogenic neural stem cells In vitro. Glia, 59(11), 1588–
1599. doi:10.1002/glia.21201

24. 21. Lauder, J. M., & Krebs, H. (1978). Serotonin as a Differentiation Signal in Early Neurogenesis.
Developmental Neuroscience, 1(1), 15–30. doi:10.1159/000112549
22. Gusel'nikova VV, Korzhevskiy DE. NeuN As a Neuronal Nuclear Antigen and Neuron Differentiation
Marker. Acta Naturae. 2015 Apr-Jun;7(2):42-7. PMID: 26085943; PMCID: PMC4463411
23. Yang Z, Wang KK. Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to
neurobiomarker. Trends Neurosci. 2015 Jun;38(6):364-74
24. Tang, Y., Yu, P., & Cheng, L. (2017). Current progress in the derivation and therapeutic application of
neural stem cells. Cell Death and Disease, 8(10), e3108. doi:10.1038/cddis.2017.504
25. Gage, F. H., & Temple, S. (2013). Neural Stem Cells: Generating and Regenerating the Brain. Neuron,
80(3), 588–601. doi:10.1016/j.neuron.2013.10.037
26. Altman, J., & Das, G. D. (1965). Autoradiographic and histological evidence of postnatal hippocampal
neurogenesis in rats. The Journal of Comparative Neurology, 124(3), 319–
335. doi:10.1002/cne.901240303
27. ALTMAN, J., & DAS, G. D. (1967). Postnatal Neurogenesis in the Guinea-pig. Nature, 214(5093),
1098–1101. doi:10.1038/2141098a0
28. Altman, J. (1969). Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell
proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis
in the olfactory bulb. The Journal of Comparative Neurology, 137(4), 433–
457. doi:10.1002/cne.901370404
29. Morest, D. K. (1970). The pattern of neurogenesis in the retina of the rat. Zeitschrift F�r Anatomie
Und Entwicklungsgeschichte, 131(1), 45–67. doi:10.1007/bf00518815
30. Kaplan, M., & Hinds, J. (1977). Neurogenesis in the adult rat: electron microscopic analysis of light
radioautographs. Science, 197(4308), 1092–1094. doi:10.1126/science.887941

25. 31. Graziadei, P. P. C., & Graziadei, G. A. M. (1979). Neurogenesis and neuron regeneration in the olfactory system of mammals. I.
Morphological aspects of differentiation and structural organization of the olfactory sensory neurons. Journal of Neurocytology,
8(1), 1–18. doi:10.1007/bf01206454
32. Goldman, S. A., & Nottebohm, F. (1983). Neuronal production, migration, and differentiation in a vocal control nucleus of the
adult female canary brain. Proceedings of the National Academy of Sciences, 80(8), 2390–2394. doi:10.1073/pnas.80.8.2390
33. Rakic, P. (1985). Limits of neurogenesis in primates. Science, 227(4690), 1054–1056. doi:10.1126/science.3975601
34. Reynolds, B., & Weiss, S. (1992). Generation of neurons and astrocytes from isolated cells of the adult mammalian central
nervous system. Science, 255(5052), 1707–1710. doi:10.1126/science.1553558
35. Kornack, D. R., & Rakic, P. (1999). Continuation of neurogenesis in the hippocampus of the adult macaque monkey.
Proceedings of the National Academy of Sciences, 96(10), 5768–5773. doi:10.1073/pnas.96.10.5768
36. Eriksson, P. S., Perfilieva, E., Björk-Eriksson, T., Alborn, A.-M., Nordborg, C., Peterson, D. A., & Gage, F. H.
(1998). Neurogenesis in the adult human hippocampus. Nature Medicine, 4(11), 1313–1317. doi:10.1038/3305
37. Kempermann, G., Gage, F. H., Aigner, L., Song, H., Curtis, M. A., Thuret, S., Kuhn, H. G., Jessberger, P. W., Frankland, P. W.,
Cameron, H. A., Gould, E., Hen, R., Abrous, D. N., Toni, N., Schinder, A. F., Zhao, X., Lucassen, P. J., Frisén, J. (2018). Human
Adult Neurogenesis: Evidence and Remaining Questions. Cell Stem Cell, 23(1), 25–30. doi:10.1016/j.stem.2018.04.004
38. Sorrells, S. F., Paredes, M. F., Cebrian-Silla, A., Sandoval, K., Qi, D., Kelley, K. W., James, D., Mayer, S., Chang, J., Auguste, K.
I., Chang, E. F., Gutierrez. A. J., Kreigstein, A. R., Mathern, G. W., Oldham, M. C., Huang, E. J., Garcia-Verdugo, J. M., Yang,
Z., Alvarez-Buylla, A. (2018). Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults.
Nature, 555(7696), 377–381. doi:10.1038/nature25975
39. Boldrini, M., Fulmore, C. A., Tartt, A. N., Simeon, L. R., Pavlova, I., Poposka, V., Rosoklija, G. B., Stankov, A., Arango, V.,
Dwork, A. J., Hen, R., Mann, J. J. (2018). Human Hippocampal Neurogenesis Persists throughout Aging. Cell Stem Cell, 22(4),
589–599.e5. doi:10.1016/j.stem.2018.03.015
40. Germain, J., Bruel-Jungerman, E., Grannec, G., Denis, C., Lepousez, G., Giros, B., Francis, F., Nosten-Bertrand, M.
(2013). Doublecortin Knockout Mice Show Normal Hippocampal-Dependent Memory Despite CA3 Lamination Defects. PLoS
ONE, 8(9), e74992. doi:10.1371/journal.pone.0074992
41. Moreno-Jiménez, E. P., Flor-García, M., Terreros-Roncal, J., Rábano, A., Cafini, F., Pallas-Bazarra, N., Ávila, J., Llorens-
Martín, M. (2019). Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients
with Alzheimer’s disease. Nature Medicine, 25(4), 554–560. doi:10.1038/s41591-019-0375-9
42. Tobin, M. K., Musaraca, K., Disouky, A., Shetti, A., Bheri, A., Honer, W. G., Kim, N., Dawe, R. J., Bennett, D. A., Arfanakis, K.,
Lazarov, O. (2019). Human Hippocampal Neurogenesis Persists in Aged Adults and Alzheimer’s Disease Patients. Cell Stem
Cell, 24(6), 974–982.e3. doi:10.1016/j.stem.2019.05.003