Examples of Histopathology from Preclinical Cellular Therapy Studies
Julia F.M. Baker, BVMS, DipRCPath, MRCVS
Charles River Laboratories Pathology Associates, Frederick, MD (julia.baker@crl.com)
Introduction
Cellular therapeutics is a branch of Regenerative Medicine and consists of the introduction of
cells into a tissue to effect a cure. It frequently focuses on degenerative or genetically-based
disorders, and may or may not include gene therapy. Strictly used, the term encompasses not
only the use of stem cells, but also the transplantation of bone marrow and other differentiated
cell types, with or without a “medical device” scaffold or capsule.
History
Swiss physician Paul Niehans is known as “the father of cell therapy” for his 1931 treatment of
an elderly woman patient (whose parathyroid had been accidentally removed by a surgeon) with
injections of homogenized calf parathyroid tissue; she lived a further 21 years. Niehans believed
that degenerative conditions could be cured by the injection of live cells removed from healthy
organs1; he went on to treat patients with a variety of conditions using cells derived from the
brain, liver, kidney, duodenum, pancreas, heart, thymus and spleen. In 1949 Niehans pioneered
the first lyophilized cell injections2, and even treated Pope Pius XII in 1953. The practice of
“fresh cell therapy” using cells derived from fetal or young animals remained popular in
Germany for decades although the practice was banned by the German government in 1997
amid growing evidence of severe immune-mediated allergic responses to the repeated
administration of heterologous antigenic material; a current Google search reveals several
German and Swiss clinics which continue to offer such treatments.
However, the first references to cellular therapeutics actually date back thousands of years; the
Egyptian Ebers papyrus of 1552 B.C. recommended preparations made from animal organs to
improve human vitality. In the 1820s, James Blundell performed the first successful human-tohuman blood transfusions at Guy’s Hospital in London3.
The Russian scientist, Alexander Maksimov, coined the term “stem cell” in 1908, referring to the
putative existence of self-renewing hematopoietic precursor cells in the bone marrow4; the
presence of such cells was confirmed in murine bone marrow in 19635. E. Donnall Thomas
performed the first successful human bone marrow transplant between identical twins in 1956,
and the first successful allogeneic bone marrow transplant was performed between siblings in
Minnesota in 1968. It was not until 1973 that a successful bone marrow transplant was
performed between an unrelated donor and recipient.
Since the first derivation of embryonic stem cells from the inner cell mass of mouse embryos in
19816, interest (and controversy) surrounding cellular therapeutics has boomed: fetal cells have
been reported to repair spinal cord injury7 and to reverse the signs of Parkinson’s disease8,9;
ectopically implanted pancreatic islets have been used in attempts to restore insulin production
in diabetes10,11,12; encapsulated chromaffin cells have been suggested for pain management13;
stem cells have been claimed to improve vision in patients with macular degeneration14; limbal
stem cell grafting has given promising results in cases of corneal surface destruction15. Several
clinical trials are ongoing using stem cells16. These include: two trials using retinal pigment
epithelium derived from human embryonic stem cells (hESC) to combat Stargardt’s macular
dystrophy in children, and age-related macular degeneration in adults; a study using human
spinal cord stem cells to treat Amyotrophic Lateral Sclerosis (Lou-Gehrig’s disease); multiple
trials using adult mesenchymal stem cells derived from bone marrow, looking at protecting beta
islet cells in cases of newly diagnosed type 1 diabetes, the repair of damaged myocardial cells
following infarct, and in the treatment of Crohn’s disease and graft-versus-host disease; and
multiple trials on the use of limbal stem cell grafting. It seems highly probable that this field of
research will continue to grow in coming years.
What do we mean by “stem cell”?
Stem cells have two defining characteristics:


The cells are unspecialized and are capable of reproducing themselves, often after long
periods of inactivity
They can be induced to differentiate into tissue-specific or organ-specific cells with
specialist properties under certain physiologic or experimental conditions
There are two major categories of stem cell:


Embryonic stem cells are derived from embryos a few days old; these cells are
“pluripotent” – they are capable of producing any type of cell found in the body.
Somatic, or “adult,” stem cells are present in many tissues, as well as in the fetus and in
cord blood. In some locations, such as the bone marrow and intestine, the cells divide
regularly, but elsewhere they may divide only under rare circumstances – heart,
pancreas, muscle, brain. These cells are “multipotent” – they can differentiate into
several pre-defined cell types; an example is the neural stem cell population in the brain,
which can produce neurons, astrocytes and oligodendrocytes.
The genes involved in the maintenance of pluripotency of mouse embryonic stem cells were
confirmed as Oct3/4, Sox2, c-myc, and Klf4 [Kruppel-like factor 4] in 200617.
Adult stem cells may be genetically modified by the transduction of such genes to produce
induced pluripotent stem cells (iPS cells)18,19. Such genetic manipulation and nuclear transfer
technology is technically challenging and recent work has shown a method of induction using a
cocktail of small molecules, to produce chemically induced pluripotent stem cells (CiPS cells)20.
