Lecture 3 - Concepts in Regenerative Medicine - Cell Based Therapies and Tissue Engineering

autologous cell based therapy

cells/tissues are obtained from the same individual (serves as donor and recipient)

knee cartilage transplantation (autologous)

find cartilage progenitor cells in unaffected knee then isolate those and grow into sheet of cartilage then place it in the affected knee of the same individual

allogeneic cell based therapy

genetically and antigenically distinct cells/tissues are obtained from different individuals of the same species

what risks are associated with allogeneic transplantation?

• the cells can be recognized as foreign by the immune system so transplant could reject

• immunosuppressive drugs are uses to avoid rejection but predispose people to infections

zeno transplantation

when genetically and antigenically distinct cells/tissues are obtained from different individuals from different species

mice research on bone marrow transplantation

showed that bone marrow transfusion could treat radiation sicknness

bone marrow transplantaion dog research

showed that recovery was improved when the donors were close relatives rather than unrelated donors

identical twin in human treatment

one twin had leukemia and the other didnt, patients were given whole body irradiation to destroy the leukemic blood cells and the bone marrow obtained from the identical twin was transplanted into the patient

what was the logic behind the identical twin transplantation study?

the bone marrow has hemopoietic stem cells in the healthy twin’s niche that could restart the hematopoiesis process with non cancerous cells in the sick twin

LT - HSC (long term reconstituting HSCs)

can regraft into bone marrow of irradiated mice to constitute their whole blood system through serial transplantation

multipotent progenitors

non self renewing but maintain developmental pluripotency of the HSC

human leukocyte antigens (HLAs)

group of polymorphic genes that encode the major histocompatibility complex

major histocompatibility complex

allows the immune system to recognize and respond differently to self vs foreign cell components

• present on every nucleated cell type in our body and could be targeted for destruction by the immune system

Graft versus host disease

when the immune cells derived from the donor bone marrow recognize the transplant recipients’ own tissues as foreign resulting in a widespread inflammation in multiple tissues

polymorphic gene

a genetic region where multiple allelic variants exist amongst individuals in a population

HLA typing and matching

uses sequence specific PCR/restriction enzyme digest at the HLA - (A, B, C, DR, DQ and DP loci)

MHC complex (typing)

have to determine which allele is present at each complex because different alleles will produce proteins with different structures

matching likelihood

25% chance of getting a sibling match or in an unrelated individual from a bone marrow registry

typing

a diagnostic test of cell/tissue DNA to determine immune compatibility

when is GvHD risk high?

when HLA typing reveals only a partial match between available HSC donors and the patient

what is a partial match donor called?

haploidentical donor HSCs

what is the standard protocol for HSC transplantation?

• stem cells are taken from the blood or bone marrow

• they are separated out and incubated with antibodies

• then stem cells that bind with antibodies are selected (FACs)

• fluid volume is reduced

• cells are cryopreserved

• cells are thawed

• cells are diluted

• cells are injected into the patient

when was the role of CCR5 first discovered?

in a patient who received a bone marrow transplant to treat leukemia and was able to clear an HIV infection at the same time

how does human immunodeficiency virus (HIV) work?

the virus primarily infects the CD4+ memory T cells using CCR5 as a coreceptor to get into the T cells

why was this patient able to clear the HIV virus?

due to a mutation in the CCR5 gene

phase 1 gene therapy clinical trial

CD4+ T cells were transplanted autologously then a mutation was introduced before T cell transplant by genome editing

IPS cells

can be used to screen potential drug candidates to select best drug to screen patient pharmacologically

genome editing

could use to correct for what’s causing disease in the patient

reprogramming process

mature patient cells are infected with transcription factors and some not all will take up the yamanaka factors and the fully reprogrammed human iPSC line carries the original background of the donor

human IPSC models of disease

• generate massive number of immune-compatible cells with patient’s own genetic sequences

• understand the molecular changes associated with disease progression (pathogenesis)

• screen drug candidates for their ability to ameliorate/reverse the disease phenotype

why is patient specific medicine using iPSCs not feasible currently?

it is too resource and time intensive and lots of screening is involved which is considering the generation of the iPSCs alone not even the differentiation stage

why are eye stem cells immunoprivileged?

because its hard for cells of the host immune system to access transplanted tissue so less likely for rejection to occur

what were the interventional studies discussed regarding patients with age-related blindness?

the first patient received an autologous transplant and the other 7 received allogeneic transplants from the autologous one

age related blindness study patients results

grafts were detectable up to a year post op and it was vascularized overtime to reduce scaring on the retina. Patients who received iPSC derived retinal pigment epithelium tissue had either slower vision loss or minor improvement in vision

multipotent adult cells

capable of differentiating into adipocytes, chondrocytes and osteoblasts

where are multipotent stromal cells found?

