Panos_NHLBI_Final Report

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

INTRODUCTION

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.

Accordingly, this project endeavored to: 1. Develop a scalable protocol that supports hematopoietic

differentiation of human iPSCs to meet the threshold cell dose of HSPCs for clinical transplantation and

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.

METHODS

Hematopoietic Differentiation from Human iPSCs

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 (Figure 1).

Figure 1. Experimental schematic for the generation of HSCs from iPSCs.

Hyperthermic Treatment and HSPC Homing

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. 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.    

RESULTS

Hematopoietic Differentiation from Human 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 (Figure 2).

Figure 2. Time course depiction of HSPC generation from iPSCs.

The initial iPSC plating density was identified to be critical for optimal hematopoietic differentiation

from human iPSCs (Figure 3).

Figure 3. Plating densities exceeding 70,000 cells/plate allowed for multiple HSPC harvests on both

Days 12 and 14.

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

(Figure 4).

70,000 iPSCs 90,000 iPSCs iPSCs

100,000 iPSCs

Figure 4. Generation of human iPSCs (CD45+CD34+) from iPSCs at various iPSC plating densities.

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.

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

Hyperthermic Treatment and HSPC Homing

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 (Figure 5).

Figure 5. Heat treatment confers a greater homing capacity among human CD34+ cells.

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

(Figure 6).

Figure 6. Heat-treatment promotes the polarization of lipid rafts.

CONCLUSIONS

Hematopoietic Differentiation of Human iPSCs

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.

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. Days 10-14 of the differentiation protocol were identified as

optimal collection times for HSPCs.

Hyperthermic Treatment and HSPC Homing

Brief hyperthermic treatment of 39.5°C of native human CD34+ cells is effective in increasing their

homing within the BM of NSG mice. Enhanced homing may be related to increased polarization of

membrane-domain, enriched in adhesion molecules. These findings provide proof-of-principle that this

approach may enhance homing and engraftment of iPSC-derived HSPCs.

REFERENCES

1. Focosi, D., Amabile, G., Di Ruscio, A., Quaranta, P., Tenen, D.G., Pistello, M. (2014).

Induced pluripotent stem cells in hematology: current and future applications. Blood

Cancer Journal, 2(114), 1-8.

2. Kaufman, D.S. (2009). Toward clinical therapies using hematopoietic cells derived from

human pluripotent stem cells. Blood, 114(7), 3513-3523.

3. Capitano, M.L., Hangoc, G., Cooper, S., Broxmeyer H.E. (2015). Mild Heat Treatment

Primes Human CD34+ Cord Blood Cells for Migration Toward SDF-1 and Enhances

Engraftment in an NSG Mouse Model. Stem Cells Express, 33, 1975-1984.