1. Development of Induced Pluripotent Stem Cell-based
Therapies for Hematopoietic Stem Cell Disorders
Joseph A. Panos, Luigi J. Alvarado Ph.D., André Larochelle M.D. Ph.D.
National Heart Lung and Blood Institute, Hematology Branch, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892
Mouse Transplantation. Immune-deficient (NSG) mice were
injected i.v. with ~1x106 human CD34+ cells or iPSC-derived
HSPCs immediately after a 4.5 hour-pre-culture at 39.5°C or
37°C. Twenty-four hours post-injection, mice were euthanized,
their BM harvested from 6 bones, stained with anti-human
CD45-FITC antibodies, and analyzed by flow cytometry.
Confocal Microscopy. For visualization of membrane domains,
cells were stained with AlexaFluor 555-conjugated Cholera toxin
subunit B and visualized with a Zeiss LSM 780 confocal
microscope.
Hematopoietic stem cells (HSCs) have the remarkable ability to
reconstitute and maintain a functional hematopoietic system for
the lifespan of an individual. In patients with inherited bone
marrow (BM) failure disorders, HSCs are insufficient. Allogeneic
HSC transplantation offers a potential cure, but there are well-
described limitations. Also, sufficient autologous HSCs for gene
therapy applications are unavailable in these patients, and
methodologies for expansion of these rare HSCs are inexistent.
Induced pluripotent stem cell (iPSC)-based therapies are a
tractable alternative for these patients because of their potential
to provide an unlimited source of autologous cells for gene
therapy applications. Several protocols have been developed to
generate hematopoietic stem and progenitor cells (HSPCs) from
iPSCs but they are inefficient at producing the quantity and
quality of HSCs required for clinical applications. Namely,
phenotypically defined HSCs derived from iPSCs are incapable
of reconstituting the hematopoietic system long-term in animal
models; the cause of this functional defect remains unclear.
We have recently shown that homing and engraftment of
HSPCs to their BM microenvironment is mediated by polarized
membrane domains enriched in adhesion molecules. The
presence of polarized membrane domains on iPSC-derived
HSCs has never been investigated as a possible cause for the
homing/engraftment defect. A recent study has suggested that
brief exposure of native HSPCs to hyperthermic conditions
enhances domain polarization, homing, and engraftment.
There are no relevant conflicts of interest to disclose.
21 days post-transplant
Introduction
Objectives
Methods
Results
Conclusions
Hyperthermic treatment of human CD34+ cells before
transplantation enhances their BM homing potential
Summary
Impact of iPSC plating density on HSPC differentiation
1. The protocol optimized in this study provides an efficient
approach for the generation of human HSPCs with a
primitive CD45lo/CD34hi phenotype from iPSCs generated
from mobilized CD34+CD38- cells.
2. Initial plating density of iPSCs was identified as a critical
factor for efficient hematopoietic differentiation, with 70,000
iPSCs as the most efficient cell density for generation of
CD34hi/CD45lo progenitors with the iPSC line utilized.
3. Days 10-14 of the differentiation protocol were identified as
optimal collection times for HSPCs.
Hyperthermic Treatment and HSPC Homing
Results
Human iPSCs were reprogrammed using Sendai viral vectors
from normal G-CSF-mobilized CD34+/CD38- cells. iPSC
colonies were cultured on Matrigel-coated plates in E8 medium.
Cells were exposed to a cocktail of mesodermal differentiation
factors for 3 days in APEL medium, followed by hematopoietic
differentiation for 10 days. iPSC-differentiated cells were
harvested from the culture supernatant starting at Day 10.
Methods
Results
Hematopoietic Differentiation from Human iPSCs
Time course depiction of HSPC generation from iPSCs
Exposure to mesodermal and hematopoietic differentiation
factors promoted the formation of a hemogenic endothelial
monolayer. As early as Day 7, spherical hematopoietic-like cells
formed superficial clusters over the endothelial monolayer.
These cells spontaneously sprouted in the culture supernatant
between Days 10 and 14, allowing easy harvest.
Optimization of iPSC plating density
The initial iPSC plating density was identified to be critical for
optimal hematopoietic differentiation from human iPSCs.
Homing of human CD34+ cells was measured as the
percentage of human CD45+ cells in the mouse BM 24 hours
after transplantation. The proportion of CD45+ cells was
significantly greater in the heat-treated group compared to the
heat-untreated group.
Future Directions
1. RNA-sequencing assays will be performed to assess the
genetic similarities between iPSC-derived HSCs and bona
fide human HSCs.
2. Functional homing experiments using iPSC-derived HSCs
will be performed with adjuvants, such as a mild
hyperthermic treatment, to assess the potential capacity of
iPSCs to serve as a viable source of HSCs for bone marrow
failure transplantation therapies.
References
[1] Focosi, D., et al. Blood Cancer J., 2014.
[2] Kaufman, D.S. Blood, 2009.
Generation of human HSPCs (CD45+CD34+)
from iPSCs at various iPSC cell density Heat-treatment promotes the polarization of lipid rafts.
Density (cells/
plate)
CD34+/CD45+
count
CD34lo/CD45hi
count
CD34hi/CD45lo
count
CD34hi /CD45lo -
CD34lo /CD45hi
Ratio
Efficiency
of HSPC
generation
70,000 151,438 15,003 30,439 2.03 6.49
90,000 17,523 819 5,291 6.56 1.71
100,000 50,607 1,701 15,485 9.10 1.51
Estimation of various HSPC counts shows that the density
condition of 70,000 cells per plate most efficiently generated the
desired CD34hi/CD45lo progenitor population for harvests
conducted at Day 12.
[3] Capitano, M., et al. Stem Cells, 2015.
[4] Larochelle, A. Blood, 2012.
1. Develop a scalable protocol that supports hematopoietic
differentiation of human iPSCs to meet the threshold cell
dose of HSPCs for clinical transplantation.
2. Investigate the presence of polarized membrane domains on
native and iPSC-derived HSPCs, and the potential role of
hyperthermia to enhance domain polarization, HSPC
homing, and engraftment.
CD34hi/CD45lo (primitive HSPCs) and CD34lo/CD45hi (more
mature HSPCs) populations, were observed in supernatant
collected from all cultures with initial iPSC plating densities
>70,000 cells per plate.
70,000 iPSCs 90,000 iPSCs 100,000 iPSCs
Hematopoietic Differentiation from Human iPSCs
Hyperthermic Treatment and HSPC Homing
Negative control 37oC treatment 39.5oC treatment
Hyperthermic Treatment and Domain Polarization
Lipid rafts on heat-treated and heat-untreated cells were
detected directly using AlexaFluor 555-conjugated Cholera toxin
subunit B, which binds specifically to ganglioside GM1. The
polarization of lipid rafts, as a result of heat treatment, may
explain enhanced CD34+ homing to the bone marrow niche.
Hyperthermic Treatment and HSPC Homing
Hematopoietic Differentiation from Human iPSCs
1. Brief hyperthermic treatment of 39.5°C of native human
CD34+ cells is effective in increasing their homing within the
BM of NSG mice.
2. Enhanced homing may be related to increased polarization
of membrane-domain, enriched in adhesion molecules.
3. These findings provide proof-of-principle that this approach
may enhance homing and engraftment of iPSC-derived
HSPCs.