Needles in haystacks – identifying administered cells with confidence
It is critical to be able to accurately identify the administered cells within the host animal. Given
that the aim of the procedure (particularly in the case of stem cell therapy) is for the
administered cells to take on the characteristics of appropriately differentiated cells for that
location, specific and robust cell markers must be identified and developed to achieve this. Such
markers may be used for immunohistochemical staining of the tissues. Species-specific markers
including human mitochondrial antigen and other commercially available markers have been
used with success in our lab.
Careful selection of animal models will also aid in identifying administered cells – for example, if
the administered cells contain pigment, it would be prudent to choose an albino model where
feasible.
Regulatory demands to retain all tissue from preclinical safety studies using stem cells result in
logistical problems and high costs for the pathology portion of the study; careful study design
strategies, such as careful tissue selection, separate consideration of different groups, and the
strategic trimming of tissue can be used to help mitigate these effects.
Aims of this presentation
Cellular therapeutics is a field of growing importance. However, it is a field associated with
controversy – particularly in the case of embryonic stem cell use, which has caused many
groups to raise ethical and religious questions. Added to that, issues of client confidentiality
make the sharing of images from preclinical cellular therapy studies problematic. In this
presentation I shall aim to show as many examples as I am able, as well as giving an overview
of some of the issues to consider when designing the pathology component for a preclinical
program using cellular therapeutics.
References
1. Niehans P. Introduction to Cellular Therapy. New York: Cooper Square Publishers Inc.,
1960.
2. Ingelheim AZ. Niehans’ cellular therapy with lyophilized cells [in German]. Hippokrates
25(17):542-4, 1954.
3. Blundell J. Observations on transfusion of blood by Dr Blundell with a description of his
gravitator. Lancet ii:321-4, 1828.
4. Igor EK. The Man behind the Unitarian Theory of Hematopoiesis. Perspect Biol Med
43:269–76, 2000.
5. Becker AJ, McCulloch EA, Till JE. Cytological demonstration of the clonal nature of
spleen colonies derived from transplanted mouse marrow cells. Nature 197:452–4,
1963.
6. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse
embryos. Nature 292:154–6, 1981.
7. McDonald JW, Liu XZ, Qu Y, Liu S, Mickey SK, Turetsky D, Gottlieb DI, Choi DW.
Transplanted embryonic stem cells survive, differentiate and promote recovery in injured
rat spinal cord. Nature Medicine 5(12):1410-2, 1999.
8. Freed CR, Breeze RE, Rosenberg NL, Schneck SA, Wells TH, Barrett JN, Grafton ST,
Huang SC, Eidelberg D, Rottenberg DA. Transplantation of human fetal dopamine cells
for Parkinson’s disease: Results at 1 year. Arch Neurol 47(5):505-12, 1990.
9. Kim JH, Auerbach JM, Rodriguez-Gomez JA, Velasco I, Gavin D, Lumelsky N, Lee SH,
Nguyen J, Sanchez-Pernaute R, Bankiewicz K, McKay R. Dopamine neurons derived
from embryonic stem cells function in an animal model of Parkinson’s disease. Nature
418:50-6, 2002.
10. Fan MY, Lum ZP, FuXW, Levesque L, Tai IT, Sun AM. Reversal of diabetes in BB rats
by transplantation of encapsulated pancreatic islets. Diabetes 39:519-22, 1990.
11. Ramiya VK, Maraist M, Arfors KE, Schatz DA, Peck AB, Cornelius JG. Reversal of
insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells.
Nature Medicine 6:278-82, 2000.
12. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R. Differentiation of
embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science
292:1389-94, 2001.
13. Sagen J, Wang H, Tresco PA, Aebischer P. Transplants of immunologically isolated
xenogeneic chromaffin cells provide a long-term source of pain-reducing neuroactive
substances. Journal of Neuroscience 13(6):2415-23, 1993.
14. Schwartz SD, Hubschman JP, Heilwell G, Franco-Cardenas V, Pan CK, Ostrick RM,
Mickunas E, Gay R, Klimanskaya I, Lanza R. Embryonic stem cell trials for macular
degeneration: a preliminary report. Lancet 379(9817):713-20, 2012.
15. Tsubota K, Satake Y, Kaido M, Shinozaki N, Shimmura S, Bissen-Miyajima H,
Shimazaki J. Treatment of severe ocular-surface disorders with corneal epithelial stemcell transplantation. N Engl J Med 340:1697-1703, 1999.
16. National Institutes of Health. Clinical trials for stem cell therapies. Stem Cell Information
http://stemcells.nih.gov/info/pages/health.aspx, 2012.
17. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic
and adult fibroblast cultures by defined factors. Cell 126:663–76, 2006.
18. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of
pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:1–12,
2007.
19. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al.
Induced pluripotent stem cell lines derived from human somatic cells. Science
318:1917–20, 2007.
20. Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, Zhao T, Ye J, Yang W, Liu K, Ge J, Xu J,
Zhang Q, Zhao Y, Deng H. Pluripotent stem cells induced from mouse somatic cells by
small-molecule compounds. Science 341:651-4, 2013.