in bone marrow, adipose, umbilicus, synovium, muscles and the perivascular region of the capillaries (anywhere in the body that has vascular beds)

what is the main function of MSCs?

they are immunomodulatory so they can modify immune function and reduce inflammation

adipocytes

fat

chondrocytes

cartilage

osteoblasts

bone

self renewal ability in MSCs is…

limited

pericyte

poorly defined mesenchymal cell population sometimes called mural cells found attached to the walls of vasculature in many organs and some are capable of becoming MSCs in response to injury

what are pericytes also referred to as?

medicinal signaling cells because some can detach from blood vessels and differentiate into MSCs in response to injury or inflammation (initiate paracrine signaling and cell-cell interactions)

donor variation

biological differences between cells derived from different individuals due to age, health, etc

what happened when MSCs were delivered into the body intravenously?

only a small percentage of the cells reached the injured tissue because they get stuck in the lung microvasculature

homing

ability of the MSCs to find their way to the site of injury where repair is needed

how long do MSCs that stay stuck in the lung microvasculature live?

2 to 3 days

how long do MSCs that home correctly live?

for multiple weeks

how does the MSC act as a cellular drugstore?

through various mechanisms to influence tissue regeneration mainly through secreted factors and exosomes/vesicles

what do terminally differentiated cell types derived from MSCs in vitro lack?

they lack paracrine and immunomodulatory functions

how do immunosuppressive effects of MSCs work?

by short range soluble factors and direct cell-cell interactions

what are the immunosuppressive effects mentioned in lecture?

• reduced B celll and T cell division

• reduced activation of the natural killer cell cytotoxicity

• inhibition of dendritic cell maturation

dendritic cell function

take up foreign antigens and present them to the immune system to make antibodies for larger immune response to the foreign protein

what strategy is promising for improving immune tolerance?

co-transplanting MSCs simultaneously with allogeneic donor tissues

what have the preclinical studies combining MSCs in cell and whole organ transplants revealed?

longer term graft stability and improved graft function

whole organ transplant

pancreatic islets, liver and kidney

cell transplants

HSC transplant (in clinical trials), spermatogonial stem cell transplant (chemo-induced infertility)

tissue repair

partial restoration of tissue architecture and function. formation of scar tissue, and may result in structural abnormalities that impair organ function

regeneration

partial restoration of tissue architecture and function, new tissue growth completely restores the damaged area, no scar tissue is formed and no permanent damage remains after healing

what is the role of ECM proteins in repair?

the ECM proteins are secreted creating fibrous tissues in the infarcted area preventing wound from getting worse

scar

fibrous tissue created during wound healing but does not fully replace the mechanical and functional properties of the tissue before injury

myocardial infarction

otherwise known as a heart attack, caused by decreased or complete cessation of blood flow to a portion of the myocardium, can go undetected

neoblasts

cells that drive new cells to be produced during whole-body regeneration or tissue homeostasis (some are pluripotent)

planarians

type of animal where if they undergo any amputation with time the complete structure will regenerate without any scar tissue

axolotl

aquatic salamanders that can regenerate severed limbs or spinal cord as well as some internal organs like the heart and lungs if only partial resection occurs

limitation of axolotl regeneration

there is a decline in regenerative ability with advanced age and when a limb is repeatedly amputated at the same location

collagen remodeling

rearranging the collagen fibers at the site of tissue repair by synthesizing and degrading the collagen matrix

scar formation

the result of mammalian wound healing marked by excessive collagen deposition

when is scar formation considered good?

in skin because the main function is to act as a barrier fromm the environment

fibroblasts

enter the wound and secrete ECM like collagen that helps protect cells migrating within or near the area wound occurred

contractile myofibroblasts

pulls together edges of the wound so it can be properly sealed

why is xenotransplantation not successful

cause its between two different species so very high rejection rate (higher than allogeneic transplantation)

heart

does not regenerate because there are no cardiac stem cells and cardiac scarring is very debilitating

important issues to consider when providing cells for cardiac repair

• source of replacement cells

• starting potency of replacement cells (muti or pluripotent)

• number of replacement cells

• localization of transplanted cells (mobility/integration)

• transplanted cell viability

• cell contamination or transformation (tumor growth)

shape memory injectable scaffolds

microfabricated elastic and biodegradable scaffold that can deliver functional tissue differentiated ex vivo with a minimally invasive single injection

how do injectable scaffolds work?

they help with cells functionally maturing before transplantation and the cells are attached to the scaffold are still viable after squishing it down and injecting it into the body

exosomes

molecules in cell captured by lipid bound spheres and exuded out of cell into external environment, they house proteins and RNA

exosome producing cell types

• cardiomyocytes

• vascular smooth muscle cells

• endothelial cells

• cardiac fibroblasts

• inflammatory cells

• resident stem cells